© 2014. Published by The Company of Biologists Ltd.
Type Iγ phosphatidylinositol phosphate kinase targets to the centrosome and restrains centriole duplication Qingwen Xua, Yuxia Zhangb, Xunhao Xionga, Yan Huanga, Jeffery L. Salisburya, Jinghua Hua,b, Kun Linga,1 a
Department of Biochemistry and Molecular Biology, and Division of Hypertension and Nephrology, Mayo Clinic, 200 First Street SW, Rochester, MN 55902.
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b
1
Correspondence Author:
Kun Ling, Ph.D. Stabile 7-06 200 First Street SW Mayo Clinic Rochester, MN 55902 Tel: 507-293-3498 [email protected]
Running Title: PIPKIγ restricts centriole biogenesis Key Words: Centriole duplication; PLK4; CEP152; CEP192
Length of the manuscript: 7,988 words.
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JCS Advance Online Article. Posted on 16 January 2014
Summary
Centriole biogenesis depends on the Polo-like kinase PLK4 and a small group of structural proteins.
The spatiotemporal regulation of these proteins at pre-existing centrioles is
critical to ensure that centriole duplication occurs once per cell cycle. Here we report that type Iγ phosphatidylinositol-4-phosphate 5-kinase (PIPKIγ) plays an important role in centriole fidelity. Depending upon an association with CEP152, PIPKIγ localized in a
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ring-like pattern in the intermediate pericentriolar materials around the proximal end of the centriole in G1, S, and G2 phases, but not in M phase. Without detaining cells in S or M phase, depletion of PIPKIγ led to centriole amplification in a PLK4/SAS-6 dependent manner. Expression of exogenous PIPKIγ reduced centriole amplification resulted from endogenous PIPKIγ depletion, hydroxyurea treatment, or PLK4 overexpression, suggesting that PIPKIγ likely functions at the PLK4 level to restrain centriole duplication. Importantly, we found that PIPKIγ bound to the cryptic Polo-Box domain of PLK4 and this binding reduced PLK4 kinase activity. Together, our findings suggest that PIPKIγ is a novel negative regulator of centriole duplication by modulating the homeostasis of PLK4 activity.
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Introduction
The centrosome, composed of one pair of centrioles and surrounding pericentriolar material (PCM), is the major microtubule organization center and plays a critical role in mitotic spindle assembly, primary ciliogenesis, and cell morphogenesis (Debec et al., 2010; Nigg and Raff, 2009). In cycling cells, the centrosome duplicates once in S-phase so that each daughter cell can inherit one centrosome after mitosis.
Abnormal number of centrosomes results in mis-assembled
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spindles that frequently gives rise to chromosome mis-segregation, genomic instability, and aneuploid daughter cells (Ganem et al., 2009), linking centrosomal anomalies to many human diseases including a variety of different aneuploid tumors (Ghadimi et al., 2000; Lingle et al., 2002; Nigg, 2006; Pihan et al., 1998; Yamamoto et al., 2004) and microcephaly syndromes (Megraw et al., 2011; Nigg and Raff, 2009). The procentriole is assembled by a small group of highly conserved proteins and is strictly limited to one per pre-existing centriole and once per cell cycle. The availability and activity of these proteins at the centrosome have to be carefully controlled to maintain the fidelity of centriole/centrosome duplication (Bettencourt-Dias et al., 2005; Eckerdt et al., 2011; Kleylein-Sohn et al., 2007; Nigg, 2007; Nigg and Stearns, 2011). It has been demonstrated that the Polo-like kinase PLK4 is a key regulator of centriole formation and excessive PLK4 activity leads to centriole amplification (Bettencourt-Dias et al., 2005; Eckerdt et al., 2011; Kleylein-Sohn et al., 2007; Nigg, 2007; Nigg and Stearns, 2011). PLK4 is recruited to the centrosome by and controls the onset of centriole assembly together with CEP152, which also provides a platform for CPAP binding to the centrosome (Cizmecioglu et al., 2010; Dzhindzhev et al., 2010). PLK4-mediated autophosphorylation and phosphorylation prime itself or its substrate like FBXW5 for ubiquitination and destruction, therefore controls the availability of itself (Brownlee and Rogers, 2012; Cunha-Ferreira et al., 2009; Guderian et al., 2010a; Holland et al., 2010; Rogers et al., 2009) or other component of centriole duplication machinery like SAS-6 (Puklowski et al., 2011) to confine centriole number. On the other hand, a protein phosphatase PP2A was reported to promote centriole formation by facilitating SAS-6 accumulation to the nascent centriole (Brownlee et al., 2011; Kitagawa et al., 2011; Song et al., -3-
2011). These results support an intrinsic, critical balance between kinase and phosphatase activities during centriole duplication, and put PLK4 into the center of the centriole assembly machinery. In spite of the importance of controlling PLK4 activity accurately, the underlying mechanism is far from fully understood. Type Iγ phosphatidylinositol-4-phosphate 5-kinase (PIPKIγ) is a lipid kinase that phosphorylates
phosphatidylinositol
4-phosphate
to
generate
phosphatidylinositol
4,5-bisphosphate (PI4,5P2). Up to date, six alternative splicing isoforms of PIPKIγ have been
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reported (Giudici et al., 2004; Giudici et al., 2006; Ishihara et al., 1998; Schill and Anderson, 2009; Xia et al., 2011). PIPKIγ isoforms target to distinct subcellular locales. By regulating the dynamics of regional PI4,5P2 pools, they are implicated in distinct cellular processes such as vesicular trafficking (Bairstow et al., 2006; Ling et al., 2007b; Sun et al., 2013; Thapa et al., 2012; Thieman et al., 2009; Xiong et al., 2012), calcium signaling (Wang et al., 2004), or cell adhesion and migration (Di Paolo et al., 2002; El Sayegh et al., 2007; Ling et al., 2007a; Ling et al., 2002; Sun et al., 2007; Thapa et al., 2012; Thieman et al., 2009; Wang et al., 2004; Xiong et al., 2012). Moreover, PIPKIγ can regulate some protein such as E-cadherin (Ling et al., 2007b) by directly interacting with it. In the present study, we report that PIPKIγ_i3 targeted to the centrosome and negatively regulated centriole duplication. This lipid kinase associated with the proximal ends of parental centrioles in a cell cycle-dependent manner, just like many other centrosomal proteins. Similar to PLK4 and CPAP, residence of PIPKIγ at the centrosome depended upon CEP152. Loss of PIPKIγ resulted in centriole amplification without blocking the S or G2/M phase, indicating that PIPKIγ is a negative regulator of centriole duplication. Indeed, further investigation revealed that PIPKIγ directly interacts with PLK4 and inhibits its activity.
These results suggested an
additional level of regulation for PLK4 activity and centriole biogenesis in addition to phosphorylation–dependent protein ubiquitination and degradation.
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Results PIPKIγ localizes at the centrosome Using the rabbit polyclonal antibodies reported previously that recognizes all of the PIPKIγ splicing isoforms, we observed that PIPKIγ localizes to multiple subcellular locales including focal adhesion, adherens junction, recycling endosome (Ling et al., 2007a; Ling et al., 2002), as well as the centrosome. To investigate the unexpected centrosome localization of PIPKIγ, we
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generated new rabbit polyclonal and monoclonal antibodies to ensure that these are not artifacts from the previous antibody. Both the purified polyclonal antibodies (Fig. S1B) and a monoclonal antibody against PIPKIγ (Fig. 1A, left panel) decorated the centrosome highlighted by the centrosomal marker Centrin2 in HeLa cells. In contrast, purified rabbit polyclonal antibodies specifically recognizing PIPKIα or PIPKIβ (Fig. S1A) did not show staining around the centrosome (Fig. 1A). The centrosomal signal picked by anti-PIPKIγ antibody was completely abolished after the treatment of a PIPKIγ siRNA targeting all PIPKIγ isoforms (Fig. 1B) or in the presence of excess PIPKIγ but not PIPKIα protein (Fig. S1B), supporting the authenticity of this PIPKIγ localization. In addition, we isolated centrosomes from HeLa cells (Fig. 1C) or mouse kidney (Fig. S1C) using sucrose gradient centrifugation and analyzed the fractions by Western blot. Similar to other centrosomal proteins those were identified using this method (Kaplan et al., 2004), PIPKIγ co-fractionated with the centrosome components human SAS-6 (referred to HsSAS-6 from now on), CPAP, and γ-tubulin, which further supports that PIPKIγ is associated with centrosomes as suggested by immunofluorescence microscopy. Moreover, nocodazole treatment had no effect on PIPKIγ signal at the centrosome, although it efficiently interrupted the microtubule integrity (Fig. S1D), indicating that the association between PIPKIγ and the centrosome is independent of the microtubule cytoskeleton. These results demonstrated that PIPKIγ was a bona fide component of the centrosome. In addition to HeLa cells, we also observed PIPKIγ at the centrosome in MDA-MB-231, U-2 OS, IMCD3 and NIH3T3 cells (Fig. S1E and unpublished data), suggesting a pervasive and potentially functional role of PIPKIγ at the centrosome.
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To determine which PIPKIγ isoform targets to centrosomes, we fused a HA tag to the N-terminus of all six known PIPKIγ splicing variants. As shown in Fig. 1D and Fig. S1F, only PIPKIγ_i3 displayed a centrosome-associated signal. Other isoforms showed vesicle-like cytoplasmic targeting or localized to focal adhesions (PIPKIγ_i2) as reported (Di Paolo et al., 2002; Ling et al., 2002). PIPKIγ_i3 contains a unique 26-amino acid insertion (a.a. 641-666) at the C-terminus between the end of PIPKIγ_i1 and the beginning of the PIPKIγ_i2 C-terminal extension (Fig. 1E).
However, the C-terminal region of PIPKIγ_i3 (a.a. 445-End) is
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predominantly cytoplasmic (Fig. 1D, right panel). The N-terminal (a.a. 1-445) fragment of PIPKIγ_i3, which is common in all PIPKIγ variants, localized to the centrosome (Fig. 1D, middle panel). These results suggest that the centrosome-targeting motif is likely within the N-terminal 445 amino acids. Since PIPKIγ_i3 is the only one that showed centrosome targeting, this raises a possibility that the unique C-terminal insertion of PIPKIγ_i3 might provoke a conformational adjustment to expose the centrosome-targeting motif embedded in the first 445 amino acids. PIPKIγ targets to the proximal end of centrioles in a cell cycle-dependent manner To examine the fine-structural details of PIPKIγ association with the centrosome, we co-labeled HeLa cells with anti-PIPKIγ antibody and antibodies against various centrosome components. The localizations of these proteins around the centrosome were analyzed using the Three Dimensional Structured Illumination Microscopy (3D-SIM). When co-stained with Ninein, a subdistal appendage marker on the mother centriole (Nakagawa et al., 2001) and Centrin2 that marks the distal end of the centriole cylinder (Paoletti et al., 1996), PIPKIγ was positioned below Ninein and Centrin2 (Fig. 2A). Further examinations implied that PIPKIγ localized between Ninein and C-Nap1, a linker protein between centrioles (Mayor et al., 2000) (Fig. 2B), as well as C-Nap1 and ODF2 that marks the distal appendage of the mature mother centriole (Piel et al., 2000) (Fig. 2C). In these images, PIPKIγ seemed distributing along the side of the centriole wall in the pericentriolar matrix and likely formed a ring around the proximal end of centrioles with a diameter smaller than Ninein. To address if the centrosomal localization of PIPKIγ is cell cycle dependent as many other -6-
centrosome proteins, we subjected HeLa cells to immunofluorescence microscopy to visualize PIPKIγ together with Centrin2 and DAPI. Based on the staining of Centrin2 and DAPI, cells were categorized as in G1-phase (2 Centrin2 foci and intact nuclear membrane), S-phase (4 clustered Centrin2 foci and intact nuclear membrane), G2-phase (2 separated pairs of Centrin2 foci and intact nuclear membrane), and variant stages of M-phase (broken nuclear membrane and condensed chromosome DNA arranged in different ways).
PIPKIγ staining around the
centrosome was observed in cells at G1-, S-, and G2-phase (Fig. S1G). At the prophase of mitosis,
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PIPKIγ signal diminished and remained absent from centrosomes until the late telophase, when PIPKIγ is recruited back to both centrosomes that would soon be inherited by each daughter cell (Fig. S1G).
Consistent with its centrosome targeting, protein level of PIPKIγ decreases
significantly as cells progress through mitosis and then recovers at mitotic exit in synchronized HeLa cells (Fig. 2D). This follows the same trend as CPAP (Tang et al., 2009) (Fig. 2D), a CEP152-interacting protein which functions early in centriole duplication, however is different from HsSAS-6 (Fig. 2D) that is degraded during the late M phase and absent in the G1-phase (Strnad et al., 2007), suggesting that PIPKIγ may have a specific function at the centrosome. PIPKIγ targeting to the centrosome is dependent on an association with CEP152 CEP152 is a scaffold protein localizing around the proximal end of centrioles and is required for the centrosomal localization of PLK4 and CPAP (Cizmecioglu et al., 2010; Dzhindzhev et al., 2010; Hatch et al., 2010). Since PIPKIγ also localizes around the proximal end of centrioles, we examined whether CEP152 was responsible for the targeting of PIPKIγ to the centrosome. The physical
association
between
PIPKIγ
and
CEP152
was
first
determined
using
co-immunoprecipitation assay. Indeed, overexpressed GFP-CEP152 pulled down HA-tagged PIPKIγ_i3 from HEK293T cells (Fig. 3A), suggesting that these two proteins have the potential to bind each other. The CEP152 antibody recognized multiple bands (Fig. 3B) that could be all knocked down by CEP152 specific siRNA (Fig. 3D), suggesting that endogenous CEP152 exists in variant forms. Endogenous PIPKIγ was co-immunoprecipitated with the endogenous CEP152 protein that migrated faster on SDS-PAGE gel (Fig. 3B). Although it is speculative at this point whether this PIPKIγ-associated CEP152 is an unmodified, truncated, or alternatively spliced form -7-
of CEP152, our results indeed supports an in vivo physical connection between these two proteins. Because the recombinant full length CEP152 expressed in E. coli is highly degraded or insoluble no matter fused to GST, His, or MBP tag, we constructed and purified the MBP-tagged CEP152 fragments and tested their interaction with His-PIPKIγ. Both N-terminal 748-aa (CEP1521-748) and C-terminal 906-aa (CEP152749-1654) pulled down PIPKIγ (Fig. S2A).
However, the
N-terminal 217 a.a. of CEP152 (CEP1521-217), where PLK4 directly binds to (Hatch et al., 2010), did not interact with PIPKIγ (Fig. S2A). These data suggest that PIPKIγ might directly bind to
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the C-terminus of CEP152, but further investigation is needed to obtain a solid conclusion because of the heavy degradation of our recombinant CEP152 polypeptides. To understand the biophysical significance of the association between PIPKIγ and CEP152, we first tested if they co-localize at the centrosome. It has been reported recently by multiple groups that centrosomal proteins including CEP152 form a ring-like structure arount the centriole (Fu and Glover, 2012b; Lawo et al., 2012; Mennella et al., 2012b; Sonnen et al., 2012b). Consistently, our 3D-SIM images supported a ring-like colocalization between PIPKIγ and Flag-CEP152 (Fig. 3C), suggesting that PIPKIγ localizes in the intermediate pericentriolar matrix around the proximal end of centrioles as CEP152 does (Fu and Glover, 2012b; Lawo et al., 2012; Mennella et al., 2012b; Sonnen et al., 2012b). More importantly, the colocalization between CEP152 and PIPKIγ reinforced the physical interaction and suggested a functional correlation between these two proteins. Indeed, siRNA-mediated depletion of CEP152 (Fig. 3D) led to a loss of PIPKIγ signal from the centrosome (Fig. 3E, H), indicating that CEP152 is necessary for the recruitment of PIPKIγ to the centrosome. Moreover, depletion of CEP152 also eliminated the centrosome targeting of exogenous PIPKIγ_i31-445 (Fig. S2B), further suggesting that CEP152 provides a necessary structural platform for the stable association of PIPKIγ with the centrosome. In addition to CEP152, CEP192 was recently shown critical for the recruitment of centrosomal proteins (Sonnen et al., 2013). In CEP192-depleted cells, the centrosomal signal of CEP152 was largely eliminated (Fig. 3G) as reported (Sonnen et al., 2013); however, PIPKIγ signal around the centrosome was mostly retained (Fig. 3G, H). Combining with our previous observation that complete loss of CEP152 blocked PIPKIγ targeting to the centrosome (Fig. 3E -8-
and 3H), these results suggest that very small amount of centrosomal CEP152 could be plenty for the centrosome recruitment of PIPKIγ. PIPKIγ negatively regulates centriole duplication The specific, cell cycle-dependent centrosomal localization of PIPKIγ led us to investigate the potential function of PIPKIγ at the centrosome. For this purpose, we knocked down PIPKIγ in HeLa cells using lentivirus-based PIPKIγ-specific shRNA (Fig. 4A). Strikingly, loss of PIPKIγ
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resulted in an augmentation of Centrin2 foci (> 4 per cell) in ~ 20% of the cells (Fig. 4B, C). Importantly, centriole amplification caused by depletion of PIPKIγ was almost fully rescued by expression of RNAi-resistant PIPKIγ (Fig. 4D, E). Similar level of Centrin2 foci increase was also observed in HeLa cells treated with two distinct PIPKIγ siRNAs (siPIPKIγ-O1 and siPIPKIγ-O2) (Fig. S3A, B), but not in PIPKIα- or PIPKIβ-depleted cells (Fig. S3F, G), further confirmed that this phenotype was caused by PIPKIγ depletion. To determine if the increased Centrin2 foci were resulted from cell cycle arrest in S phase or G2/M phase, we examined the cell cycle distribution of PIPKIγ-depleted cells. BrdU incorporation (Fig. S3C) and PCNA staining (Fig. S3D) were used to determine the S phase and phospho-Histone H3 (Fig. S3E) was used to determine the G2/M phase. As shown in Fig. S3C-E, no significant difference was observed between cells treated with siPIPKIγ or cells treated with control siRNA. Therefore, the excessive Centrin2 foci in PIPKIγ-depleted cells were not likely resulted from a sustained S, G2, or M phase. Furthermore, depletion of PIPKIγ but not PIPKIα or PIPKIβ caused an increase of centrosome number (>2 γ–tubulin loci per cell) in other types of cells like NIH 3T3 (Fig. S3H, I) and renal collecting duct epithelial IMCD3 (Fig. S3J). These results suggest that PIPKIγ might inhibit centriole duplication. To test this possibility, we examined if expression of PIPKIγ could reverse centrosome amplification caused by hydroxyurea (HU) treatment. As shown in Fig. 4F, U2 OS cells were treated with HU for 24 hr before being transfected with or without wild-type or kinase-dead HA-PIPKIγ. This experimental design limits the impact of overexpressed PIPKIγ in S phase and excluded the potential influence on other phases of cell cycle (Hemerly et al., 2009). As shown in Fig. 4G, HU-induced centrosome amplification in U-2 OS cells was significantly suppressed by -9-
overexpressed HA-PIPKIγ, suggesting that PIPKIγ is a negative regulator of centrosome duplication.
Expression of both wild type and kinase dead PIPKIγ could rescue centriole
overduplication caused by PIPKIγ depletion or HU treatment, suggesting that PI4,5P2 might not play a significant role in centriole biogenesis. To further characterize these excessive Centrin2 foci in PIPKIγ-depleted cells, we checked the localization of several centriolar proteins including CP110 (localizes at the distal ends of centrioles (Kleylein-Sohn et al., 2007)), Centrin2 (localizes at the distal ends of centrioles),
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CEP135 (localizes at the proximal end of centrioles (Kleylein-Sohn et al., 2007)), and CPAP (localizes at the proximal ends of centrioles (Kleylein-Sohn et al., 2007)). At 48 hr after HeLa cells were infected by lentivirus carrying control or PIPKIγ shRNA, we observed excessive CP110 and Centrin2 foci (>4 per cell) that associated with each other in PIPKIγ-depleted cells; however, the centrosome number (represented by γ–tubulin foci) in these cells was normal (2 per cell). The numbers of CEP135 and CPAP foci were also normal (2 per cell) in PIPKIγ-depleted cells as in control cells (Fig. 5A). These results suggest that the additional Centrin2 and CP110 foci in PIPKIγ-depleted cells might correspond to overduplicated procentrioles that still engaged with the parental centrioles. Indeed, 72 hours after lentivirus infection when the disengagement of nascent centrioles occurred, excessive CEP135, CPAP, and γ–tubulin foci (>2 per cell) were observed in PIPKIγ-depleted cells (Fig. 5B).
Moreover, transmission electron microscopy analysis of
serial-sectioned PIPKIγ-depleted HeLa cells showed two procentrioles (P1 and P2, Fig. 5D, left and middle panels) attached to the proximal wall of a mother centriole identified by the distal appendages (Fig. 5D, right panel), further supporting that lack of PIPKIγ leads to procentriole amplification. It’s been reported that overexpression of PLK4 (Habedanck et al., 2005) or HsSAS-6 (Leidel et al., 2005; Strnad et al., 2007) leads to biogenesis of multiple procentrioles around one pre-existing centriole. PIPKIγ depletion also yielded more than one procentrioles per parental centriole, suggesting a connection with PLK4 or HsSAS-6. To test this, we designed specific siRNAs to knock down PLK4 (Fig. S3K) or HsSAS-6 (Fig. S3L). When PLK4 or HsSAS-6 was co-depleted with PIPKIγ, the centriole overduplication phenotype caused by PIPKIγ exhaustion - 10 -
was completely rescued (Fig. 5E), suggesting that PIPKIγ functions against PLK4 and/or HsSAS-6 at the early stage of centriole biogenesis. In agreement with this, depletion of PIPKIγ, without affecting the level of PLK4 (Fig. S3M), synergically enhanced centriole amplification resulted from PLK4 overexpression (Fig. S3N). In addition, Myc-PLK4 overexpression induced centriole amplification in U-2 OS cells was significantly suppressed by expression of either wild type or kinase dead HA-PIPKIγ (Fig. 5F), further suggesting that PIPKIγ might counteract PLK4
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function. The cryptic Polo-Box domain of PLK4 directly interacts with PIPKIγ PLK4, the key regulator of centriole duplication, also localizes in the PCM around the proximal end of centrioles in a CEP152-dependent manner (Cizmecioglu et al., 2010; Dzhindzhev et al., 2010; Hatch et al., 2010), like PIPKIγ does. To investigate the functional correlation between PIPKIγ and PLK4, we first tested whether they physically associate with each other. Since the endogenous level of PLK4 is undetectable by immunoblotting, we examined if endogenous PIPKIγ associates with overexpressed GFP-tagged PLK4.
PIPKIγ indeed pulled down
GFP-PLK4, indicating that they form a complex in vivo (Fig. 6A). To understand the spatial correlation between PIPKIγ and PLK4, we developed a mouse monoclonal PLK4 antibody. This antibody specifically recognized purified MBP-PLK4 but not MBP or MBP-CEP1521-748 (Fig. S4A) and stains PLK4 at the centrosome that was abolished by two independent PLK4-specific siRNAs (Fig. S4B), indicating that this new PLK4 antibody can be used to visualize endogenous PLK4 by immunofluorescence microscopy. Images obtained by 3D-SIM showed that our PLK4 antibody nicely decorated the centrosome with the similar pattern as endogenous CEP152, the binding partner of PLK4 (Fig. S4C), further endorsing the authenticity of this antibody. Colocalization of endogenous PIPKIγ with endogenous PLK4 at the centrosome was observed (Fig. 6B), suggesting that the working spaces of these two proteins are overlapped. Both PIPKIγ and PLK4 displayed a ring-like structure with similar shape and diameter as CEP152 (Fig. 3C and Fig. 6B) (Sir et al., 2011). This is in line with the notion that PLK4 and CEP152 interact and colocalize (Cizmecioglu et al., 2010; Dzhindzhev et al., 2010; Hatch et al., 2010), and positioned PIPKIγ to the inner to intermediate region of the PCM (Sonnen et al., 2012a). - 11 -
Next we tested the direct interaction between PIPKIγ and PLK4 with in vitro pull-down assays using purified MBP-PLK4 and His-PIPKIγ. PIPKIγ clearly shows direct binding to PLK4 (Fig. 6C). Using a series of truncated recombinant PLK4 fragments (Fig. 6F), we found that the Cryptic Polo-Box (CPB) domain of PLK4 is necessary and sufficient for PIPKIγ interaction (Fig. 6D). Using truncated PIPKIγ proteins, we narrowed down the PLK4-binding region within PIPKIγ to the kinase domain (Fig. 6E, F). The N- or C- terminus of PIPKIγ alone could not bind PLK4 and the PIPKIγ N-terminus is not necessary for PLK4 binding (Fig. 6E, F). These results
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strongly support the physical association between PIPKIγ and PLK4, and suggested a functional consequence of this interaction. PIPKIγ impairs PLK4 activity Since both CEP152 and PIPKIγ bind to the CPB domain of PLK4, we tested if PIPKIγ and CEP152 compete for the interaction with PLK4. Because the full length or fragments of CEP152 were highly degraded when purified from E. coli, we produced the 35S-labeled CEP152 N-terminal
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fragment (35S-CEP1521-748) by in vitro translation. Consistent with the literature (Cizmecioglu et al., 2010; Dzhindzhev et al., 2010; Hatch et al., 2010), MBP-tagged full length PLK4 pulled down 35
S-CEP1521-748 nicely. Although PIPKIγ could still be pulled down by MBP-PLK4, addition of
increasing amount of PIPKIγ did not affect CEP152 binding to PLK4 (Fig. 7A), suggesting that PIPKIγ and CEP152 do not bind to the same region of PLK4. Well-controlled activity of PLK4 is essential for centriole duplication and fidelity (Bettencourt-Dias et al., 2005; Eckerdt et al., 2011; Kleylein-Sohn et al., 2007; Nigg, 2007; Nigg and Stearns, 2011). PLK4 can phosphorylate itself (Brownlee and Rogers, 2012; Cunha-Ferreira et al., 2009; Guderian et al., 2010a; Holland et al., 2010; Rogers et al., 2009) and its potential in vivo substrate CEP152 (Cizmecioglu et al., 2010; Dzhindzhev et al., 2010; Hatch et al., 2010). Therefore, we overexpressed Flag-tagged CEP1521-748 in HeLa cells and purified this protein by immunoprecipitation. The precipitates obtained by normal mouse IgG or by anti-Flag antibody were incubated with purified MBP-PLK4 to perform kinase assay.
Results from these
experiments demonstrated that PLK4 could phosphorylate itself and CEP152 (Fig. 7B) but not PIPKIγ (Fig. 7C), indicating that PIPKIγ would not compete with PLK4 substrates. We next - 12 -
tested whether the interaction of PIPKIγ influences PLK4 activity (Fig. 7D). Because PIPKIγ and PLK4 are both kinases and they might impair each other’s activity by competing for ATP, we compared the PLK4 activity with or without variant amounts of the wild type (WT) or kinase dead (KD) PIPKIγ that does not bind ATP (Kunz et al., 2000) but binds PLK4 in vitro at a comparable level as its wild-type counterpart (unpublished data). Both PIPKIγ-WT and PIPKIγ-KD inhibited PLK4 activity in a dose-dependent manner (Fig. 7D-F). When the same amount of PIPKIγ as PLK4 was added, PIPKIγ-WT or PIPKIγ-KD resulted in a ~20% inhibition of PLK4
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autophosphorylation and CEP152 phosphorylation (Fig. 7D-F). When the amount of PIPKIγ-WT or PIPKIγ-KD was increased to three-fold of PLK4, ~40% of PLK4 autophosphorylation and ~50% of CEP1521-748 phosphorylation were inhibited (Fig. 7D-F). PIPKIγ-WT and PIPKIγ-KD consistently repressed PLK4 at a comparable level, suggesting that this inhibition was not caused by competition for ATP. These results argue that PIPKIγ by binding to PLK4 regulates PLK4 activity. To explore how PIPKIγ inhibits PLK4 activity, we determined if binding of PIPKIγ affects PLK4 dimerization, which is important for the self-activation of PLK4 (Guderian et al., 2010b). As shown in Fig. 7G, overexpressed Myc-PLK4 formed dimer with purified MBP-PLK4. This dimerization was not interrupted by the addition of increasing amount of recombinant PIPKIγ (Fig. 7G), indicating more complicated mechanism for future exploration. Nevertheless, in the context that PLK4 activity is critical for it to initiate procentriole biogenesis and achieve self-regulation, our results suggest that PIPKIγ could restrain centriole duplication by binding to and inhibiting PLK4 kinase activity.
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Discussion How centrioles are built and how the centriole assembly is limited to one daughter per mother in a single cell cycle are associated puzzles that have not been fully resolved. In addition to the key players identified in recent years (Brito et al., 2012; Nigg and Stearns, 2011), here we report PIPKIγ as a new component of centriole duplication machinery that restricts centriole formation by potentially limiting PLK4 activity. As a negative regulator of centriole biogenesis, association
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of PIPKIγ with the centrosome likely is sensitive and carefully regulated, which could be a reason that PIPKIγ was not described in the recent centrosome proteome studies (Andersen et al., 2003; Keck et al., 2011; Muller et al., 2011; Muller et al., 2010; Nogales-Cadenas et al., 2009; Ren et al., 2010; Zellner et al., 2011). Nevertheless, our results from multiple experiments (Fig. 1 and Fig. 2) provided solid evidence supporting the physical association of endogenous PIPKIγ with the centrosome. The overall levels and centrosome association of PIPKIγ are regulated through the cell cycle as many other centriolar proteins, however, in a rather unique way. Comparing to the positive regulators of centriole biogenesis like PLK4, SAS-6, and STIL whose levels are highest in M phase and drop sharply at mitotic exit, PIPKIγ is regulated oppositely: it is down-regulated during mitosis but increases vigorously right after mitosis. In addition, association of PIPKIγ with a centriole is first seen at G1/S boundary when the procentriole starts to emerge from this centriole (Fig. S1G), which is similar to Centrin2, SAS-6, and CPAP that are recruited to centrioles in G1/S phase to support procentriole assembly (Kleylein-Sohn et al., 2007; Paoletti et al., 1996; Strnad et al., 2007; Tang et al., 2009). Although it is not fully understood why PLK4, SAS-6, and STIL/SAS-5 accumulate in M phase when the budding of the procentriole has completed, the cell cycle-dependent expression and centrosome association of PIPKIγ serves its function well: it counteracts the centriole assembly machinery and inhibits the formation of excessive procentrioles. Although only PIPKIγ_i3 when overexpressed can target to the centrosome, the unique insert sequence in PIPKIγ_i3, which distinguishes it from other PIPKIγ alternative splicing isoforms, does not contain a centrosome-targeting sequence (Fig. 1E).
Instead, the
centrosome-targeting signal seems locates in the kinase domain that commonly presents in other - 14 -
PIPKIγ splicing isoforms and is highly conserved among all type I PIPK members; however none of them shows centrosomal localization. This suggests that the centrosome-targeting signal in PIPKIγ is normally hidden and needs certain modification to get exposed. It is plausible that dimerization of PIPKIγ (Rao et al., 1998) covers the centrosome-targeting motif in the kinase domain. The unique C-terminal insertion of PIPKIγ_i3 might alter the protein conformation and expose the centrosome-targeting motif. Since only a fraction of PIPKIγ_i3 expressing cells showed PIPKIγ_i3 at the centrosome, it is also possible that other modification mechanism, which
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could have been exhausted by overexpressed PIPKIγ_i3, is necessary to achieve a more efficient exposure of the centrosome-targeting motif in PIPKIγ. Similar as other centrosomal proteins (Fu and Glover, 2012a; Mennella et al., 2012a; Pelletier and Yamashita, 2012; Sonnen et al., 2012a), both endogenous and overexpressed PIPKIγ exhibited toroid-shaped distribution around the centriole in most cells, suggesting that the association of PIPKIγ with the centrosome follows the same common mechanism as other centrosomal proteins. This was reinforced by the observation that the stable association between PIPKIγ and the centrosome strictly relies on the existence of CEP152 but not CEP192, which is likely achieved via a physical association between PIPKIγ and CEP152. Indeed, the PIPKIγ toroid overlapped nicely with the CEP152 toroid, indicating that PIPKIγ is an intermediate PCM protein like CEP152 (Sonnen et al., 2012a). However, PIPKIγ is absent from the centrosome in M phase when CEP152 still presents, suggesting that CEP152 might not be sufficient to recruit PIPKIγ to the centrosome. More investigations need to be done in the future to decipher the signal code that governs the cell-cycle dependent association of PIPKIγ with the centrosome. Traditionally, PIPKIγ regulates variant cellular processes by targeting to specific subcellular locales and providing PI4,5P2 to the proximate downstream effector. This leads to the question if PI4,5P2 has a role in centriole duplication. Using GFP-tagged PLCδ PH domain, we failed to see clear GFP signal at the centrosome due to the strong membrane and cytosolic background (data not shown). PI4,5P2 antibody also failed to highlight any specific subcellular locations including the ones where PI4,5P2 plays important roles, such as focal adhesions and clathrin-coated pits (data not shown). However, it has been reported that there are membrane - 15 -
vesicles around the centrosome (Foraker et al., 2012; Westlake et al., 2011) and phosphoinositide-binding proteins like clathrin (Ford et al., 2001) and FYVE-CENT (Sagona et al., 2010) localize at the centrosome and regulate centrosome integrity (Foraker et al., 2012), suggesting that phosphoinositides including PI4,5P2 might exist and function at the centrosome. Nevertheless, in our hands both the wild type and kinase dead forms of PIPKIγ could similarly released the centriole/centrosome amplification caused by PIPKIγ depletion, HU treatment, or PLK4 overexpression, suggesting that PI4,5P2 is not essential in limiting centriole duplication.
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production. For example, PIPKIγ functions as a scaffold between E-cadherin and clathrin adaptor
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We reported previously that PIPKIγ can regulate some cellular process independent of PI4,5P2
the highly restricted expression of endogenous PLK4 (Guderian et al., 2010a; Holland et al., 2010;
complex AP1B, therefore facilitates the transport of E-cadherin from the trans-Golgi network to the recycling endosome (Ling et al., 2007a). In this process, the physical interactions of PIPKIγ with E-cadherin and AP1B, but not the PI4,5P2 generated by PIPKIγ, is critical. We propose that binding of PIPKIγ to PLK4 might impair the conformation and/or the access of ATP or substrates of PLK4 so that weakens PLK4 activity. Although this model has not been tested in vivo due to
Sillibourne et al., 2010), it is in agreement with our observation that loss of PIPKIγ causes PLK4-dependent centriole amplification without affecting cell cycle progression. The auto-phosphorylation of PLK4 triggers both activation and degradation of PLK4 (Guderian et al., 2010a; Holland et al., 2010; Sillibourne et al., 2010). It has been shown that the degradation of PLK4 requires multisite phosphorylation, which leads to a model that PLK4 is destructed once its activity reaches certain threshold level (Guderian et al., 2010a; Holland et al., 2010; Sillibourne et al., 2010). Our results suggest that PIPKIγ by binding to PLK4 inhibits its activity, providing another level of regulation in addition to phosphorylation. This seems critical to maintain the appropriate activity of centriole duplication machinery, since loss of PIPKIγ leads to generation of multiple procentrioles and overexpression of PIPKIγ blocks centrosome overduplication. Our results suggest that CEP152, by engaging both the positive regulator PLK4 and its negative regulator PIPKIγ around the proximal ends of parental centrioles, provides a physical platform for the accurate control of centriole assembly and homeostasis. - 16 -
Future studies
will be necessary to provide great details about the functional consequence of the physical interaction between PIPKIγ and PLK4. It is also important to investigate whether PLK4 is the only centrosomal protein regulated by PIPKIγ and whether PI4,5P2 has a functional role in other
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centrosome-related cellular processes.
- 17 -
Materials and methods Cell Culture and Transfection. HeLa, U-2 OS, MDA-MB-231, NIH3T3 and HEK293T cells were maintained in DMEM containing 10% FBS. IMCD3 cells were cultured in DMEM:F12 containing 10% FBS. HeLa and HEK293T cells were transfected using Lipofectamine 2000 (Invitrogen) or FuGENE 6 Transfection Reagent (Promega). U-2 OS cells were transfected using X-tremeGENE 9 (Roche) or Amaxa Cell Line Nucleofector® Kit V (Lonza). siRNA duplexes were introduced into cells using Lipofectamine RNAiMAX (Invitrogen).
HeLa cells were
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synchronized with standard double-thymidine block [two 16-hour thymidine (Sigma, 2 mM) treatments separated by a 9-hour release].
U-2 OS cells were synchronized with 16 mM
hydroxyurea (Sigma) for 24 h. The tetracyclin-inducible cell-line expressing Myc-tagged PLK4 was kindly provided by Dr. Erich Nigg (Kleylein-Sohn et al., 2007). Myc-PLK4 expression was induced by the addition of 1 μg/ml of Doxycycline (Clontech). Antibodies and Constructs. Rabbit monoclonal antibody against PIPKIγ was generated at Epitomics Inc. by immunizing rabbits with recombinant His-tagged PIPKIγ. Before collecting the spleens, anti-sera were harvested and purified against affinity column conjugated with MBP-tagged PIPKIγ C-terminus (a.a. 469-668) to obtain polyclonal PIPKIγ antibody. Rabbit polyclonal antibodies against PIPKIα and PIPKIβ were obtained by immunizing rabbits with recombinant GST-His-tagged PIPKIα or PIPKIβ. Anti-sera were purified with affinity column conjugated with MBP-tagged C-terminus of PIPKIα (a.a. 451-562) or PIPKIβ (a.a. 397-540). Mouse monoclonal anti-PLK4 antibody was generated at Abmart, Inc. Rabbit polyclonal antibodies against the following proteins were used: phospho-Histone H3 (Ser10) (Millipore), CPAP (Proteintech), β-Actin, CEP135, GFP (Abcam), CEP152 (Bethyl Laboratories, A302-480A), CEP192 (a generous gift from Dr. Erich Nigg) (Schmidt et al., 2009), and CP110 (Proteintech). Mouse antibodies against the following proteins were used: Centrin2 (20H5, a generous gift from Dr. Jeffrey Salisbury), ODF2 (Abnova), C-Nap1 (BD Biosciences), His (HIS.H8, Millipore), GFP (Roche); α-Tubulin (DM1A), γ-Tubulin (GTU-88), FLAG (M2), and HA (HA-7) are from Sigma; Myc (9E10), HsSAS-6 (91.390.21), and cyclin B (GNS1) are from Santa Cruz. Anti-Ninein (Y-16) antibdy is from Santa Cruz and Maltose Binding Protein antibody (7G4) is from Sigma. - 18 -
cDNAs encoding full length or truncated human PIPKIγ (Ling et al., 2002), PLK4, and CEP152 were obtained by PCR and sub-cloned into pCMV-HA, pcDNA3-Flag, pGEX-4T-1, pET-28, pET-42, or pMAL-C2X constructs.
pcDNA3-Myc-PLK4 was obtained from Addgene.
HA-PIPKIγ kinase dead (KD), RNAi resistant HA-PIPKIγ-WT or -KD which contains mismatches to the HsPIPKIγ-O1 siRNA was generated using QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies). All constructs were verified by sequencing.
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Lentiviruses. Lentiviruses carrying shRNA were constructed by cloning shRNA oligonucleotides (Invitrogen) into the pLKO.1 vector (AddGene). The shRNA sequence targeting human PIPKIγ was 5′-GTCGTGGTCATGAACAACA-3′. An interfering RNA sequence targeting luciferase (5′-GTACCTGTACTTCATGCAG-3′) was used as negative control.
All constructs were
confirmed by sequencing. HEK293T cells were co-transfected with pLKO.1-puro carrying shRNA, pCMV-VSVG, and pCMV-R8.91 (gifts from Dr. Gaoxiang Ge, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, China) at 2:1:1 ratio using FuGENE 6 (Promega).
Medium supernatant was collected 48-72 h after transfection.
Subconfluent HeLa Cells were infected in the presence of 8 μg/mL polybrene (Sigma).
siRNAs. All siRNA oligonucleotides were obtained from Invitrogen. HsPIPKIγ-O1: 5'- GCGTGGTCAAGATGCACCTCAAGTT-3'; HsPIPKIγ-O2: 5'-CCTACAGGTTCATCAAGAAACTGGA-3'; mouse PIPKIγ: 5'-GCGAGAGAGAGGATGTGCAGTATGA-3'. PLK4-O1: 5'-CACTGGTTTGGAAGTTGCAATCAAA-3'; PLK4-O2: 5'- AGGAGGTGTGTGTGGAGCTTGTAAA-3'; HsSAS-6: 5'-AGAAAAGCACGTTAATCAGCTACAA-3' (Strnad et al., 2007); CEP152-O1: 5'-CAGAACAACTGAAATGGCTCTGGAA-3'; CEP152-O2: 5'- CAGCGTTTGCTGGGTAGCAACTCAA-3'; CEP192-O1: 5'- CCCAAAGGAAGACATTTTCATCTCT-3'; CEP192-O2: 5'- ATCAGACAGAGGAATCAATAATAAA-3' (Gomez-Ferreria et al., 2007; Sonnen et al., 2013; Zhu et al., 2008); - 19 -
HsPIPKIα-O1: 5'-TTGAAAGGTGCCATCCAGTTAGGCA-3'; HsPIPKIα-O2: 5'-TGTTAAGAAGTTGGAGCACTCTTGG-3' (Mellman et al., 2008); HsPIPKIβ: 5'-CAGCAAAGGGTTACCTTCCAGTTCA-3'; Stealth RNAi Negative Control siRNA (Invitrogen) was used as negative control. Similar results were got by using both siRNA oligonucleotides (O1, O2) for depletion of HsPIPKIγ, PLK4, CEP152, CEP192 or HsPIPKIα, respectively. Unless stated otherwise, results for oligonucleotide
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No.1 (O1) are shown. Immunoprecipitation and Microscopy. Immunoprecipitation was performed as previously described (Ling et al., 2002) using IP buffer [20 mM Hepes-KOH, pH7.2, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 0.5% NP-40, Complete Protease Inhibitor Cocktail (Roche), PhosSTOP Phosphatase Inhibitor Cocktail (Roche)].
For indirect
immunofluorescence, cells were grown on glass coverslips, fixed with ice-cold methanol for 10 min, permeablized, and stained as previously described (Ling et al., 2002). Fluorescence images were acquired using Nikon TE 2000-U with Metamorph (Molecular Devices). Z-Series were taken at 0.1-μm steps.
Three Dimensional Structured Illumination Microscopy (3D-SIM) was
performed on ELYRA Superresolution Microscopy system (Zeiss) equipped with alpha “Plan-Apochromat” 100x/1,46 Oil DIC oil immersion objective and Andor iXon 885 EMCCD camera, following standard protocol. Sections were acquired at 0.125 mm z-steps.
Color
channels were aligned using alignment parameter from control measurements with 0.5 μm diameter multispectral fluorescent beads (Zeiss). Structured illumination reconstruction and image processing was performed with the ZEN software package (Zeiss). Final image processing was done using Adobe Photoshop. In vitro Protein Pull-down Assay. GST or MBP-fused proteins were incubated with His-tagged proteins and Glutathione-Sepharose beads (GE Healthcare) or Amylose resin (New England Biolabs) in binding buffer (25mM Tris, pH 7.6, 150 mM NaCl, 1 mM DTT, 0.5% Triton X-100, 10% Glycerol) for 2 h at 4 °C. The precipitates were washed five times with binding buffer before analyzed by immunoblotting. CEP152 N-terminus (1-748 a.a.) protein was generated using the TNT Quick Coupled Transcription/Translation System (Promega), in presence of - 20 -
35
S-methionine (Perkin-Elmer), used for pull-down assay and analyzed by autoradiography.
Transmission Electron Microscopy. Cells were fixed for 1 h using Trump's fixative (Electron Microscopy Sciences).
Embedding and serial sectioning of cell samples was performed
according to standard procedures at the Electron Microscopy Core Facility, Mayo Clinic. Specimens were observed in a JEOL 1400 transmission electron microscope (JEOL) operating at 80 kV.
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PLK4 Kinase Assay. PLK4 activity was determined following a published approach (Hatch et al., 2010; Holland et al., 2010).
Briefly, Flag-tagged CEP152 fragment (CEP1521-748) was
overexpressed in HeLa cells and immunoprecipitated by anti-Flag antibody. 0.2 μg purified MBP-PLK4 was incubated with or without recombinant CEP152 N-terminus and/or appropriate amount of wild type (WT) or kinase dead (KD) His-PIPKIγ in kinase buffer with 5 μCi 32P-ATP (Perkin Elmer) and 33 μM ATP (New England Biolabs) for 30 min at room temperature. ImageJ (National Institutes of Health, Bethesda, MD) was used to quantify autoradiographs. Centrosome Isolation. Centrosomes were isolated from HeLa cells or mouse kidneys, based on a published approach (Meigs and Kaplan, 2008). Statistical Analyses. Data represent means and standard deviations (SD) from at least three independent experiments. Significance was calculated by Student’s t-test using Excel software (Microsoft). p
Type Iγ phosphatidylinositol phosphate kinase targets to the centrosome and restrains centriole duplication Qingwen Xua, Yuxia Zhangb, Xunhao Xionga, Yan Huanga, Jeffery L. Salisburya, Jinghua Hua,b, Kun Linga,1 a
Department of Biochemistry and Molecular Biology, and Division of Hypertension and Nephrology, Mayo Clinic, 200 First Street SW, Rochester, MN 55902.
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b
1
Correspondence Author:
Kun Ling, Ph.D. Stabile 7-06 200 First Street SW Mayo Clinic Rochester, MN 55902 Tel: 507-293-3498 [email protected]
Running Title: PIPKIγ restricts centriole biogenesis Key Words: Centriole duplication; PLK4; CEP152; CEP192
Length of the manuscript: 7,988 words.
-1-
JCS Advance Online Article. Posted on 16 January 2014
Summary
Centriole biogenesis depends on the Polo-like kinase PLK4 and a small group of structural proteins.
The spatiotemporal regulation of these proteins at pre-existing centrioles is
critical to ensure that centriole duplication occurs once per cell cycle. Here we report that type Iγ phosphatidylinositol-4-phosphate 5-kinase (PIPKIγ) plays an important role in centriole fidelity. Depending upon an association with CEP152, PIPKIγ localized in a
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ring-like pattern in the intermediate pericentriolar materials around the proximal end of the centriole in G1, S, and G2 phases, but not in M phase. Without detaining cells in S or M phase, depletion of PIPKIγ led to centriole amplification in a PLK4/SAS-6 dependent manner. Expression of exogenous PIPKIγ reduced centriole amplification resulted from endogenous PIPKIγ depletion, hydroxyurea treatment, or PLK4 overexpression, suggesting that PIPKIγ likely functions at the PLK4 level to restrain centriole duplication. Importantly, we found that PIPKIγ bound to the cryptic Polo-Box domain of PLK4 and this binding reduced PLK4 kinase activity. Together, our findings suggest that PIPKIγ is a novel negative regulator of centriole duplication by modulating the homeostasis of PLK4 activity.
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Introduction
The centrosome, composed of one pair of centrioles and surrounding pericentriolar material (PCM), is the major microtubule organization center and plays a critical role in mitotic spindle assembly, primary ciliogenesis, and cell morphogenesis (Debec et al., 2010; Nigg and Raff, 2009). In cycling cells, the centrosome duplicates once in S-phase so that each daughter cell can inherit one centrosome after mitosis.
Abnormal number of centrosomes results in mis-assembled
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spindles that frequently gives rise to chromosome mis-segregation, genomic instability, and aneuploid daughter cells (Ganem et al., 2009), linking centrosomal anomalies to many human diseases including a variety of different aneuploid tumors (Ghadimi et al., 2000; Lingle et al., 2002; Nigg, 2006; Pihan et al., 1998; Yamamoto et al., 2004) and microcephaly syndromes (Megraw et al., 2011; Nigg and Raff, 2009). The procentriole is assembled by a small group of highly conserved proteins and is strictly limited to one per pre-existing centriole and once per cell cycle. The availability and activity of these proteins at the centrosome have to be carefully controlled to maintain the fidelity of centriole/centrosome duplication (Bettencourt-Dias et al., 2005; Eckerdt et al., 2011; Kleylein-Sohn et al., 2007; Nigg, 2007; Nigg and Stearns, 2011). It has been demonstrated that the Polo-like kinase PLK4 is a key regulator of centriole formation and excessive PLK4 activity leads to centriole amplification (Bettencourt-Dias et al., 2005; Eckerdt et al., 2011; Kleylein-Sohn et al., 2007; Nigg, 2007; Nigg and Stearns, 2011). PLK4 is recruited to the centrosome by and controls the onset of centriole assembly together with CEP152, which also provides a platform for CPAP binding to the centrosome (Cizmecioglu et al., 2010; Dzhindzhev et al., 2010). PLK4-mediated autophosphorylation and phosphorylation prime itself or its substrate like FBXW5 for ubiquitination and destruction, therefore controls the availability of itself (Brownlee and Rogers, 2012; Cunha-Ferreira et al., 2009; Guderian et al., 2010a; Holland et al., 2010; Rogers et al., 2009) or other component of centriole duplication machinery like SAS-6 (Puklowski et al., 2011) to confine centriole number. On the other hand, a protein phosphatase PP2A was reported to promote centriole formation by facilitating SAS-6 accumulation to the nascent centriole (Brownlee et al., 2011; Kitagawa et al., 2011; Song et al., -3-
2011). These results support an intrinsic, critical balance between kinase and phosphatase activities during centriole duplication, and put PLK4 into the center of the centriole assembly machinery. In spite of the importance of controlling PLK4 activity accurately, the underlying mechanism is far from fully understood. Type Iγ phosphatidylinositol-4-phosphate 5-kinase (PIPKIγ) is a lipid kinase that phosphorylates
phosphatidylinositol
4-phosphate
to
generate
phosphatidylinositol
4,5-bisphosphate (PI4,5P2). Up to date, six alternative splicing isoforms of PIPKIγ have been
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reported (Giudici et al., 2004; Giudici et al., 2006; Ishihara et al., 1998; Schill and Anderson, 2009; Xia et al., 2011). PIPKIγ isoforms target to distinct subcellular locales. By regulating the dynamics of regional PI4,5P2 pools, they are implicated in distinct cellular processes such as vesicular trafficking (Bairstow et al., 2006; Ling et al., 2007b; Sun et al., 2013; Thapa et al., 2012; Thieman et al., 2009; Xiong et al., 2012), calcium signaling (Wang et al., 2004), or cell adhesion and migration (Di Paolo et al., 2002; El Sayegh et al., 2007; Ling et al., 2007a; Ling et al., 2002; Sun et al., 2007; Thapa et al., 2012; Thieman et al., 2009; Wang et al., 2004; Xiong et al., 2012). Moreover, PIPKIγ can regulate some protein such as E-cadherin (Ling et al., 2007b) by directly interacting with it. In the present study, we report that PIPKIγ_i3 targeted to the centrosome and negatively regulated centriole duplication. This lipid kinase associated with the proximal ends of parental centrioles in a cell cycle-dependent manner, just like many other centrosomal proteins. Similar to PLK4 and CPAP, residence of PIPKIγ at the centrosome depended upon CEP152. Loss of PIPKIγ resulted in centriole amplification without blocking the S or G2/M phase, indicating that PIPKIγ is a negative regulator of centriole duplication. Indeed, further investigation revealed that PIPKIγ directly interacts with PLK4 and inhibits its activity.
These results suggested an
additional level of regulation for PLK4 activity and centriole biogenesis in addition to phosphorylation–dependent protein ubiquitination and degradation.
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Results PIPKIγ localizes at the centrosome Using the rabbit polyclonal antibodies reported previously that recognizes all of the PIPKIγ splicing isoforms, we observed that PIPKIγ localizes to multiple subcellular locales including focal adhesion, adherens junction, recycling endosome (Ling et al., 2007a; Ling et al., 2002), as well as the centrosome. To investigate the unexpected centrosome localization of PIPKIγ, we
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generated new rabbit polyclonal and monoclonal antibodies to ensure that these are not artifacts from the previous antibody. Both the purified polyclonal antibodies (Fig. S1B) and a monoclonal antibody against PIPKIγ (Fig. 1A, left panel) decorated the centrosome highlighted by the centrosomal marker Centrin2 in HeLa cells. In contrast, purified rabbit polyclonal antibodies specifically recognizing PIPKIα or PIPKIβ (Fig. S1A) did not show staining around the centrosome (Fig. 1A). The centrosomal signal picked by anti-PIPKIγ antibody was completely abolished after the treatment of a PIPKIγ siRNA targeting all PIPKIγ isoforms (Fig. 1B) or in the presence of excess PIPKIγ but not PIPKIα protein (Fig. S1B), supporting the authenticity of this PIPKIγ localization. In addition, we isolated centrosomes from HeLa cells (Fig. 1C) or mouse kidney (Fig. S1C) using sucrose gradient centrifugation and analyzed the fractions by Western blot. Similar to other centrosomal proteins those were identified using this method (Kaplan et al., 2004), PIPKIγ co-fractionated with the centrosome components human SAS-6 (referred to HsSAS-6 from now on), CPAP, and γ-tubulin, which further supports that PIPKIγ is associated with centrosomes as suggested by immunofluorescence microscopy. Moreover, nocodazole treatment had no effect on PIPKIγ signal at the centrosome, although it efficiently interrupted the microtubule integrity (Fig. S1D), indicating that the association between PIPKIγ and the centrosome is independent of the microtubule cytoskeleton. These results demonstrated that PIPKIγ was a bona fide component of the centrosome. In addition to HeLa cells, we also observed PIPKIγ at the centrosome in MDA-MB-231, U-2 OS, IMCD3 and NIH3T3 cells (Fig. S1E and unpublished data), suggesting a pervasive and potentially functional role of PIPKIγ at the centrosome.
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To determine which PIPKIγ isoform targets to centrosomes, we fused a HA tag to the N-terminus of all six known PIPKIγ splicing variants. As shown in Fig. 1D and Fig. S1F, only PIPKIγ_i3 displayed a centrosome-associated signal. Other isoforms showed vesicle-like cytoplasmic targeting or localized to focal adhesions (PIPKIγ_i2) as reported (Di Paolo et al., 2002; Ling et al., 2002). PIPKIγ_i3 contains a unique 26-amino acid insertion (a.a. 641-666) at the C-terminus between the end of PIPKIγ_i1 and the beginning of the PIPKIγ_i2 C-terminal extension (Fig. 1E).
However, the C-terminal region of PIPKIγ_i3 (a.a. 445-End) is
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predominantly cytoplasmic (Fig. 1D, right panel). The N-terminal (a.a. 1-445) fragment of PIPKIγ_i3, which is common in all PIPKIγ variants, localized to the centrosome (Fig. 1D, middle panel). These results suggest that the centrosome-targeting motif is likely within the N-terminal 445 amino acids. Since PIPKIγ_i3 is the only one that showed centrosome targeting, this raises a possibility that the unique C-terminal insertion of PIPKIγ_i3 might provoke a conformational adjustment to expose the centrosome-targeting motif embedded in the first 445 amino acids. PIPKIγ targets to the proximal end of centrioles in a cell cycle-dependent manner To examine the fine-structural details of PIPKIγ association with the centrosome, we co-labeled HeLa cells with anti-PIPKIγ antibody and antibodies against various centrosome components. The localizations of these proteins around the centrosome were analyzed using the Three Dimensional Structured Illumination Microscopy (3D-SIM). When co-stained with Ninein, a subdistal appendage marker on the mother centriole (Nakagawa et al., 2001) and Centrin2 that marks the distal end of the centriole cylinder (Paoletti et al., 1996), PIPKIγ was positioned below Ninein and Centrin2 (Fig. 2A). Further examinations implied that PIPKIγ localized between Ninein and C-Nap1, a linker protein between centrioles (Mayor et al., 2000) (Fig. 2B), as well as C-Nap1 and ODF2 that marks the distal appendage of the mature mother centriole (Piel et al., 2000) (Fig. 2C). In these images, PIPKIγ seemed distributing along the side of the centriole wall in the pericentriolar matrix and likely formed a ring around the proximal end of centrioles with a diameter smaller than Ninein. To address if the centrosomal localization of PIPKIγ is cell cycle dependent as many other -6-
centrosome proteins, we subjected HeLa cells to immunofluorescence microscopy to visualize PIPKIγ together with Centrin2 and DAPI. Based on the staining of Centrin2 and DAPI, cells were categorized as in G1-phase (2 Centrin2 foci and intact nuclear membrane), S-phase (4 clustered Centrin2 foci and intact nuclear membrane), G2-phase (2 separated pairs of Centrin2 foci and intact nuclear membrane), and variant stages of M-phase (broken nuclear membrane and condensed chromosome DNA arranged in different ways).
PIPKIγ staining around the
centrosome was observed in cells at G1-, S-, and G2-phase (Fig. S1G). At the prophase of mitosis,
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PIPKIγ signal diminished and remained absent from centrosomes until the late telophase, when PIPKIγ is recruited back to both centrosomes that would soon be inherited by each daughter cell (Fig. S1G).
Consistent with its centrosome targeting, protein level of PIPKIγ decreases
significantly as cells progress through mitosis and then recovers at mitotic exit in synchronized HeLa cells (Fig. 2D). This follows the same trend as CPAP (Tang et al., 2009) (Fig. 2D), a CEP152-interacting protein which functions early in centriole duplication, however is different from HsSAS-6 (Fig. 2D) that is degraded during the late M phase and absent in the G1-phase (Strnad et al., 2007), suggesting that PIPKIγ may have a specific function at the centrosome. PIPKIγ targeting to the centrosome is dependent on an association with CEP152 CEP152 is a scaffold protein localizing around the proximal end of centrioles and is required for the centrosomal localization of PLK4 and CPAP (Cizmecioglu et al., 2010; Dzhindzhev et al., 2010; Hatch et al., 2010). Since PIPKIγ also localizes around the proximal end of centrioles, we examined whether CEP152 was responsible for the targeting of PIPKIγ to the centrosome. The physical
association
between
PIPKIγ
and
CEP152
was
first
determined
using
co-immunoprecipitation assay. Indeed, overexpressed GFP-CEP152 pulled down HA-tagged PIPKIγ_i3 from HEK293T cells (Fig. 3A), suggesting that these two proteins have the potential to bind each other. The CEP152 antibody recognized multiple bands (Fig. 3B) that could be all knocked down by CEP152 specific siRNA (Fig. 3D), suggesting that endogenous CEP152 exists in variant forms. Endogenous PIPKIγ was co-immunoprecipitated with the endogenous CEP152 protein that migrated faster on SDS-PAGE gel (Fig. 3B). Although it is speculative at this point whether this PIPKIγ-associated CEP152 is an unmodified, truncated, or alternatively spliced form -7-
of CEP152, our results indeed supports an in vivo physical connection between these two proteins. Because the recombinant full length CEP152 expressed in E. coli is highly degraded or insoluble no matter fused to GST, His, or MBP tag, we constructed and purified the MBP-tagged CEP152 fragments and tested their interaction with His-PIPKIγ. Both N-terminal 748-aa (CEP1521-748) and C-terminal 906-aa (CEP152749-1654) pulled down PIPKIγ (Fig. S2A).
However, the
N-terminal 217 a.a. of CEP152 (CEP1521-217), where PLK4 directly binds to (Hatch et al., 2010), did not interact with PIPKIγ (Fig. S2A). These data suggest that PIPKIγ might directly bind to
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the C-terminus of CEP152, but further investigation is needed to obtain a solid conclusion because of the heavy degradation of our recombinant CEP152 polypeptides. To understand the biophysical significance of the association between PIPKIγ and CEP152, we first tested if they co-localize at the centrosome. It has been reported recently by multiple groups that centrosomal proteins including CEP152 form a ring-like structure arount the centriole (Fu and Glover, 2012b; Lawo et al., 2012; Mennella et al., 2012b; Sonnen et al., 2012b). Consistently, our 3D-SIM images supported a ring-like colocalization between PIPKIγ and Flag-CEP152 (Fig. 3C), suggesting that PIPKIγ localizes in the intermediate pericentriolar matrix around the proximal end of centrioles as CEP152 does (Fu and Glover, 2012b; Lawo et al., 2012; Mennella et al., 2012b; Sonnen et al., 2012b). More importantly, the colocalization between CEP152 and PIPKIγ reinforced the physical interaction and suggested a functional correlation between these two proteins. Indeed, siRNA-mediated depletion of CEP152 (Fig. 3D) led to a loss of PIPKIγ signal from the centrosome (Fig. 3E, H), indicating that CEP152 is necessary for the recruitment of PIPKIγ to the centrosome. Moreover, depletion of CEP152 also eliminated the centrosome targeting of exogenous PIPKIγ_i31-445 (Fig. S2B), further suggesting that CEP152 provides a necessary structural platform for the stable association of PIPKIγ with the centrosome. In addition to CEP152, CEP192 was recently shown critical for the recruitment of centrosomal proteins (Sonnen et al., 2013). In CEP192-depleted cells, the centrosomal signal of CEP152 was largely eliminated (Fig. 3G) as reported (Sonnen et al., 2013); however, PIPKIγ signal around the centrosome was mostly retained (Fig. 3G, H). Combining with our previous observation that complete loss of CEP152 blocked PIPKIγ targeting to the centrosome (Fig. 3E -8-
and 3H), these results suggest that very small amount of centrosomal CEP152 could be plenty for the centrosome recruitment of PIPKIγ. PIPKIγ negatively regulates centriole duplication The specific, cell cycle-dependent centrosomal localization of PIPKIγ led us to investigate the potential function of PIPKIγ at the centrosome. For this purpose, we knocked down PIPKIγ in HeLa cells using lentivirus-based PIPKIγ-specific shRNA (Fig. 4A). Strikingly, loss of PIPKIγ
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resulted in an augmentation of Centrin2 foci (> 4 per cell) in ~ 20% of the cells (Fig. 4B, C). Importantly, centriole amplification caused by depletion of PIPKIγ was almost fully rescued by expression of RNAi-resistant PIPKIγ (Fig. 4D, E). Similar level of Centrin2 foci increase was also observed in HeLa cells treated with two distinct PIPKIγ siRNAs (siPIPKIγ-O1 and siPIPKIγ-O2) (Fig. S3A, B), but not in PIPKIα- or PIPKIβ-depleted cells (Fig. S3F, G), further confirmed that this phenotype was caused by PIPKIγ depletion. To determine if the increased Centrin2 foci were resulted from cell cycle arrest in S phase or G2/M phase, we examined the cell cycle distribution of PIPKIγ-depleted cells. BrdU incorporation (Fig. S3C) and PCNA staining (Fig. S3D) were used to determine the S phase and phospho-Histone H3 (Fig. S3E) was used to determine the G2/M phase. As shown in Fig. S3C-E, no significant difference was observed between cells treated with siPIPKIγ or cells treated with control siRNA. Therefore, the excessive Centrin2 foci in PIPKIγ-depleted cells were not likely resulted from a sustained S, G2, or M phase. Furthermore, depletion of PIPKIγ but not PIPKIα or PIPKIβ caused an increase of centrosome number (>2 γ–tubulin loci per cell) in other types of cells like NIH 3T3 (Fig. S3H, I) and renal collecting duct epithelial IMCD3 (Fig. S3J). These results suggest that PIPKIγ might inhibit centriole duplication. To test this possibility, we examined if expression of PIPKIγ could reverse centrosome amplification caused by hydroxyurea (HU) treatment. As shown in Fig. 4F, U2 OS cells were treated with HU for 24 hr before being transfected with or without wild-type or kinase-dead HA-PIPKIγ. This experimental design limits the impact of overexpressed PIPKIγ in S phase and excluded the potential influence on other phases of cell cycle (Hemerly et al., 2009). As shown in Fig. 4G, HU-induced centrosome amplification in U-2 OS cells was significantly suppressed by -9-
overexpressed HA-PIPKIγ, suggesting that PIPKIγ is a negative regulator of centrosome duplication.
Expression of both wild type and kinase dead PIPKIγ could rescue centriole
overduplication caused by PIPKIγ depletion or HU treatment, suggesting that PI4,5P2 might not play a significant role in centriole biogenesis. To further characterize these excessive Centrin2 foci in PIPKIγ-depleted cells, we checked the localization of several centriolar proteins including CP110 (localizes at the distal ends of centrioles (Kleylein-Sohn et al., 2007)), Centrin2 (localizes at the distal ends of centrioles),
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CEP135 (localizes at the proximal end of centrioles (Kleylein-Sohn et al., 2007)), and CPAP (localizes at the proximal ends of centrioles (Kleylein-Sohn et al., 2007)). At 48 hr after HeLa cells were infected by lentivirus carrying control or PIPKIγ shRNA, we observed excessive CP110 and Centrin2 foci (>4 per cell) that associated with each other in PIPKIγ-depleted cells; however, the centrosome number (represented by γ–tubulin foci) in these cells was normal (2 per cell). The numbers of CEP135 and CPAP foci were also normal (2 per cell) in PIPKIγ-depleted cells as in control cells (Fig. 5A). These results suggest that the additional Centrin2 and CP110 foci in PIPKIγ-depleted cells might correspond to overduplicated procentrioles that still engaged with the parental centrioles. Indeed, 72 hours after lentivirus infection when the disengagement of nascent centrioles occurred, excessive CEP135, CPAP, and γ–tubulin foci (>2 per cell) were observed in PIPKIγ-depleted cells (Fig. 5B).
Moreover, transmission electron microscopy analysis of
serial-sectioned PIPKIγ-depleted HeLa cells showed two procentrioles (P1 and P2, Fig. 5D, left and middle panels) attached to the proximal wall of a mother centriole identified by the distal appendages (Fig. 5D, right panel), further supporting that lack of PIPKIγ leads to procentriole amplification. It’s been reported that overexpression of PLK4 (Habedanck et al., 2005) or HsSAS-6 (Leidel et al., 2005; Strnad et al., 2007) leads to biogenesis of multiple procentrioles around one pre-existing centriole. PIPKIγ depletion also yielded more than one procentrioles per parental centriole, suggesting a connection with PLK4 or HsSAS-6. To test this, we designed specific siRNAs to knock down PLK4 (Fig. S3K) or HsSAS-6 (Fig. S3L). When PLK4 or HsSAS-6 was co-depleted with PIPKIγ, the centriole overduplication phenotype caused by PIPKIγ exhaustion - 10 -
was completely rescued (Fig. 5E), suggesting that PIPKIγ functions against PLK4 and/or HsSAS-6 at the early stage of centriole biogenesis. In agreement with this, depletion of PIPKIγ, without affecting the level of PLK4 (Fig. S3M), synergically enhanced centriole amplification resulted from PLK4 overexpression (Fig. S3N). In addition, Myc-PLK4 overexpression induced centriole amplification in U-2 OS cells was significantly suppressed by expression of either wild type or kinase dead HA-PIPKIγ (Fig. 5F), further suggesting that PIPKIγ might counteract PLK4
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function. The cryptic Polo-Box domain of PLK4 directly interacts with PIPKIγ PLK4, the key regulator of centriole duplication, also localizes in the PCM around the proximal end of centrioles in a CEP152-dependent manner (Cizmecioglu et al., 2010; Dzhindzhev et al., 2010; Hatch et al., 2010), like PIPKIγ does. To investigate the functional correlation between PIPKIγ and PLK4, we first tested whether they physically associate with each other. Since the endogenous level of PLK4 is undetectable by immunoblotting, we examined if endogenous PIPKIγ associates with overexpressed GFP-tagged PLK4.
PIPKIγ indeed pulled down
GFP-PLK4, indicating that they form a complex in vivo (Fig. 6A). To understand the spatial correlation between PIPKIγ and PLK4, we developed a mouse monoclonal PLK4 antibody. This antibody specifically recognized purified MBP-PLK4 but not MBP or MBP-CEP1521-748 (Fig. S4A) and stains PLK4 at the centrosome that was abolished by two independent PLK4-specific siRNAs (Fig. S4B), indicating that this new PLK4 antibody can be used to visualize endogenous PLK4 by immunofluorescence microscopy. Images obtained by 3D-SIM showed that our PLK4 antibody nicely decorated the centrosome with the similar pattern as endogenous CEP152, the binding partner of PLK4 (Fig. S4C), further endorsing the authenticity of this antibody. Colocalization of endogenous PIPKIγ with endogenous PLK4 at the centrosome was observed (Fig. 6B), suggesting that the working spaces of these two proteins are overlapped. Both PIPKIγ and PLK4 displayed a ring-like structure with similar shape and diameter as CEP152 (Fig. 3C and Fig. 6B) (Sir et al., 2011). This is in line with the notion that PLK4 and CEP152 interact and colocalize (Cizmecioglu et al., 2010; Dzhindzhev et al., 2010; Hatch et al., 2010), and positioned PIPKIγ to the inner to intermediate region of the PCM (Sonnen et al., 2012a). - 11 -
Next we tested the direct interaction between PIPKIγ and PLK4 with in vitro pull-down assays using purified MBP-PLK4 and His-PIPKIγ. PIPKIγ clearly shows direct binding to PLK4 (Fig. 6C). Using a series of truncated recombinant PLK4 fragments (Fig. 6F), we found that the Cryptic Polo-Box (CPB) domain of PLK4 is necessary and sufficient for PIPKIγ interaction (Fig. 6D). Using truncated PIPKIγ proteins, we narrowed down the PLK4-binding region within PIPKIγ to the kinase domain (Fig. 6E, F). The N- or C- terminus of PIPKIγ alone could not bind PLK4 and the PIPKIγ N-terminus is not necessary for PLK4 binding (Fig. 6E, F). These results
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strongly support the physical association between PIPKIγ and PLK4, and suggested a functional consequence of this interaction. PIPKIγ impairs PLK4 activity Since both CEP152 and PIPKIγ bind to the CPB domain of PLK4, we tested if PIPKIγ and CEP152 compete for the interaction with PLK4. Because the full length or fragments of CEP152 were highly degraded when purified from E. coli, we produced the 35S-labeled CEP152 N-terminal
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fragment (35S-CEP1521-748) by in vitro translation. Consistent with the literature (Cizmecioglu et al., 2010; Dzhindzhev et al., 2010; Hatch et al., 2010), MBP-tagged full length PLK4 pulled down 35
S-CEP1521-748 nicely. Although PIPKIγ could still be pulled down by MBP-PLK4, addition of
increasing amount of PIPKIγ did not affect CEP152 binding to PLK4 (Fig. 7A), suggesting that PIPKIγ and CEP152 do not bind to the same region of PLK4. Well-controlled activity of PLK4 is essential for centriole duplication and fidelity (Bettencourt-Dias et al., 2005; Eckerdt et al., 2011; Kleylein-Sohn et al., 2007; Nigg, 2007; Nigg and Stearns, 2011). PLK4 can phosphorylate itself (Brownlee and Rogers, 2012; Cunha-Ferreira et al., 2009; Guderian et al., 2010a; Holland et al., 2010; Rogers et al., 2009) and its potential in vivo substrate CEP152 (Cizmecioglu et al., 2010; Dzhindzhev et al., 2010; Hatch et al., 2010). Therefore, we overexpressed Flag-tagged CEP1521-748 in HeLa cells and purified this protein by immunoprecipitation. The precipitates obtained by normal mouse IgG or by anti-Flag antibody were incubated with purified MBP-PLK4 to perform kinase assay.
Results from these
experiments demonstrated that PLK4 could phosphorylate itself and CEP152 (Fig. 7B) but not PIPKIγ (Fig. 7C), indicating that PIPKIγ would not compete with PLK4 substrates. We next - 12 -
tested whether the interaction of PIPKIγ influences PLK4 activity (Fig. 7D). Because PIPKIγ and PLK4 are both kinases and they might impair each other’s activity by competing for ATP, we compared the PLK4 activity with or without variant amounts of the wild type (WT) or kinase dead (KD) PIPKIγ that does not bind ATP (Kunz et al., 2000) but binds PLK4 in vitro at a comparable level as its wild-type counterpart (unpublished data). Both PIPKIγ-WT and PIPKIγ-KD inhibited PLK4 activity in a dose-dependent manner (Fig. 7D-F). When the same amount of PIPKIγ as PLK4 was added, PIPKIγ-WT or PIPKIγ-KD resulted in a ~20% inhibition of PLK4
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autophosphorylation and CEP152 phosphorylation (Fig. 7D-F). When the amount of PIPKIγ-WT or PIPKIγ-KD was increased to three-fold of PLK4, ~40% of PLK4 autophosphorylation and ~50% of CEP1521-748 phosphorylation were inhibited (Fig. 7D-F). PIPKIγ-WT and PIPKIγ-KD consistently repressed PLK4 at a comparable level, suggesting that this inhibition was not caused by competition for ATP. These results argue that PIPKIγ by binding to PLK4 regulates PLK4 activity. To explore how PIPKIγ inhibits PLK4 activity, we determined if binding of PIPKIγ affects PLK4 dimerization, which is important for the self-activation of PLK4 (Guderian et al., 2010b). As shown in Fig. 7G, overexpressed Myc-PLK4 formed dimer with purified MBP-PLK4. This dimerization was not interrupted by the addition of increasing amount of recombinant PIPKIγ (Fig. 7G), indicating more complicated mechanism for future exploration. Nevertheless, in the context that PLK4 activity is critical for it to initiate procentriole biogenesis and achieve self-regulation, our results suggest that PIPKIγ could restrain centriole duplication by binding to and inhibiting PLK4 kinase activity.
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Discussion How centrioles are built and how the centriole assembly is limited to one daughter per mother in a single cell cycle are associated puzzles that have not been fully resolved. In addition to the key players identified in recent years (Brito et al., 2012; Nigg and Stearns, 2011), here we report PIPKIγ as a new component of centriole duplication machinery that restricts centriole formation by potentially limiting PLK4 activity. As a negative regulator of centriole biogenesis, association
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of PIPKIγ with the centrosome likely is sensitive and carefully regulated, which could be a reason that PIPKIγ was not described in the recent centrosome proteome studies (Andersen et al., 2003; Keck et al., 2011; Muller et al., 2011; Muller et al., 2010; Nogales-Cadenas et al., 2009; Ren et al., 2010; Zellner et al., 2011). Nevertheless, our results from multiple experiments (Fig. 1 and Fig. 2) provided solid evidence supporting the physical association of endogenous PIPKIγ with the centrosome. The overall levels and centrosome association of PIPKIγ are regulated through the cell cycle as many other centriolar proteins, however, in a rather unique way. Comparing to the positive regulators of centriole biogenesis like PLK4, SAS-6, and STIL whose levels are highest in M phase and drop sharply at mitotic exit, PIPKIγ is regulated oppositely: it is down-regulated during mitosis but increases vigorously right after mitosis. In addition, association of PIPKIγ with a centriole is first seen at G1/S boundary when the procentriole starts to emerge from this centriole (Fig. S1G), which is similar to Centrin2, SAS-6, and CPAP that are recruited to centrioles in G1/S phase to support procentriole assembly (Kleylein-Sohn et al., 2007; Paoletti et al., 1996; Strnad et al., 2007; Tang et al., 2009). Although it is not fully understood why PLK4, SAS-6, and STIL/SAS-5 accumulate in M phase when the budding of the procentriole has completed, the cell cycle-dependent expression and centrosome association of PIPKIγ serves its function well: it counteracts the centriole assembly machinery and inhibits the formation of excessive procentrioles. Although only PIPKIγ_i3 when overexpressed can target to the centrosome, the unique insert sequence in PIPKIγ_i3, which distinguishes it from other PIPKIγ alternative splicing isoforms, does not contain a centrosome-targeting sequence (Fig. 1E).
Instead, the
centrosome-targeting signal seems locates in the kinase domain that commonly presents in other - 14 -
PIPKIγ splicing isoforms and is highly conserved among all type I PIPK members; however none of them shows centrosomal localization. This suggests that the centrosome-targeting signal in PIPKIγ is normally hidden and needs certain modification to get exposed. It is plausible that dimerization of PIPKIγ (Rao et al., 1998) covers the centrosome-targeting motif in the kinase domain. The unique C-terminal insertion of PIPKIγ_i3 might alter the protein conformation and expose the centrosome-targeting motif. Since only a fraction of PIPKIγ_i3 expressing cells showed PIPKIγ_i3 at the centrosome, it is also possible that other modification mechanism, which
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could have been exhausted by overexpressed PIPKIγ_i3, is necessary to achieve a more efficient exposure of the centrosome-targeting motif in PIPKIγ. Similar as other centrosomal proteins (Fu and Glover, 2012a; Mennella et al., 2012a; Pelletier and Yamashita, 2012; Sonnen et al., 2012a), both endogenous and overexpressed PIPKIγ exhibited toroid-shaped distribution around the centriole in most cells, suggesting that the association of PIPKIγ with the centrosome follows the same common mechanism as other centrosomal proteins. This was reinforced by the observation that the stable association between PIPKIγ and the centrosome strictly relies on the existence of CEP152 but not CEP192, which is likely achieved via a physical association between PIPKIγ and CEP152. Indeed, the PIPKIγ toroid overlapped nicely with the CEP152 toroid, indicating that PIPKIγ is an intermediate PCM protein like CEP152 (Sonnen et al., 2012a). However, PIPKIγ is absent from the centrosome in M phase when CEP152 still presents, suggesting that CEP152 might not be sufficient to recruit PIPKIγ to the centrosome. More investigations need to be done in the future to decipher the signal code that governs the cell-cycle dependent association of PIPKIγ with the centrosome. Traditionally, PIPKIγ regulates variant cellular processes by targeting to specific subcellular locales and providing PI4,5P2 to the proximate downstream effector. This leads to the question if PI4,5P2 has a role in centriole duplication. Using GFP-tagged PLCδ PH domain, we failed to see clear GFP signal at the centrosome due to the strong membrane and cytosolic background (data not shown). PI4,5P2 antibody also failed to highlight any specific subcellular locations including the ones where PI4,5P2 plays important roles, such as focal adhesions and clathrin-coated pits (data not shown). However, it has been reported that there are membrane - 15 -
vesicles around the centrosome (Foraker et al., 2012; Westlake et al., 2011) and phosphoinositide-binding proteins like clathrin (Ford et al., 2001) and FYVE-CENT (Sagona et al., 2010) localize at the centrosome and regulate centrosome integrity (Foraker et al., 2012), suggesting that phosphoinositides including PI4,5P2 might exist and function at the centrosome. Nevertheless, in our hands both the wild type and kinase dead forms of PIPKIγ could similarly released the centriole/centrosome amplification caused by PIPKIγ depletion, HU treatment, or PLK4 overexpression, suggesting that PI4,5P2 is not essential in limiting centriole duplication.
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production. For example, PIPKIγ functions as a scaffold between E-cadherin and clathrin adaptor
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We reported previously that PIPKIγ can regulate some cellular process independent of PI4,5P2
the highly restricted expression of endogenous PLK4 (Guderian et al., 2010a; Holland et al., 2010;
complex AP1B, therefore facilitates the transport of E-cadherin from the trans-Golgi network to the recycling endosome (Ling et al., 2007a). In this process, the physical interactions of PIPKIγ with E-cadherin and AP1B, but not the PI4,5P2 generated by PIPKIγ, is critical. We propose that binding of PIPKIγ to PLK4 might impair the conformation and/or the access of ATP or substrates of PLK4 so that weakens PLK4 activity. Although this model has not been tested in vivo due to
Sillibourne et al., 2010), it is in agreement with our observation that loss of PIPKIγ causes PLK4-dependent centriole amplification without affecting cell cycle progression. The auto-phosphorylation of PLK4 triggers both activation and degradation of PLK4 (Guderian et al., 2010a; Holland et al., 2010; Sillibourne et al., 2010). It has been shown that the degradation of PLK4 requires multisite phosphorylation, which leads to a model that PLK4 is destructed once its activity reaches certain threshold level (Guderian et al., 2010a; Holland et al., 2010; Sillibourne et al., 2010). Our results suggest that PIPKIγ by binding to PLK4 inhibits its activity, providing another level of regulation in addition to phosphorylation. This seems critical to maintain the appropriate activity of centriole duplication machinery, since loss of PIPKIγ leads to generation of multiple procentrioles and overexpression of PIPKIγ blocks centrosome overduplication. Our results suggest that CEP152, by engaging both the positive regulator PLK4 and its negative regulator PIPKIγ around the proximal ends of parental centrioles, provides a physical platform for the accurate control of centriole assembly and homeostasis. - 16 -
Future studies
will be necessary to provide great details about the functional consequence of the physical interaction between PIPKIγ and PLK4. It is also important to investigate whether PLK4 is the only centrosomal protein regulated by PIPKIγ and whether PI4,5P2 has a functional role in other
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centrosome-related cellular processes.
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Materials and methods Cell Culture and Transfection. HeLa, U-2 OS, MDA-MB-231, NIH3T3 and HEK293T cells were maintained in DMEM containing 10% FBS. IMCD3 cells were cultured in DMEM:F12 containing 10% FBS. HeLa and HEK293T cells were transfected using Lipofectamine 2000 (Invitrogen) or FuGENE 6 Transfection Reagent (Promega). U-2 OS cells were transfected using X-tremeGENE 9 (Roche) or Amaxa Cell Line Nucleofector® Kit V (Lonza). siRNA duplexes were introduced into cells using Lipofectamine RNAiMAX (Invitrogen).
HeLa cells were
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synchronized with standard double-thymidine block [two 16-hour thymidine (Sigma, 2 mM) treatments separated by a 9-hour release].
U-2 OS cells were synchronized with 16 mM
hydroxyurea (Sigma) for 24 h. The tetracyclin-inducible cell-line expressing Myc-tagged PLK4 was kindly provided by Dr. Erich Nigg (Kleylein-Sohn et al., 2007). Myc-PLK4 expression was induced by the addition of 1 μg/ml of Doxycycline (Clontech). Antibodies and Constructs. Rabbit monoclonal antibody against PIPKIγ was generated at Epitomics Inc. by immunizing rabbits with recombinant His-tagged PIPKIγ. Before collecting the spleens, anti-sera were harvested and purified against affinity column conjugated with MBP-tagged PIPKIγ C-terminus (a.a. 469-668) to obtain polyclonal PIPKIγ antibody. Rabbit polyclonal antibodies against PIPKIα and PIPKIβ were obtained by immunizing rabbits with recombinant GST-His-tagged PIPKIα or PIPKIβ. Anti-sera were purified with affinity column conjugated with MBP-tagged C-terminus of PIPKIα (a.a. 451-562) or PIPKIβ (a.a. 397-540). Mouse monoclonal anti-PLK4 antibody was generated at Abmart, Inc. Rabbit polyclonal antibodies against the following proteins were used: phospho-Histone H3 (Ser10) (Millipore), CPAP (Proteintech), β-Actin, CEP135, GFP (Abcam), CEP152 (Bethyl Laboratories, A302-480A), CEP192 (a generous gift from Dr. Erich Nigg) (Schmidt et al., 2009), and CP110 (Proteintech). Mouse antibodies against the following proteins were used: Centrin2 (20H5, a generous gift from Dr. Jeffrey Salisbury), ODF2 (Abnova), C-Nap1 (BD Biosciences), His (HIS.H8, Millipore), GFP (Roche); α-Tubulin (DM1A), γ-Tubulin (GTU-88), FLAG (M2), and HA (HA-7) are from Sigma; Myc (9E10), HsSAS-6 (91.390.21), and cyclin B (GNS1) are from Santa Cruz. Anti-Ninein (Y-16) antibdy is from Santa Cruz and Maltose Binding Protein antibody (7G4) is from Sigma. - 18 -
cDNAs encoding full length or truncated human PIPKIγ (Ling et al., 2002), PLK4, and CEP152 were obtained by PCR and sub-cloned into pCMV-HA, pcDNA3-Flag, pGEX-4T-1, pET-28, pET-42, or pMAL-C2X constructs.
pcDNA3-Myc-PLK4 was obtained from Addgene.
HA-PIPKIγ kinase dead (KD), RNAi resistant HA-PIPKIγ-WT or -KD which contains mismatches to the HsPIPKIγ-O1 siRNA was generated using QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies). All constructs were verified by sequencing.
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Lentiviruses. Lentiviruses carrying shRNA were constructed by cloning shRNA oligonucleotides (Invitrogen) into the pLKO.1 vector (AddGene). The shRNA sequence targeting human PIPKIγ was 5′-GTCGTGGTCATGAACAACA-3′. An interfering RNA sequence targeting luciferase (5′-GTACCTGTACTTCATGCAG-3′) was used as negative control.
All constructs were
confirmed by sequencing. HEK293T cells were co-transfected with pLKO.1-puro carrying shRNA, pCMV-VSVG, and pCMV-R8.91 (gifts from Dr. Gaoxiang Ge, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, China) at 2:1:1 ratio using FuGENE 6 (Promega).
Medium supernatant was collected 48-72 h after transfection.
Subconfluent HeLa Cells were infected in the presence of 8 μg/mL polybrene (Sigma).
siRNAs. All siRNA oligonucleotides were obtained from Invitrogen. HsPIPKIγ-O1: 5'- GCGTGGTCAAGATGCACCTCAAGTT-3'; HsPIPKIγ-O2: 5'-CCTACAGGTTCATCAAGAAACTGGA-3'; mouse PIPKIγ: 5'-GCGAGAGAGAGGATGTGCAGTATGA-3'. PLK4-O1: 5'-CACTGGTTTGGAAGTTGCAATCAAA-3'; PLK4-O2: 5'- AGGAGGTGTGTGTGGAGCTTGTAAA-3'; HsSAS-6: 5'-AGAAAAGCACGTTAATCAGCTACAA-3' (Strnad et al., 2007); CEP152-O1: 5'-CAGAACAACTGAAATGGCTCTGGAA-3'; CEP152-O2: 5'- CAGCGTTTGCTGGGTAGCAACTCAA-3'; CEP192-O1: 5'- CCCAAAGGAAGACATTTTCATCTCT-3'; CEP192-O2: 5'- ATCAGACAGAGGAATCAATAATAAA-3' (Gomez-Ferreria et al., 2007; Sonnen et al., 2013; Zhu et al., 2008); - 19 -
HsPIPKIα-O1: 5'-TTGAAAGGTGCCATCCAGTTAGGCA-3'; HsPIPKIα-O2: 5'-TGTTAAGAAGTTGGAGCACTCTTGG-3' (Mellman et al., 2008); HsPIPKIβ: 5'-CAGCAAAGGGTTACCTTCCAGTTCA-3'; Stealth RNAi Negative Control siRNA (Invitrogen) was used as negative control. Similar results were got by using both siRNA oligonucleotides (O1, O2) for depletion of HsPIPKIγ, PLK4, CEP152, CEP192 or HsPIPKIα, respectively. Unless stated otherwise, results for oligonucleotide
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No.1 (O1) are shown. Immunoprecipitation and Microscopy. Immunoprecipitation was performed as previously described (Ling et al., 2002) using IP buffer [20 mM Hepes-KOH, pH7.2, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 0.5% NP-40, Complete Protease Inhibitor Cocktail (Roche), PhosSTOP Phosphatase Inhibitor Cocktail (Roche)].
For indirect
immunofluorescence, cells were grown on glass coverslips, fixed with ice-cold methanol for 10 min, permeablized, and stained as previously described (Ling et al., 2002). Fluorescence images were acquired using Nikon TE 2000-U with Metamorph (Molecular Devices). Z-Series were taken at 0.1-μm steps.
Three Dimensional Structured Illumination Microscopy (3D-SIM) was
performed on ELYRA Superresolution Microscopy system (Zeiss) equipped with alpha “Plan-Apochromat” 100x/1,46 Oil DIC oil immersion objective and Andor iXon 885 EMCCD camera, following standard protocol. Sections were acquired at 0.125 mm z-steps.
Color
channels were aligned using alignment parameter from control measurements with 0.5 μm diameter multispectral fluorescent beads (Zeiss). Structured illumination reconstruction and image processing was performed with the ZEN software package (Zeiss). Final image processing was done using Adobe Photoshop. In vitro Protein Pull-down Assay. GST or MBP-fused proteins were incubated with His-tagged proteins and Glutathione-Sepharose beads (GE Healthcare) or Amylose resin (New England Biolabs) in binding buffer (25mM Tris, pH 7.6, 150 mM NaCl, 1 mM DTT, 0.5% Triton X-100, 10% Glycerol) for 2 h at 4 °C. The precipitates were washed five times with binding buffer before analyzed by immunoblotting. CEP152 N-terminus (1-748 a.a.) protein was generated using the TNT Quick Coupled Transcription/Translation System (Promega), in presence of - 20 -
35
S-methionine (Perkin-Elmer), used for pull-down assay and analyzed by autoradiography.
Transmission Electron Microscopy. Cells were fixed for 1 h using Trump's fixative (Electron Microscopy Sciences).
Embedding and serial sectioning of cell samples was performed
according to standard procedures at the Electron Microscopy Core Facility, Mayo Clinic. Specimens were observed in a JEOL 1400 transmission electron microscope (JEOL) operating at 80 kV.
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PLK4 Kinase Assay. PLK4 activity was determined following a published approach (Hatch et al., 2010; Holland et al., 2010).
Briefly, Flag-tagged CEP152 fragment (CEP1521-748) was
overexpressed in HeLa cells and immunoprecipitated by anti-Flag antibody. 0.2 μg purified MBP-PLK4 was incubated with or without recombinant CEP152 N-terminus and/or appropriate amount of wild type (WT) or kinase dead (KD) His-PIPKIγ in kinase buffer with 5 μCi 32P-ATP (Perkin Elmer) and 33 μM ATP (New England Biolabs) for 30 min at room temperature. ImageJ (National Institutes of Health, Bethesda, MD) was used to quantify autoradiographs. Centrosome Isolation. Centrosomes were isolated from HeLa cells or mouse kidneys, based on a published approach (Meigs and Kaplan, 2008). Statistical Analyses. Data represent means and standard deviations (SD) from at least three independent experiments. Significance was calculated by Student’s t-test using Excel software (Microsoft). p
