KIF3B phosphorylation regulates kinesin-II interaction with IFT-B to control IFT entry and turnaround.

Please cite this article in press as: Liang et al., FLA8/KIF3B Phosphorylation Regulates Kinesin-II Interaction with IFT-B to Control IFT Entry and Turnaround, Developmental Cell (2014), http://dx.doi.org/10.1016/j.devcel.2014.07.019

Developmental Cell

Article FLA8/KIF3B Phosphorylation Regulates Kinesin-II Interaction with IFT-B to Control IFT Entry and Turnaround Yinwen Liang,1,2,3 Yunong Pang,2,3 Qiong Wu,1,2 Zhangfeng Hu,1,2 Xue Han,1,2 Yisheng Xu,2 Haiteng Deng,2 and Junmin Pan1,2,* 1MOE

Key Laboratory of Protein Science, Tsinghua University, Beijing 100084, China of Life Sciences, Tsinghua University, Beijing 100084, China 3Co-first author *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2014.07.019 2School

SUMMARY

The assembly and maintenance of cilia depends on intraflagellar transport (IFT). Activated IFT motor kinesin-II enters the cilium with loaded IFT particles comprising IFT-A and IFT-B complexes. At the ciliary tip, kinesin-II becomes inactivated, and IFT particles are released. Moreover, the rate of IFT entry is dynamically regulated during cilium assembly. However, the regulatory mechanism of IFT entry and loading/unloading of IFT particles remains elusive. We show that the kinesin-II motor subunit FLA8, a homolog of KIF3B, is phosphorylated on the conserved S663 by a calcium-dependent kinase in Chlamydomonas. This phosphorylation disrupts the interaction between kinesin-II and IFT-B, inactivates kinesin-II and inhibits IFT entry, and is also required for IFT-B unloading at the ciliary tip. Furthermore, our data suggest that the IFT entry rate is controlled by regulation of the cellular level of phosphorylated FLA8. Therefore, FLA8 phosphorylation acts as a molecular switch to control IFT entry and turnaround.

INTRODUCTION Intraflagellar transport (IFT), a microtubule-based bidirectional transport system between the cell body and the cilium, delivers ciliary precursors for cilium assembly and maintenance and regulatory components for signaling, such as hedgehog signaling (Goetz and Anderson, 2010; Rosenbaum and Witman, 2002; Scholey, 2008). IFT undergoes dynamic regulation. During ciliary entry, activated heterotrimeric kinesin-2 (kinesin-II), which was first identified in the sea urchin (Cole et al., 1993), enters into the cilium loaded with IFT particles comprising complex A (IFT-A) and complex B (IFT-B) and inactive cytoplasmic dynein. At the ciliary tip, several events occur, including inactivation of kinesin-II and IFT particle dissociation from kinesin-II and reassociation with the activated dynein (Pedersen and Rosenbaum, 2008). In addition, the IFT entry rate undergoes dynamic changes during cilium assembly (Engel et al., 2009; Ludington et al.,

2013). However, the molecular mechanisms underlying the activation/inactivation of kinesin-II, the interaction between kinesinII and IFT particles, and the regulation of the IFT entry rate are not well understood. Available evidence suggests that IFT may be regulated by protein phosphorylation. The null mutant of LF2, a homolog of cell cycle-related kinase, exhibits unequal length of flagella with accumulation of IFT particles at the flagellar tip (Tam et al., 2007). A group of conserved mitogen-activated protein (MAP) kinase subfamily members, including Chlamydomonas LF4, worm DYF-5, and mammalian MAK and ICK, are involved in IFT regulation (Berman et al., 2003; Burghoorn et al., 2007; Chaya et al., 2014; Omori et al., 2010; Yang et al., 2013). An increased IFT injection rate is observed in the lf4 mutant (Ludington et al., 2013). The null mutant of MAK induces the accumulation of IFT-B at the ciliary tip (Omori et al., 2010). In the dyf-5 mutant, kinesin-II does not dissociate from IFT particles at the end of the middle segment of cilia as in the wild-type. Rather, it travels further, to the distal segment, indicating that DYF-5 regulates the interaction of kinesin-II with IFT particles (Burghoorn et al., 2007). It has been shown recently that KIF3A is phosphorylated by ICK and that either loss or overexpression of ICK causes the accumulation of IFT-B at the ciliary tip (Chaya et al., 2014). Calcium is a critical regulator of cilium assembly and disassembly in Chlamydomonas and mammalian cells (Besschetnova et al., 2010; Cheshire and Keller, 1991). A link between calcium and IFT regulation has been revealed during flagellar gliding in Chlamydomonas. An absence of calcium induces the accumulation of IFT20 (an IFT-B component) at the flagellar tip, and an elevation of calcium initiates retrograde IFT transport (Collingridge et al., 2013). Because of the link between calcium and IFT regulation and because phosphorylation by CaMKII of KIF17, a homodimeric kinesin-2 in mammalian cells disrupts motor-cargo interaction during neuronal transport (Guillaud et al., 2008), we reasoned that calcium-mediated protein phosphorylation may regulate IFT particle-kinesin-II interaction to control IFT entry and turnaround. The protein kinases that are most similar to CaMKII in Chlamydomonas are calcium-dependent protein kinases (CDPKs). In this work, we show that CrCDPK1 is localized at the basal body and proximal flagella in steady-state cells. During flagellar assembly, CrCDPK1 is enriched at the flagellar tip, whereas it disappears at proximal flagella. In vitro and in vivo studies

Developmental Cell 30, 1–13, September 8, 2014 ª2014 Elsevier Inc. 1


Developmental Cell IFT Regulation by Protein Phosphorylation

show that CrCDPK1 phosphorylates FLA8/KIF3B, a motor subunit of kinesin-II on S663, a conserved residue among its homologs. This phosphorylation disrupts kinesin-II interaction with IFT-B and regulates IFT entry at the flagellar base and IFT turnaround at the tip. We further show that the cellular level of phosphorylated FLA8 increases gradually along with flagellar elongation during flagellar assembly, suggesting a role in regulating the IFT entry rate. RESULTS CrCDPK1 Is Localized to the Basal Body and Proximal Flagella CrCDPK1, first identified in the Chlamydomonas flagellar proteome (Pazour et al., 2005), is a calcium-dependent protein kinase with four EF hands following the protein kinase domain and a C2 domain at the N terminus (Figure S1A available online) (Liang and Pan, 2013). Fractionation of cell bodies and flagella followed by immunoblotting with an anti-CrCDPK1 antibody confirmed that CrCDPK1 is a flagellar protein, although it was mainly distributed in the cell body (Figure 1A; Figure S1A). In the flagellar compartment, CrCDPK1 was distributed in the membrane/matrix as well as in the axoneme (Figure 1B). Immunostaining analysis revealed that CrCDPK1 was localized to the basal body and also at proximal flagella (Figure 1C). The localization of CrCDPK1 was further confirmed by coimmunostaining of CrCDPK1 with anti-centrin antibody (Figure 1D) and by analyzing isolated cell bodies and flagella (Figure 1E). Finally, CrCDPK1 knockdown by RNAi removed the basal body and proximal flagellar staining (Figures 1F and 1G). These results indicate that CrCDPK1 is localized in the basal body through to the very proximal flagella. Translocation of CrCDPK1 from Proximal Flagella to the Flagellar Tip during Flagellar Assembly We next examined whether CrCDPK1 was involved in flagellar assembly. Although immunoblot analysis of cells undergoing flagellar regeneration failed to detect changes of the cellular level of CrCDPK1 (Figure 2A), CrCDPK1 was found to be enriched in regenerating flagella, similar to IFT proteins represented by IFT139 (Figure 2B) (Marshall et al., 2005), providing the first link of CrCDPK1 to flagellar assembly. Chlamydomonas flagella are assembled rapidly within 60 min after deflagellation, followed by a decreasing rate of assembly, leading to completion of flagellar assembly within 90–120 min (Rosenbaum et al., 1969). An immunostaining assay was used to track flagellar changes of CrCDPK1 during flagellar regeneration (Figure 2C). Consistent with the immunoblot analysis, increased immunostaining of CrCDPK1 was observed in the rapidly assembling flagella. Interestingly, CrCDPK1 was also enriched at the flagellar tip, whereas its location at proximal flagella disappeared. Toward the completion of flagellar assembly, the percentage of CrCDPK1 tip localization decreased gradually, and proximal flagellar location of CrCDPK1 was recovered (Figures 2C and 2D). The tip localization of CrCDPK1 in regenerating flagella is consistent with a report showing that CrCDPK1 is a candidate for flagellar tip proteins (Satish Tammana et al., 2013). To determine whether flagellar tip localization of CrCDPK1 is a function of flagellar assembly rather than flagellar length, cells

were induced to shorten their flagella by adding sodium pyrophosphate (NaPPi) (Lefebvre et al., 1978). When the flagella shortened, however, flagellar CrCDPK1 did not increase, nor was it enriched at the flagellar tip (Figure 2E). By removing sodium pyrophosphate to allow flagellar regrowth, CrCDPK1 was increased in the flagella with enrichment at the tip and loss at the proximal flagella. Therefore, the flagellar localization of CrCDPK1 undergoes dynamic changes that are tightly associated with the flagellar assembly process. CrCDPK1 possesses a lipid binding C2 domain at the amino terminus that may regulate the membrane association of CrCDPK1 (Corbalan-Garcia and Go´mez-Ferna´ndez, 2014) (Figure S1A). Indeed, biochemical and cell biological experiments showed that the localization of CrCDPK1 at proximal flagella and at the flagellar tip required membrane association mediated by the C2 domain (data not shown). CrCDPK1 Functions in Flagellar Assembly by Affecting IFT The dynamic localization of CrCDPK1 at proximal flagella and at the flagellar tip during flagellar regeneration implicates its possible function in flagellar assembly (Figure 2C). Examination of two representative RNAi strains revealed that depletion of CrCDPK1 caused shorter flagella (Figure 3A) and a reduced rate of flagellar regeneration (Figures S2A and S2B). We noticed that the time taken to regenerate half-length flagella in the RNAi strains was similar to that of wild-type cells, which may reflect a complex regulation of flagellar length and assembly. Nevertheless, these data demonstrate that CrCDPK1 is required for proper flagellar assembly. To examine whether this defect in flagellar assembly is related to IFT, flagella from the wild-type and from the RNAi strains were isolated and processed for immunoblotting with antibodies against IFT proteins (Figure 3B). Consistent with knockdown of CrCDPK1 (Figure 1F), the flagellar level of CrCDPK1 in the RNAi strains was decreased. IFT144 and IFT139 from IFT-A, IFT172, and IFT81 from IFT-B were all reduced compared with those in the wild-type. Similarly, FLA8, the KIF3B homolog of the kinesin-II motor subunit, was also reduced, as shown by using a peptide antibody (Figure 3B; Figures S2C and S2D). In addition, CrCDPK1 affected the proper localization of IFT proteins. In steady-state wild-type cells, FLA8 was enriched at the basal body region but not at the flagellar tip (Figures 3C and 3D). In contrast, around 70% of cells showed tip localization of FLA8 in the CrCDPK1 RNAi strains. Examination of IFT-A (represented by IFT139) and IFT-B (represented by IFT172) showed similar results (Figures 3E and 3F). The tip localization for IFT139 and 172 was 76.45% and 66.64% in the RNAi strains, respectively (Figures 3G and 3H). Taken together, CrCDPK1 regulates IFT entry and turnaround at the flagellar tip. S663 of FLA8 Is Phosphorylated by CrCDPK1 The mammalian homolog of CrCDPK1, CaMKII, has been shown to regulate cargo release by phosphorylation of homodimeric kinesin-2 (Guillaud et al., 2008). That alteration of CrCDPK1 expression results in tip accumulation of kinesin-II and IFT complexes suggests that CrCDPK1 may regulate cargo (IFT complex) release from the kinesin-II motor at the flagellar tip during IFT turnaround. Analysis of phosphopeptides in the flagellar

2 Developmental Cell 30, 1–13, September 8, 2014 ª2014 Elsevier Inc.



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Figure 1. CrCDPK1 Is a Flagellar Protein with Localization at the Basal Body through to Proximal Flagella (A) Whole cells (WC) were fractionated into the cell body (CB) and flagella, followed by immunoblot analysis with anti-CrCDPK1 antibody. Equal (13 F) or 50 proportions (503 F) of flagella relative to whole cells were loaded. a-tubulin was used as a loading control. (B) Immunoblot analysis of CrCDPK1 in fractionated flagellar membrane/matrix (M/M) and axonemal (Axo) fractions. The flagellar membrane protein FMG1b and a-tubulin were used as controls. Fla, flagella. (C) Coimmunostaining of steady-state cells with anti-CrCDPK1 or preimmune serum (immunoglobulin G [IgG]) and anti-a-tubulin antibodies. The insets show higher magnification views of the basal body region. (D) Coimmunostaining of steady-state cells with anti-CrCDPK1 and anti-centrin antibodies. The insets are as shown in (C). (E) Immunostaining of an isolated cell body and flagella with anti-CrCDPK1 and anti-a-tubulin antibodies showing CrCDPK1 localization at the basal body and proximal flagella (arrowheads). (F) Immunoblot analysis of wild-type and two representative CrCDPK1 RNAi strains with anti-CrCDPK1 antibody. CrCDPK3 was used as a control. WT, wild-type; 1–91 and 2–12, RNAi strains. (G) Immunostaining analysis of RNAi cells showing depletion of CrCDPK1 at the basal body and proximal flagella. Scale bars represent 5 mm. See also Figure S1.

proteome (Pazour et al., 2005) showed that S663 in the C-terminal domain of FLA8 was phosphorylated (G. Pazour, personal communication). Alignment of FLA8 with its homologs shows that this site is conserved in various organisms except for C. elegans (Figure 4A). Furthermore, this phosphorylation site

falls within the consensus phosphorylation sequence R-X-X-S of CaMKII (White et al., 1998). To confirm FLA8 phosphorylation on S633 and attempt to identify other phosphorylation sites in kinesin-II, we performed mass spectrometry on immunoprecipitated subunits. FLA8 S663 is indeed phosphorylated, and S476




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Figure 2. Dynamic Change of Flagellar Localization of CrCDPK1 during Flagellar Assembly (A) Predeflagellated cells (pdf) and cells undergoing flagellar assembly at different times after deflagellation by pH shock were analyzed by immunoblotting with anti-CrCDPK1 antibody. a-tubulin was used as a loading control. (B) Full-length flagella (flf) from steady-state cells and assembling flagella (asf) from cells undergoing flagellar assembly (30 min after deflagellation) were isolated and analyzed by immunoblotting of equal amounts of protein with anti-CrCDPK1 antibody. (C) Immunostaining of predeflagellated cells and cells undergoing flagellar regeneration at different times after deflagellation. Note the changes of CrCDPK1 staining at proximal flagella and flagellar tips (arrows). (D) Percentages of flagellar tip localization of CrCDPK1 from predeflagellated cells and cells undergoing flagellar regeneration at different times after deflagellation. Data are presented as mean ± SD (150 cells, n = 3 for each time point). ***p < 0.0001 (Student’s t test). (E) Flagellar localization of CrCDPK1 during flagellar resorption followed by flagellar regrowth. 20 mM NaPPi was added to cells to induce flagellar resorption, followed by removal of NaPPi to allow flagellar regrowth. The cells were coimmunostained with anti-CrCDPK1 and anti-a-tubulin antibodies. Note the changes of CrCDPK1 at flagellar tips (arrow and insets) and proximal flagella. Scale bars represent 5 mm.

might be a minor phosphorylated residue (Figures S3A and S3B). Analysis of coimmunoprecipitated FLA10 and KAP did not identify any phosphoresidues on KAP, whereas FLA10 was phosphorylated on S401 (Figure S3C). S401 of FLA10 is not conserved in other species (data not shown), suggesting that S663 of FLA8 is likely the major site for kinesin-II regulation by phosphorylation. We generated a polyclonal antibody (pFLA8 antibody) against phosphorylated S663. Immunoblot analysis showed that this antibody was specific and recognized phosphorylated FLA8 (pFLA8) in Chlamydomonas steady-state cells (Figure 4B). Mass spectrometry analysis of immunoprecipitated FLA8 showed that 15.99% was phosphorylated. CrCDPK1 knock-

down reduced the amount of phosphorylated but not total FLA8, as examined by immunoblotting, indicating that FLA8 phosphorylation is regulated by CrCDPK1 (Figure 4C). Further experiments suggest that FLA8 at S663 is directly phosphorylated by CrCDPK1. Sucrose gradient analysis showed that part of CrCDPK1 comigrated with the kinesin-II motor subunits FLA8 and FLA10 (Figure 4D). To demonstrate a physical interaction between FLA8 and CrCDPK1, hemagglutinin-tagged (HA-tagged) FLA8 was transformed into an aflagellate fla8 mutant (CC-829) with V(12)E mutation (Dutcher et al., 2012). Immunoprecipitation with an anti-HA antibody pulled down CrCDPK1 from the cell lysate of the transformed strain but not from that of the wild-type (Figure 4E). Finally, an




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Figure 3. Depletion of CrCDPK1 by RNAi Inhibits Flagellar Regeneration and Affects IFT (A) Flagellar length distribution of WT cells and two CrCDPK1 RNAi strains. (B) Immunoblot analysis of IFT proteins in the flagella of WT and CrCDPK1 RNAi strains. (C) Flagellar tip localization of FLA8 in CrCDPK1 RNAi cells. WT and RNAi cells were coimmunostained with anti-FLA8 and anti-a-tubulin antibodies. The insets show higher magnification views of the flagellar tips (arrows). (D, G, and H) Percentage of cells with flagellar tip localization in CrCDPK1 RNAi and WT cells of FLA8 (D), IFT139 (G), and IFT172 (H). Data are presented as mean ± SD (150 cells, n = 3). ***p < 0.0001 (Student’s t test). (E and F) Immunostaining of CrCDPK1 RNAi and WT cells with anti-IFT139 (E) or anti-IFT172 antibodies (F). Differential interference contrast (DIC) images are presented to show the flagella. Arrows and insets are as shown in (C). Scale bars represent 5 mm. See also Figure S2.

in vitro phosphorylation assay demonstrated that CrCDPK1 phosphorylates FLA8 at S663 (Figure 4F). Taken together, FLA8 is a phosphoprotein and phosphorylated on S663 by CrCDPK1.

Cellular and Flagellar Levels of Phosphorylated FLA8 on S663 Are Tightly Associated with Flagellar Assembly Flagellar regeneration was associated with an increase of pFLA8 in the flagella, as shown by analysis of different regenerating




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(A) FLA8 S663 (red) is highly conserved among its homologs. The genetic identity is as follows: human, 3913958; mouse, 1060923; zebrafish, 53945806; C. elegans, 9800183; sea urchin, 47551265; Chlamydomonas, 158274949; and Tetrahymena, 118360030. The line indicates the consensus sequence for CaMKII phosphorylation. B C Note that this consensus sequence is not conserved in KRP95 in C. elegans, which has a F sequence of RPPIS (serine at 680) instead of RXXS. X, any amino acid. (B) FLA8 is a phosphoprotein. Shown is immunoblotting with anti-pFLA8 antibody of cell lysates from the WT or a fla8 null mutant (left). Cell lysates from wild-type cells were treated with or without protein phosphatase (PPase), followed by immunoblotting with anti-pFLA8 and anti-FLA8 D antibodies, respectively (right). In the center blot, FLA8 phosphopeptides were added in the primary antibody reaction. (C) Immunoblotting of cell lysates from WT and CrCDPK1 RNAi strains shows that the cellular level of pFLA8 was reduced by depletion of CrCDPK1. (D) Chlamydomonas cell lysates were separated in a sucrose gradient, followed by immunoblotting with the indicated antibodies. (E) Coimmunoprecipitation of FLA8 and CrCDPK1. Cell lysates from WT cells or cells expressing FLA8-HA was immunoprecipitated (IP) with anti-HA antibody followed by immunoblotting with anti-HA and anti-CrCDPK1 antibodies, respectively. (F) In vitro phosphorylation assay. Bacterially expressed WT or S663A (SA) of the FLA8 C-terminal domain (FLA8 CTD) was incubated with GST-CrCDPK1. Because CaMKII can use ATP-g-S as a phosphodonor to thiophosphorylate its substrate (Allen et al., 2005), the phosphorylation assay was performed with ATP-g-S as a phosphodonor and anti-thiophosphate ester antibody to detect substrate phosphorylation (Allen et al., 2007). The final reaction was probed by immunoblotting with antibodies as indicated. Thio, thiophosphate ester. See also Figure S3.

flagella (Figure 5A). The ratio of pFLA8 to FLA8 in 6 mm flagella was increased around 4-fold (3.7 ± 1.4, n = 3) compared with that of full-length flagella. Therefore, active flagellar assembly is coupled with increased phosphorylation of FLA8 in the flagella, which is consistent with flagellar enrichment of CrCDPK1 during flagellar assembly. Cell fractionation analysis showed, however, that flagellar pFLA8 was present at a minimal level relative to whole-cell pFLA8 in steady-state cells (Figure 5B). Interestingly, pFLA8 was barely detected in the cell bodies. When cells undergoing flagellar regeneration were fractionated and analyzed, a similar result for pFLA8 in the cell bodies was obtained, although we observed an increased level of pFLA8 in the flagella (Figure 5C). The decrease in the pFLA8 level after flagellar loss is reminiscent of CALK, whose T193 phosphorylation level is regulated by flagellar length during flagellar assembly (Cao et al., 2013). Immunoblot analysis of cells during flagellar regeneration showed that the cellular level of pFLA8 was decreased drastically after deflagellation and increased gradually to the predeflagellation level when flagella reached full length, although total FLA8 was not changed (Figures 5D and 5E). Taken together, the cellular level of pFLA8 is intimately linked with flagellar assembly, indicating that IFT motor kinesin-II is regulated during flagellar assembly as flagellar length increases. The localization of pFLA8 was attempted by immunostaining with anti-pFLA8 antibody. Unfortunately, this antibody showed unspecific staining at the basal body, as assessed by using a fla8 null mutant (Figure S4). Because the cell body FLA8 was concentrated in the basal body region (Figure 5F) and no other

strong staining with anti-pFLA8 antibody was detected in the cell body except for the basal body region (Figure 5G), pFLA8 in the cell body was likely localized to the basal body region. In steady-state cells, pFLA8 was also localized to proximal flagella. In contrast, in cells undergoing flagellar regeneration, the proximal flagellar staining disappeared, and flagellar tip staining was observed that mirrored the staining pattern of CrCDPK1 (Figure 5G). The above data suggest that pFLA8 is regulated by CrCDPK1 during flagellar assembly and in steady-state cells. FLA8 S663 Phosphorylation Disrupts the Interaction between Kinesin-II and IFT-B and Inactivates Kinesin-II Next we determined whether alteration of FLA8 S663 phosphorylation affected flagellar assembly. HA-tagged wild-type FLA8, a phosphodefective (S663A) mutant, or a phosphomimetic (S663D) mutant was transformed into an aflagellate fla8 mutant (CC-829). Wild-type FLA8 fully complemented the flagellar phenotype, whereas S663A mutant cells possessed shorter flagella with a slower rate of flagellar regeneration, and S663D mutant cells were aflagellate (Figure 6A and Figures S5A and S5B). Therefore, proper phosphorylation of FLA8 is required for flagellar assembly. To test whether FLA8 phosphorylation affected kinesin-II transport, HA-tagged FLA8 wild-type and phosphomutants were transformed into wild-type strains. Unlike wild-type FLA8, the S663D mutant was not detected in the flagella analyzed by immunoblotting, whereas the S663A mutant was targeted to the flagella with an increasing amount (Figure 6B).




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Figure 5. FLA8 Phosphorylation Is Tightly Linked with Flagellar Assembly (A) Flagella were isolated from cells at different stages during flagellar assembly, followed by immunoblotting. The lengths of isolated flagella are indicated. (B and C) Steady-state cells (B) or cells undergoing flagellar assembly for 30 min after deflagellation (C) were fractionated, followed by immunoblotting. Equal proportions of whole cells (WC), cell body (CB), flagella (13 F), or 503 flagella (503 F) were loaded for analysis. (D) Changes of the cellular level of pFLA8 during flagellar assembly. Cells were collected before deflagellation (pdf) and at different times during flagellar assembly after deflagellation, followed by immunoblotting. Exposures (Exp) of the same pFLA8 blot with two different times are shown. (E) The ratio of pFLA8 versus total FLA8 determined by densitometry is plotted with flagellar length against time after deflagellation. Data are presented as mean ± SD from four independent experiments. (F and G) Immunostaining of steady-state cells and cells undergoing flagellar assembly with anti-FLA8 (F) or anti-pFLA8 (G) and anti-a-tubulin antibodies. The insets show higher magnification views of the flagellar tip. Scale bars represent 5 mm. See also Figure S4.

Immunostaining analysis with anti-HA antibody was consistent with these data (Figure 6C). Therefore, phosphorylated FLA8 inhibits kinesin-II flagellar entry, whereas nonphosphorylated FLA8 stimulates it. Because kinesin-II carries IFT particles into the flagellum, we predicted an increase of IFT proteins in the S663A mutant flagella. Indeed, immunoblot analysis of isolated flagella showed that components from both IFT-A and IFT-B increased in the S663A mutant flagella compared with those of the wild-type (Figure 6D). Kinesin-II nonmotor subunit KAP, which was recognized by a polyclonal antibody (Figure S5C), and FLA8 were also

increased (Figure 6D). The loss of flagellar entry of FLA8-S663D suggests that kinesin-II with phosphorylated FLA8 is inactive. We reasoned that phosphorylation of FLA8 might disrupt kinesin-II binding to IFT particles and, simultaneously, result in its inactivation. To demonstrate this, HA-tagged wild-type FLA8 and its phosphomutants were immunoprecipitated and analyzed by immunoblot analysis (Figure 6E). Wild-type FLA8 and its two phosphomutants pulled down FLA10 and KAP, indicating that the phosphorylation status of FLA8 does not affect the integrity of the kinesin-II motor. Wild-type FLA8 and the S663A mutant pulled down IFT172 and IFT81 of IFT complex




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Figure 6. FLA8 Phosphorylation Regulates the Interaction between Kinesin-II and IFT-B to Control IFT Entry (A) The aflagellate fla8 mutant (CC-829) was transformed with HA-tagged wild-type FLA8, a phosphodefective (S663A) or phosphomimetic (S663D) mutant. The flagellar length of the wild-type (21 gr), the fla8 mutant, and transformants was measured. Data are presented as mean ± SD. ***p < 0.0001 (Student’s t test). (B) Wild-type cells transformed with HA-tagged FLA8-HA, FLA8-S663A, or FLA8-S663D were fractionated into cell body (CB) and flagella (F), followed by immunoblotting analysis with anti-HA and anti-a-tubulin antibodies. WC, whole cells. (C) Immunostaining of transformants in (B) with anti-HA and anti-a-tubulin antibodies showing the S663D mutation preventing FLA8 entry to the flagellum while the S663A mutation stimulating the entry. Scale bar represents 5 mm. (D) Immunoblot analysis of isolated flagella from wild-type (21 gr) and the fla8 mutant transformed with HA-tagged wild-type FLA8 (WT) or the S663A (SA) mutant with the indicated antibodies. (E) Cells from (B) were used for immunoprecipitation with anti-HA antibody, followed by immunoblotting with the indicated antibodies. Wild-type cells (21 gr) not expressing HA-tagged constructs were used as a negative control. See also Figure S5.

B, although IFT139 of IFT complex A was not pulled down. In contrast, no IFT components were pulled down with the S663D mutant. These data demonstrate that FLA8 phosphorylation at S663 dissociates IFT-B from the kinesin-II motor and imply that kinesin-II activation is regulated by binding of IFT particles. FLA8 S663 Phosphorylation Mediates IFT Turnaround at the Flagellar Tip At the flagellar tip, IFT particles dissociate from kinesin-II (Pedersen and Rosenbaum, 2008). It would be expected that inhibition of FLA8 phosphorylation at the flagellar tip prevents dissociation

of IFT particles from kinesin-II and leads to an abnormal accumulation of IFT proteins. To demonstrate this, we examined the distribution of IFT proteins in the aflagellate fla8 mutant (CC-829) transformed with HA-tagged FLA8 or FLA8-S663A. In FLA8 cells, both IFT-B components, IFT172 and IFT81, did not accumulate at the flagellar tip (Figures 7A and 7B). In contrast, in S663A mutant cells, IFT172 and IFT81 accumulated at the flagellar tip with 89.2% and 66.4%, respectively, whereas the IFT-A component IFT139 did not (Figures 7C and 7D). As expected, FLA8-S663A increased in the flagella and accumulated at the tip (Figure S6). Therefore, FLA8 phosphorylation mediates the dissociation of IFT-B from kinesin-II.




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Protein PK CrCDPK1 PP phosphotase

Figure 7. FLA8 Phosphorylation Regulates IFT Turnaround at the Flagellar Tip (A–C) Immunostaining of cells expressing FLA8-HA or FLA8-S663A-HA with anti-IFT172 (A), anti-IFT81 (B), and anti-IFT139 (C) antibodies. DIC images are also presented. The insets show higher magnification views of the flagellar tip (arrow). Scale bars represent 5 mm. (D) Percentage of flagellar tip localization of IFT proteins in transformants expressing FLA8-HA or FLA8-S663A-HA. Data are presented as mean ± SD (150 cells, n = 3). ***p < 0.0001 (Student’s t test). ns., statistically not significant. (E) Working model of IFT regulation by FLA8/KIF3B phosphorylation. At the ciliary base, the phosphorylation state of FLA8 is controlled by CrCDPK1, or its counterpart in other organisms, and an unknown phosphatases. Nonphosphorylated FLA8 allows IFT-B to bind to the kinesin-II motor, which is then activated and carries IFT particles into the cilium. At the ciliary tip, tip-localized CrCDPK1 phosphorylates FLA8 and disrupts the interaction between kinesin-II and IFT particles. Remodeled IFT particles and, possibly, inactive kinesin-II are loaded onto cytoplasmic dynein and return to the cell body to complete the IFT cycle. During cilium assembly, an increasing level of pFLA8 as cilia elongate would gradually inhibit the loading of IFT particles and kinesin-II activity, leading to a decreased IFT entry rate. See also Figure S6.




DISCUSSION As presented in Figure 7E, we demonstrated that cells employ the regulation of a single molecule, FLA8/KIF3B, to control three fundamental processes of IFT: association and dissociation of IFT particles with the kinesin-II motor, activation and inactivation of kinesin-II, and control of the IFT entry rate. CrCDPK1 Regulates FLA8 Phosphorylation and IFT Although a few cases have shown that protein phosphorylation may regulate IFT (Burghoorn et al., 2007; Omori et al., 2010; Tam et al., 2007), our work is significant because we identified the target of CrCDPK1 as the kinesin-II motor subunit FLA8 and identified S663 as the key phosphorylation residue. Second, we showed that the FLA8 phosphorylation state is dynamically controlled during flagellar assembly. Third, we demonstrated that the phosphorylation state of FLA8 affects the motor-cargo interaction and kinesin-II activity. In mammalian cells, the closest homologs of CrCDPK1 are CaMKs. CaMKII-dependent phosphorylation has been shown to regulate cargo interaction with kinesin motors and has been suggested to be a general mechanism for controlling motor-cargo interaction (Guillaud et al., 2008; Verhey and Hammond, 2009). CaMKs have been found in the cilia proteome (Liu et al., 2007). In zebrafish embryos, CaMKII is found at the base of the cilia of Kupffer’s vesicle (Francescatto et al., 2010), and CaMKII deficiency destabilizes kidney cilia (Rothschild et al., 2011), which is consistent with our results showing that depletion of CrCDPK1 results in shorter flagella. It is likely that ciliary CaMKII in animals performs similar functions as CrCDPK1. Depletion of CrCDPK1 by RNAi decreases the cellular level of pFLA8 but reduces IFT protein levels in the flagellum, which is inconsistent with the model hypothesizing that a reduced level of pFLA8 would increase IFT proteins in the flagellum. One interpretation is that CrCDPK1 may have additional functions besides regulation of FLA8. Indeed, both IFT-A and IFT-B are accumulated at the flagellar tip after depletion of CrCDPK1, whereas only IFT-B accumulates in the S663A mutant of FLA8. It is likely that proper interaction of IFT-A and IFT-B mediated by CrCDPK1 affects IFT entry into the flagellum. CrCDPK1 exhibits three distinct locations: basal body, proximal flagella, and flagellar tip. The localization of CrCDPK1 at the basal body is consistent with the accumulation of IFT particles at the transition fibers, where the loading of IFT particles to the kinesin-II motor might occur (Deane et al., 2001). The enrichment of CrCDPK1 at the flagellar tip during vigorous flagellar assembly may reflect the requirement for higher CrCDPK1 activity at the tip because IFT is expected to turn around more rapidly in the short assembling flagella because the IFT amount in the flagella is constant (Marshall et al., 2005). The localization of CrCDPK1 at proximal flagella in cells with completed flagellar assembly is intriguing. CrCDPK1 at this location may serve as a secondary control to prevent the ‘‘overshooting’’ of IFT particles into the flagella in cells with full-length flagella. Proximal flagellar localization has been shown for a NIMA-related protein kinase, FA2, which is consistent with its role in deflagellation (Mahjoub et al., 2004). Interestingly, LF5, a CDK-like protein kinase that regulates flagellar length, and the smoothened-Evc2 complex that regulates hedgehog signaling

are also localized at the very proximal flagella or cilia (Dorn et al., 2012; Tam et al., 2013). Therefore, the proximal flagellar region may be an important location for signaling events involved in flagellar assembly or cilia-based signaling. Kinesin-II motors regulate various transport process within the cytoplasm in addition to IFT in cilia (Scholey, 2013). IFT components are delivered to the base of the cilium after synthesis. It will be interesting to determine whether CrCDPK1 also regulates the delivery of IFT complexes within the cytoplasm. IFT-B and Kinesin-II Interaction and Regulation of IFT Turnaround IFT exhibits anterograde and retrograde transport and turns around at the ciliary base and tip. IFT-B has been proposed to be linked with anterograde transport (Pedersen and Rosenbaum, 2008; Scholey, 2008). However, the evidence for a direct interaction between IFT-B and the kinesin-2 motor is rare. It has been reported that kinesin-II associates with IFT-B in an ATPdependent manner, which is consistent with this work, and that IFT20 interacts directly with KIF3B (Baker et al., 2003). In C. elegans, kinesin-II associates with IFT-A, whereas OSM-3 interacts with IFT-B. IFT70 (DYF-1), a complex B subunit, is required for docking OSM-3 to IFT particles (Fan et al., 2010; Ou et al., 2005). We show that phosphorylation of FLA8 disrupts the interaction between kinesin-II and IFT-B, indicating that IFTB interacts directly with kinesin-II during anterograde transport. Because FLA8 phosphorylation does not change the integrity of the kinesin-II complex, this phosphorylation may induce conformation changes of kinesin-II to affect IFT-B binding. IFT is regulated at the level of ciliary entry and tip turnaround. We show that the phosphomimetic mutant S663D of FLA8 disrupts the interaction between kinesin-II and IFT-B, whereas the phosphomutant S663A results in accumulation of IFT-B at the ciliary tip. This suggests that dephosphorylation of FLA8 is required for loading of IFT-B to kinesin-II at the ciliary base and that phosphorylation is needed for unloading IFT cargo at the ciliary tip. Therefore, FLA8 phosphorylation serves as a molecular switch to control loading and unloading of IFT particles. Interestingly, it has been shown recently that KIF3A phosphorylation regulates ciliogenesis (Chaya et al., 2014). However, no direct evidence has been presented for the involvement of KIF3A phosphorylation in IFT regulation. In C. elegans, DYF-5 encodes a MAP kinase and has been proposed to phosphorylate kinesin-II and to dissociate the motor from IFT particles at the middle segment of the cilium (Burghoorn et al., 2007), which is consistent with our data in that protein phosphorylation is a key regulation process to control the interaction between motor and IFT complexes. IFT-A and IFT-B undergo dissociation and reassociation at the ciliary tip. IFT172 in Chlamydomonas, BBSome, and IFT144 in C. elegans have been shown to be critical for this interaction (Pedersen et al., 2005; Wei et al., 2012). The induced accumulation of both IFT-A and IFT-B by depletion of CrCDPK1 (this work) or ICK (Chaya et al., 2014) demonstrates that this interaction is under regulatory control. Regulation of the Rate of IFT Entry The loss of kinesin-II activity (i.e., ciliary entry) and coincident loss of interaction between kinesin-II and IFT-B suggests that kinesin-II is inactive in the absence of cargo loading and activated




by cargo loading. It has been shown that kinesin-2 may exist as active and inactive forms (Scholey, 2008) and that the autoinhibited OSM-3 is activated after attaching to beads in an optical trap (Imanishi et al., 2006). We show that phosphorylated kinesin-II does not enter the cilium, indicating that it is not active when phosphorylated. Future studies are needed to determine the in vitro motility of kinesin-II affected by protein phosphorylation. More importantly, the rate of kinesin-II-driven motility studied to date in other systems is 0.3–0.6 mm/s, whereas the rate of kinesin-II-driven IFT in Chlamydomonas is 2 mm/s (reviewed in Scholey, 2008). Such an in vitro assay is expected to resolve this discrepancy. The rate of IFT entry or injection decreases as cilium length increases during cilium assembly because the IFT amount in the cilium is constant regardless of cilium length (Engel et al., 2012; Marshall et al., 2005). Imaging analysis and modeling of the IFT injection dynamics show that IFT injection fits with an avalanching model in which signaling regulation is not involved. The injection rate is predicted to be controlled by regulated recruitment of IFT materials to the ciliary base, possibly through a cilium length-dependent RanGTP signaling gradient (Ludington et al., 2013). Our data suggest an alternative mechanism for the control of IFT injection. We show that phosphorylation of FLA8 inhibits kinesin-II ciliary entry, therefore preventing IFT entry, whereas nonphosphorylated FLA8 stimulates IFT entry. Interestingly, the cellular level of pFLA8 increases with cilium elongation during cilium assembly. Therefore, cells may control the cellular level of pFLA8 to regulate the IFT entry rate. We have shown previously that the cellular level of phosphorylated CALK is regulated by cilium length during cilium assembly (Cao et al., 2013; Luo et al., 2011). This, plus the new finding of regulated phosphorylation of FLA8, strengthens the idea that cells possess a cilium length sensing system (Chan and Marshall, 2012) that would regulate the FLA8 phosphorylation level to control the IFT entry rate. EXPERIMENTAL PROCEDURES Cell Culture and Strains C. reinhardtii was grown in TAP medium for transformation or in M medium for other experiments in 250 ml Erlenmeyer flasks with aeration at 23 C on a 14:10 hr light-dark cycle as described previously (Liang and Pan, 2013). Wild-type strain 21 gr (CC-1690), fla8 (CC-829), and a strain expressing KAP-GFP (CC-4296) were obtained from the Chlamydomonas Resource Center (University of Minnesota). For characterization of antibodies against FLA8, a fla8 null mutant generated in our genetic screen was used. Flagellar Regeneration, Length Measurement, Flagellar Isolation, and Fractionation Cells were deflagellated by pH shock and allowed to regenerate flagella at the indicated times as described previously (Liang and Pan, 2013). For flagellar length measurement, cells were fixed in 1% glutaraldehyde followed by imaging with a Zeiss Axio Observer Z1 microscope (Zeiss) equipped with an electron microscope charge-coupled device camera (QuantEM512SC, Photometrics). Flagellar length was measured in at least 50 cells using ImageJ software (NIH) with calibration with a micrometer. Flagellar length results were graphed using GraphPad Prism version 5.0, and statistical analysis was performed by Student’s t test. Flagellar isolation and fractionation were performed essentially as described previously (Liang and Pan, 2013). Briefly, flagella were isolated after deflagellation by pH shock followed by sucrose gradient purification. The isolated flagella were dissolved in HMDEK buffer (50 mM HEPES [pH 7.2], 5 mM

MgCl2, 0.5 mM EDTA, 1 mM dithiothreitol [DTT], and 25 mM KCl) containing protease inhibitor cocktail (complete-mini EDTA-free, Roche) and 25 mg/ml ALLN (Sigma) and fresh-frozen in liquid nitrogen. The flagellar matrix fraction was obtained by freezing and thawing of flagella followed by centrifugation, and the pellets were dissolved in HMEDK buffer containing 0.5% NP-40 and stayed on ice for 15 min. The supernatant and pellet were collected after centrifugation as membrane and axonemal fractions, respectively. Gene Silencing and Ectopic Gene Expression in Chlamydomonas General molecular techniques were employed to make different constructs for expression in bacteria or Chlamydomonas that were verified by sequencing. Knockdown of CrCDPK1 was achieved by using artificial microRNA essentially as previously described (Liang and Pan, 2013; Molnar et al., 2009). The following constructs with HA-tagging at the C terminus were made for expression in Chlamydomonas: pCrCDPK1-HA, pCrCDPK1DC2-HA, pFLA8-HA, pFLA8S663A-HA, and pFLA8S663D-HA. The detailed procedures are reported in the Supplemental Experimental Procedures. Bacterial Protein Expression and Generation of Antibodies The cDNAs of CrCDPK1, CrKAP, and FLA8 used for bacterial protein expression were amplified from a Chlamydomonas cDNA library (Takara). E. coli ER2566 cells were used for protein expression. Other procedures and information regarding the generation of antibodies against CrCDPK1, FLA8, pFLA8, and CrKAP are reported in the Supplemental Experimental Procedures. In Vitro Phosphorylation Assay A mixture (30 ml) contained 100 ng of GST-CrCDPK1 or GST in a buffer (10 mM HEPES [pH 7.2], 150 mM NaCl, 10 mM MgCl2, 5 mM DTT, and 1 mM ATP-g-S). 2 mg FLA8-CTD or its mutant S663A was added as substrate. After incubation at room temperature for 30 min, 1.5 ml of 50 mM p-nitrobenzylmesylate (catalog no. ab138910, Abcam) in DMSO was added. The reaction was stopped by adding SDS sample buffer and boiling after incubation for another 2 hr. Protein phosphorylation was detected by immunoblotting with anti-thiophosphate ester antibody. Immunoprecipitation and Sucrose Gradient Analysis Cells were lysed in buffer A_IP (20 mM HEPES [pH 7.2], 5 mM MgCl2, 1 mM DTT, 1 mM EDTA [pH 7.5], 150 mM NaCl, 20 mM b-glycerol phosphate, 0.1 mM Na3VO4, 10 mM NaF, and 5% glycerol). The lysates were precleared by adding protein A Sepharose beads (GE Healthcare) followed by rotating at 4 C for 10 min. The precleared cell lysates were incubated with anti-HA affinity matrix for 3 hr at 4 C. After washing four times with buffer A_IP containing 0.1% Triton X-100 and 0.1% NP-40, samples were resolved by SDS-PAGE and analyzed by immunoblotting. For sucrose gradient analysis, 100–150 ml cell lysates in buffer A (50 mM Tris [pH 7.5], 10 mM MgCl2, 1 mM EDTA, and 1 mM DTT) containing the protease inhibitors described above was loaded on a 2 ml 5–20% sucrose gradient made in 10 mM HEPES (pH 7.2) followed by centrifugation (200,000 3 g for 4 hr at 4 C on a TLS-55 rotor, Beckman). Approximately 25–26 fractions were collected. Immunoblotting and Immunofluorescence Immunoblotting and immunofluorescence were performed essentially as described previously (Liang and Pan, 2013). Information regarding the primary antibodies used as well as procedures for immunoblotting and immunofluorescence can be found in the Supplemental Experimental Procedures. Phosphorylation Analysis by Mass Spectrometry Immunoprecipitates of anti-FLA8 antibody were analyzed by SDS-PAGE followed by Coomassie blue staining. The protein bands were excised from the gel and subjected to liquid chromatography-tandem mass spectrometry analysis as described previously (Wang et al., 2013). SUPPLEMENTAL INFORMATION Supplemental information includes Supplemental Experimental Procedures and six figures and can be found with this article online at http://dx.doi.org/ 10.1016/j.devcel.2014.07.019.




AUTHOR CONTRIBUTIONS Y.L. and Y.P. carried out most of the experiments. Q.W. helped with the analysis of pFLA8. Z.H. generated the fla8 null mutant. Y.X. and H.D. performed the mass spectrometry analysis. X.H. generated the anti-KAP antibody. J.P. designed the study and wrote the paper with contributions from Y.L. and the other authors. ACKNOWLEDGMENTS We thank Gregory Pazour for sharing unpublished data, discussion, and critical review of the manuscript and Guangshuo Ou for discussion. We also thank Douglas Cole, Dennis Diener, Joel Rosenbaum, Robert Bloodgood, Jeffrey Salisbury, and Marry Porter for providing antibodies and strains. We also thank the Core Facility of Center of Biomedical Analysis (Tsinghua University) for assistance with cell imaging analysis. This work was supported by National Basic Research Program of China (973 program) Grants 2013CB910703 and 2012CB945003), by National Natural Science Foundation of China Grant 31330044, and by Ministry of Education Grant 20110002110054 (to J.P.). Received: May 23, 2014 Revised: July 15, 2014 Accepted: July 23, 2014 Published: August 28, 2014

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Phosphorylation on TRPV4 Serine 824 Regulates Interaction with STIM1.

Serine Phosphorylation of HIV-1 Vpu and Its Binding to Tetherin Regulates Interaction with Clathrin Adaptors.

Boarder control on the IFT train.

Control of interaction of spectrin and actin by phosphorylation.

Store-operated Ca(2+) entry regulates glioma cell migration and invasion via modulation of Pyk2 phosphorylation.

Translational control by eIF2α phosphorylation regulates vulnerability to the synaptic and behavioral effects of cocaine.

Phosphorylation of LRRK2 by casein kinase 1α regulates trans-Golgi clustering via differential interaction with ARHGEF7.

Effects of computerized physician order entry on medication turnaround time and orders requiring pharmacist intervention.

Preparing a cell for nuclear envelope breakdown: Spatio-temporal control of phosphorylation during mitotic entry.

[Erratum to: Teleradiological report turnaround times. An internal efficiency and quality control analysis].

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Tau phosphorylation regulates the interaction between BIN1's SH3 domain and Tau's proline-rich domain.

Phosphorylation regulates the Star-PAP-PIPKIα interaction and directs specificity toward mRNA targets.

Phosphorylation regulates mycobacterial proteasome.

IL-32θ negatively regulates IL-1β production through its interaction with PKCδ and the inhibition of PU.1 phosphorylation.

Dynamic interaction of SARAF with STIM1 and Orai1 to modulate store-operated calcium entry.

Orai1 regulates calcium entry into dendritic spines.

SQSTM1 regulates PB1 domain interaction partner binding.

JASMS to 12 issues means faster turnaround.

Interaction of benzoquinones with mitochondria interferes with oxidative phosphorylation characteristics.

Matrix elasticity regulates lamin-A,C phosphorylation and turnover with feedback to actomyosin.

STIM1 Phosphorylation at Y361 Recruits Orai1 to STIM1 Puncta and Induces Ca2+ Entry.

Overview: phosphorylation and translation control.

Bax mitochondrial relocation is linked to its phosphorylation and its interaction with Bcl-xL.