Journal of Structural Biology 185 (2014) 366–374
Contents lists available at ScienceDirect
Journal of Structural Biology journal homepage: www.elsevier.com/locate/yjsbi
Structure of MST2 SARAH domain provides insights into its interaction with RAPL Guoguang Liu a,1, Zhubing Shi b,1, Shi Jiao b,1, Zhenzhen Zhang a, Wenjia Wang b, Cuicui Chen b, Qiao Hao b, Meng Zhang b, Miao Feng b, Liang Xu b, Zhen Zhang b, Zhaocai Zhou b,⇑, Min Zhang a,⇑ a b
School of Life Sciences, Anhui University, Hefei, Anhui 230039, China State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
a r t i c l e
i n f o
Article history: Received 31 August 2013 Received in revised form 13 January 2014 Accepted 20 January 2014 Available online 25 January 2014 Keywords: MST2 RAPL/RASSF5/NORE1 SARAH domain Coiled coil Apoptosis
a b s t r a c t The STE20 kinases MST1 and MST2 are key players in mammalian Hippo pathway. The SARAH domains of MST1/2 act as a platform to mediate homodimerization and hetero-interaction with a range of adaptors including RASSFs and Salvador, which also possess SARAH domains. Here, we determined the crystal structure of human MST2 SARAH domain, which forms an antiparallel homodimeric coiled coil. Structural comparison indicates that SARAH domains of different proteins may utilize a shared dimerization module to form homodimer or heterodimer. Structure-guided mutational study identified specific interface residues critical for MST2 homodimerization. MST2 mutations disrupting its homodimerization also impaired its hetero-interaction with RAPL (also named RASSF5 and NORE1), which is mediated by their SARAH domains. Further biochemical and cellular assays indicated that SARAH domain-mediated homodimerization and hetero-interaction with RAPL are required for full activation of MST2 and therefore apoptotic functions in T cells. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction The STE20 like kinases MST1 and MST2 are core components of mammalian Hippo signaling pathway, which plays a fundamental role in organ size control and tumor suppression (Avruch et al., 2006; O’Neill and Kolch, 2005; O’Neill et al., 2004; Song et al., 2010). MST1/2 in conjunction with an adaptor protein Salvador phosphorylates and activates LATS1/2 kinases, which in turn associates with adaptor protein MOB1, to phosphorylate and prevent nuclear translocation of the downstream transcription coactivator Yes-associated protein (YAP), and thereby inhibit cell proliferation and promote apoptosis (Oka et al., 2008; Wu et al., 2003). MST1 and MST2 share high sequence homology with identical N-terminal kinase domains for catalysis and distinct C-terminal SARAH (Salvador, RASSF and Hpo homology) domains for homodimerization or hetero-interaction with Salvador and RASSF (Ras association domain family) proteins (Fig. 1A) (Pfeifer et al., 2010; Scheel and Hofmann, 2003). Functional redundancy between MST1 and MST2 has been evidenced by animal studies showing ⇑ Corresponding authors. Address: State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, 320 Yueyang Road, Shanghai 200031, China. Fax: +86 21 54921291 (Z. Zhou). E-mail addresses: [email protected] (Z. Zhou), [email protected] (M. Zhang). 1 These authors contribute equally to this work. http://dx.doi.org/10.1016/j.jsb.2014.01.008 1047-8477/Ó 2014 Elsevier Inc. All rights reserved.
that MST1/2 double knockout causes embryonic lethality while single knockout of either MST1 or MST2 does not impair organ development (Oh et al., 2009; Zhou et al., 2009). Ablation of both MST1 and MST2 caused liver tumorigenesis through largely abolishing YAP phosphorylation and increasing YAP nuclear localization (Zhao et al., 2009). On the other hand, despite of high degree sequence homology and functional redundancy, MST1 and MST2 displayed differential regulatory properties in certain aspects such as susceptibility to protein phosphatases (Deng et al., 2003). The structure of MST1 SARAH domain has been determined by NMR, showing an antiparallel dimeric conformation (Hwang et al., 2007), yet the structure of MST2 SARAH domain has not been studied in detail. In addition to their critical functions in development, MST1/2 kinases have been implicated in immune regulation including T cell proliferation, activation, and homeostasis, lymphocyte chemotaxis and trafficking, as well as inflammatory responses (Dong et al., 2009; Katagiri et al., 2009; Mou et al., 2012; Yun et al., 2011; Zhou et al., 2008). Crosstalk of MST1/2 with Akt signaling may antagonize Akt1 activity and therefore modulate T cell costimulation and NFjB target gene transcription (Cinar et al., 2007). In MST1 and MST2 double knockout mice, mature T cells could not efficiently migrate from thymus to the circulation and secondary lymphoid organs, indicating that MST1/2 may control lymphocyte trafficking
G. Liu et al. / Journal of Structural Biology 185 (2014) 366–374
and migration, which is important for efficient immunosurveillance and effective immune responses (Katagiri et al., 2009; Mou et al., 2012). Consistently, mice lacking MST1 exhibit a variety of T cell abnormalities (Mou et al., 2012); while patients lacking MST1 displayed primary immunodeficiency like features (Abdollahpour et al., 2012). Recent studies suggest that a group of RASSF proteins including RASSF1-6 may interact with MST1/2 to differentially regulate Hippo signaling (Hwang et al., 2007; Ikeda et al., 2009; Pfeifer et al., 2010; Romano et al., 2010; Vichalkovski et al., 2008). The specific mechanism of RASSF-mediated differential regulation of MST1/2, and its functional role in immune regulation remains largely unknown. The regulator for cell adhesion and polarization enriched in lymphoid tissues (RAPL), also named RASSF5 and NORE1, has been found to associate with MST1 to induce cell polarity and adhesion of lymphocytes (Katagiri et al., 2006). RAPL is an effector for small GTPase Rap1 that is essential for lymphocyte trafficking. During TCR stimulated induction of polarized morphology and clustering of the integrin LFA-1 in T cells, RAPL can associate with MST1 to alter its cellular localization and kinase activity (Katagiri et al., 2009). Meanwhile, animal study suggested that RAPL-MST1 complex negatively regulate naïve T cell proliferation and that MST1 is required for the maintenance of RAPL level in lymphoid cells (Zhou et al., 2008). It is generally understood that RAPL may associate with MST1/2 through their SARAH domains. Here, we performed structural and biochemical studies of MST2-RAPL signaling. Our crystallographic analysis of MST2 SARAH domain revealed an antiparallel conformation similar to that of MST1 with local variations. SARAH domains are sufficient for MST2-RAPL stable association. Mutations of the SARAH domain interface impaired MST2 homodimerization and interaction with RAPL, preventing full activation of MST2. RAPL stimulates the kinase activity of MST2 in vitro, and promotes its apoptotic function in vivo. Mutation disrupting MST2 association with RAPL abrogated their cooperative function in T cell apoptosis.
367
2.2. Crystallization, structure determination and refinement The native or Se-Met-substituted MST2 SARAH domain was concentrated to 16 mg/ml and crystallized by sitting drop vapor diffusion method at 16 °C, in a drop with 1 ll protein solution and 1 ll reservoir solution. The MST2 SARAH domain was crystallized in 0.4 M calcium chloride dihydrate, 0.1 M sodium acetate trihydrate pH 4.6, 5% v/v 2-propanol. Diffraction date were collected from a flash cooled crystal at 100 K on beamline BL17U of Shanghai Synchrotron Radiation facility (SSRF), China, and processed in HKL2000 (Otwinowski and Minor, 1997). The structure of MST2 SARAH domain was solved using single-wavelength anomalous diffraction method from a Se-Met derivatized with program AutoSol in Phenix (Adams et al., 2010). The model building was performed in Coot (Emsley et al., 2010) and the structure was refined using REFMAC5 (CCP4, 1994; Murshudov et al., 2011). 2.3. Immunoprecipitation HEK293T cells were maintained in DMEM medium with 5% (vol/vol) fetal calf serum, 2 mM L-glutamine, penicillin (100 units/ ml), and streptomycin (100 lg/ml) in 5% CO2/95% humidified air at 37 °C at a density of 106 cells per ml. Cells were then transiently with expression vectors encoding Flag-tagged wild-type MST2 and Myc-tagged MST2 mutants. Cell extracts were prepared in lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 10% glycerol, 0.1% Triton X-100, 0.5 mg/ml BSA and protease inhibitor cocktail), and incubated with anti-Flag antibody (sigma) for 4 h at 4 °C followed by overnight incubation with Protein A/G agarose (Santa Cruz). The immunoprecipitates were washed three times in the lysis buffer before loading onto SDS–PAGE gels. The samples separated by SDS–PAGE is blotted and developed with anti-Myc antibodies. 2.4. Cross-linking experiment
2. Materials and methods 2.1. Cloning, protein expression and purification Human MST2 SARAH domain (amino acids 436–484) were cloned into HT-pET-28a and expressed in Escherichia coli (E. coli) BL21(DE3) codon plus cells. The expression of proteins was induced by 0.5 mM isopropyl b-D-thiogalactopyranoside in LuriaBertani medium. After cultured for 12 h at 16 °C, bacterial cells were harvested by centrifugation and suspended with lysis buffer (20 mM Hepes, 500 mM NaCl, 5% glycerol, 1 mM DTT and 20 mM imidazole, pH 7.5), and then lysed. The soluble cell lysate was fractionated after centrifuged at 18,000g for 40 min. The protein was purified with affinity column pre-charged with Ni2+. The proteins was eluted with elution buffer (20 mM Hepes, 500 mM NaCl, 5% glycerol, 1 mM DTT and 300 mM imidazole, pH 7.5) and digested by TEV protease to remove the N-terminal His-tag. Then the proteins were concentrated and loaded on HiLoad 16/60 Superdex 200 column in 20 mM Hepes, 100 mM NaCl, 1 mM DTT, pH 7.5. The purity of proteins was monitored by SDS–PAGE. Selenomethionine (Se-Met)-substituted MST2 SARAH domain was purified as described above, except that E. coli cells were cultured in M9 minimal medium containing amino-acid supplement (lysine, phenylalanine, threonine to final concentration of 100 mg/ l, isoleucine, leucine, valine to 50 mg/l, and L-Se-Met to 60 mg/l). Other MST2 mutants and mouse RAPL fragments were constructed and purified by similar procedures described above. For cell-based assays, Flag-tagged MST2, Myc-tagged MST2 and Flagtagged human RAPL were subcloned into BamHI/XhoI sites of pcDNA3.1.
Purified 1 mg/ml wild-type or mutant MST2 was incubated with 0 and 0.005% (vol/vol) glutaraldehyde (GA) in conjugation buffer (20 mM Hepes, 100 mM NaCl, 1 mM DTT, pH 7.5) at room temperature for 1 h. After the reaction was quenched with 50 mM Tris–Cl pH 8.0 for 30 min, the cross-linked samples were analyzed on a 8% SDS–PAGE. 2.5. Kinase assay The activity of purified wild-type or mutant MST2 was assayed by using myelin basic protein (MBP) as substrate. Kinase activity of wild-type or mutant MST2 was measured in a total assay volume of 40 ll consisting of 50 mM Tris–HCl pH 7.5, 0.1 mM EGTA, 1 mM DTT, 10 mM magnesium acetate, 0.1 mM [c-32P]ATP (200 c.p.m./pmol) and 1 mg/ml MBP, with 18.5 lM MST2 and 92.5 lM RAPL. The assays were carried out at 30 °C and were terminated after 20 min by spotting the 40 ll reaction mixture onto P81 membranes. The membranes were washed in phosphoric acid for 3 times, and the incorporated radioactivity was measured by scintillation counting (Beckman LS6500) (Hastie et al., 2006). 2.6. GST pulldown assay GST-tagged MST2 and its SARAH domain protein coupled on glutathione-Sepharose beads were mixed with RAPL and its SARAH domain respectively at 4 °C for 1 h in 20 mM Hepes pH7.5, 100 mM NaCl, 1 mM DTT, and washed three times. The input and output samples were boiled and loaded on 12% SDS–PAGE followed by Coomassie blue staining.
368
G. Liu et al. / Journal of Structural Biology 185 (2014) 366–374
2.7. Kinetic assay
Table 1 Data collection and refinement statistics.
Interaction analysis was performed using an Octet Red 96 instrument (ForteBio). RAPL was labeled by biotin in 20 mM Hepes pH 7.5, 100 mM NaCl, 1 mM DTT, and biotinylated proteins were immobilized on streptavidin (SA) biosensors and incubated with 800 nM wild-type or mutant MST2 in 1 kinetics buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4, 0.01% BSA, and 0.002% Tween 20). All binding experiment were performed in 1 kinetics buffer at 25 °C. The experiments comprised 5 steps: (1) Baseline acquisition; (2) Biotinylated proteins loading onto SA biosensor; (3) Second baseline acquisition; (4) Association of interacting protein for kon measurement; (5) Dissociation of interacting protein for koff measurement. Data were analyzed using Octet Data Analysis Software 7.0 (ForteBio).
MST2 SARAH Native
SeMet
Data collection Wavelength (Å) Space group
0.97930 C2221
0.97915 C2221
Cell dimensions a, b, c (Å) a, b, c (°) Resolution (Å) Rmerge I/rI Completeness (%) Redundancy
42.7, 45.3, 208.8 90.0, 90.0, 90.0 50.00–2.00 (2.00–2.03)a 0.145 (0.600) 14.9 (4.5) 96.9 (99.9) 4.5 (5.6)
42.2, 44.6, 209.2 90.0, 90.0, 90.0 50.00–1.50 (1.53–1.50) 0.125 (0.660) 17.1 (3.8) 92.3 (85.7) 5.3 (5.7)
Refinement Resolution (Å) No. reflections Rwork/Rfree No. atoms Protein Ligand/ion Water
2.8. Flow cytometry Jurkat cells were cultured in RPMI 1640 medium and then transfected with different constructs encoding Myc-tagged MST2 and Flag-tagged RAPL. The cells were incubated with 50 lg/ml propidium iodide (PI) in PBS with 0.2 mg/ml RNase A and 0.1% Triton X-100, and analyzed by flow cytometer. siRNA of MST2 and RAPL were bought from Santa Cruz. 3. Results and discussion 3.1. MST2 SARAH domain forms an antiparallel dimeric coiled coil a
34.86–1.50 29481 0.19/0.23 1751 – 239
B-factors Protein Ligand/ion Water
16.9 – 23.9
R.m.s. deviations Bond lengths (Å) Bond angles (°)
0.007 1.179
Values in parentheses are for highest-resolution shell.
Human MST2 comprises a kinase domain (amino acids 1–297), an inhibitory domain (amino acids 327–392), and a C-terminal
Fig.1. Structure of human MST2 SARAH domain. (A) Schematic illustration of domain organization for human MST2. (B) MST2 SARAH domain forms an antiparallel dimeric coiled coil. Two chains (monomers) of MST2 SARAH domain are shown in cartoon and colored slate and green, respectively. The interaction between helices 2 of the two chains is mainly mediated by hydrophobic interactions. (C) Helix 1 of one MST2 monomer interacts with helix 2 of the other via hydrophobic interactions. (D) The heptad repeats of MST2 SARAH domain coiled coil. Two tetrad transitions are colored red.
G. Liu et al. / Journal of Structural Biology 185 (2014) 366–374
SARAH domain (amino acids 436–491) (Creasy et al., 1996) (Fig. 1A). We purified and crystallized MST2 SARAH domain (amino acids 436–484). The structure was solved by single-wavelength anomalous diffraction method from a Se-Met derivative crystal with statistics summarized in Table 1. There are four copies of MST2 SARAH domain in the asymmetric unit of the crystals, forming two symmetric dimeric coiled coil. Each monomer is composed of two helices, helix 1 (amino acids 436–442) and helix 2 (amino acids 445–484) (Fig. 1B and C). These two dimers are very similar except the head of helix 1. The r.m.s.d
369
values of superimposition for all atoms and Ca only are 1.118 Å and 0.995 Å, respectively. Two chains of MST2 SARAH domains form an antiparallel dimeric coiled coil (Fig. 1B). A classical coiled coil usually adopts a heptad repeat in its sequence, consisting a–g positions. Amino acid residues at positions a and d often mediate core packing or interlocking of the alpha helical chains. However, unlike the ideal coiled coil conformation, the MST2 SARAH domain has two transitions in periodicity from heptad (a–g) to tetrad (d–g, Met459-Glu462 and Ile477-Ala479) (Fig. 1D), which are punctuated by two prolines
Fig.2. Structural comparison of SARAH domains. (A and B) The structures of MST2 SARAH domain colored green (A) and slate (B, PDB code 4L0N), respectively. (C) Comparison of two structures of the MST2 SARAH domain. (D) The structure of MST1 SARAH domain (PDB code 2JO8) colored yellow. (E) The structure of RAPL SARAH domain (PDB code 2YMY) colored cyan. (F) The heterodimeric structure of MST2 and RAPL SARAH domains (PDB code 4LGD) colored magenta. (G) Structural comparison of SARAH domains. (H) Sequence alignment of SARAH domains of MST1/2, RASSF1–6 and Salvador SARAH domains. The heptad repeats are labeled. Resides in a and d positions are colored red.
370
G. Liu et al. / Journal of Structural Biology 185 (2014) 366–374
(Pro457 and Pro476) (Fig. 1B). The two tetrads result in a decreased degree of supercoiling. The coiled coil formed by MST2 SARAH domain buries a total of 1,439 Å2 surface area. Hydrophobic interactions dominates the association between helices 2 of two chains, involving residues Leu445, Leu448, Leu452, Leu455, Met459, Ile463, Leu466, Tyr470, Lys473, Ile477, Met481 and Lys484 in helix 2 (Fig. 1B). The homodimer is further stabilized by hydrogen bonding between residues Glu462 and Tyr470, and salt bridges formed by residues Asp456, Glu460, Arg467 and Arg474. Helix 1 of one chain folds back to contact helix 2 of the other chain (Fig. 1C). Residues Leu440 and Phe437 of helix 1 interact with residues Pro476, Ile477, Ala480, and Ala483 from helix 2 via hydrophobic interaction. 3.2. Structural comparison of SARAH domains During the preparation of this manuscript, Structural Genomics Consortium also determined the structure of MST2 SARAH domain (PDB code 4L0N). The helices 2 have similar conformations with a root-mean-square deviation (r.m.s.d) value of 1.071 Å for Ca superimposition of structure 4L0N and the structure determined in this work (Fig. 2A–C). However, positions of the helix 1 shows derivation relative to helix 2, resulting in an r.m.s.d value of 1.940 Å for Ca superimposition of the coiled coil. This is partially due to the flexible loop between the helix 1 and the helix 2. Relative position between two chains in two structures also changes, although the hydrophobic interface of coiled coil, as well as
residues in the interface, is the same as that in our structure. These differences between the two structures indicate the flexibility of SARAH domain. The structure of MST1 SARAH domain, which was previously determined by nuclear magnetic resonance, folds into an antiparallel dimer (Hwang et al., 2007). The monomeric structure of MST2 SARAH domain resembles that of MST1, with r.m.s.d values of 1.853 Å and 1.300 Å for overlapping Ca atoms of the whole SARAH domain and only helix 2, respectively (Fig. 2A and D). Like differences between two structures of the MST2 SARAH domain, the conformation of MST1 and MST2 homodimers varies. When helices 2 of MST1 and MST2 SARAH domains are superimposed, the position of helix 1 is altered relative to the helix 2, and one chain shows a bend with different degree relative to the other chain, although sequences, especially residues involved in core packing of coiled coil, are highly conserved and the interfaces are very similar (Fig. 2G). The conformational differences of MST1 and MST2 SARAH domains further indicate that structures of SARAH domains are dynamic. Recently, structures of RAPL SARAH domain alone and its complex with MST2 were determined (Makbul et al., 2013; Ni et al., 2013). Although there are certain degrees of conformational variations, the overall structure of MST2-RAPL SARAH domain heterodimer is very similar to that of MST2 or RAPL SARAH domain homodimer (Fig. 2E–G). Residues at a and d positions of the coiled coils are highly conserved in these SARAH domains (Fig. 2H). Transitions in periodicity from heptad to tetrad are also present in MST1 and RAPL SARAH domains. Besides MST1/2 and RAPL, other
Fig.3. Interface mutations impair MST2 SARAH domain-mediated homodimerization and its kinase activity. (A) The indicated constructs of Myc-tagged MST2 and Flagtagged MST2 were co-transfected into HEK293T cells. Cells were lysed, and lysate were immunoprecipitated (IP) with anti-Flag antibody and subjected to immunoblot (IB) analysis. (B) The relative association was calculated by ratio of Myc and Flag gray level in IBs and the association between wild-type Myc-tagged MST2 and Flag-tagged MST2 was set to 1.0. (C) Cross-linking experiment of wild-type or mutant MST2. 1 mg/ml wild-type or mutant MST2 was incubated with 0% and 0.005% (vol/vol) glutaraldehyde (GA) at room temperature for 1 h. The cross-linked samples were analyzed on 8% SDS–PAGE. MST2 oligomer was indicated by asterisk. (D) Purified wild-type and mutant MST2 were assayed for determining the ability of MST2 to phosphorylate MBP. Error bars represent SD of data obtained in three independent experiments.
G. Liu et al. / Journal of Structural Biology 185 (2014) 366–374
SARAH domain-containing proteins, such as RASSF1–4, RASSF6 and Salvador, also possess these sequence features (Fig. 2H). Together, these observations suggest that SARAH domains of MST1/2 and their adaptor proteins share a common dimerization module with conserved overall conformation for the formation of either homodimer or heterodimer. 3.3. Kinase activity of MST2 is regulated by SARAH domain-mediated homodimerization Following our structural analysis of MST2 SARAH domain, we performed biochemical and cellular assays to identify specific amino acids most critical for MST2 SARAH domain-mediated homodimerization. Mutations M459A and L466A, but not L452A, partially disrupted MST2 homodimerization as shown by immunoprecipitation (IP) assay in HEK293T cells (Fig. 3A and B). Moreover, we perform cross-linking experiment using purified recombinant proteins of wild-type and mutant MST2. MST2 M452A mutant formed oligomer as does wild-type. However, 0.005% glutaraldehyde didn’t induce band shift of mutant M459A, consistent with IP results (Fig. 3C). In the structure of MST2 SARAH domain, residues Met459 and Leu466 locate in the center of SARAH domain, suggesting that they would play a key role in homodimerization of MST2. These binding assays further confirmed this notion. To further assess the function of MST2 SARAH domain, we used purified proteins to measure the kinase activity of wild-type and mutant MST2 towards MBP, a general kinase substrate. Mutations M459A and L466A, which disrupted SARAH domain-mediated MST2 homodimerization in vitro and in vivo, impaired the kinase activity of MST2 (Fig. 3D). Residue Leu452 is in the distal end of MST2 SARAH domain and may contribute less to its association. However, mutation of L452A also impaired MST2 kinase activity (Fig. 3D). This could be explained, in part, by mutation-induced local conformational change even though the overall association remain unbroken. As a negative control, the kinase dead mutant of
371
MST2 (D164A) did not show activity (Fig. 3D). These results indicated that hydrophobic interactions mediated by Met459 and Leu466 are critical for SARAH domain-mediated homodimerization and full activation of MST2. 3.4. SARAH domains mediates hetero-interaction between MST2 and RAPL As mentioned, RAPL functions as an adaptor protein for the small GTPase RAP1, and is required for efficient immune cell trafficking. It has been reported that MST1 may act as a key participant in the control of cell polarization and adhesion ‘downstream’ of RAP1 and RAPL activation triggered by chemokine and antigen stimulation (Katagiri et al., 2006). To verify and map the interacting domains between MST2 and RAPL, we performed pulldown and gel filtration assays using purified proteins of MST2 and RAPL. In addition to the full-length MST2 and its SARAH domain, two fragments of RAPL proteins were used, corresponding to its SARAH domain (amino acids 365–413) and Ras association (RA) domain plus SARAH domain (amino acids 200–413), respectively. As shown in Fig. 4A, RAPL and its SARAH domain could be pulled down by GST-tagged MST2 and MST2 SARAH domain. Moreover, mixtures of full-length MST2 or MST2 SARAH domain and RAPL or RAPL SARAH domain were eluted in one peak in gel filtration assay (Fig. 4B–E), indicating that MST2 and RAPL directly interact with each other through their SARAH domains. These results are consistent with a recent structural study of MST2 in complex with RAPL SARAH domain (Ni et al., 2013). To further dissect the interaction between MST2 and RAPL, purified RAPL and wild-type or mutant MST2 were used to detect their binding affinities using bio-layer interferometry (BLI). RAPL binds to wild-type MST2 with an equilibrium dissociation constant (Kd) of 6.54 nM. Single mutation of MST2 M459A or L466A, which impaired its homodimerization, substantially weakened its interaction with RAPL (Fig. 5A). In addition, mutation L452A of MST2
Fig.4. MST2 interacts with RAPL through their SARAH domains. (A) The indicated proteins of GST-tagged MST2 were coupled on glutathione Sepharose beads, and then mixed with RAPL and its SARAH domain, respectively. The input and output samples were loaded on SDS–PAGE followed by Coomassie blue staining. RAPL was indicated by asterisk. (B–E) MST2 or MST2 SARAH domain and RAPL or RAPL SARAH domain were mixed and analyzed by gel filtration using Superdex 200 10/300 GL column. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
372
G. Liu et al. / Journal of Structural Biology 185 (2014) 366–374
Fig.5. Disruption of MST2 SARAH domain homodimerization affects its interaction with RAPL. (A) Sensorgrams for wild-type or mutant MST2 on dimerization interface binding to RAPL. Binding affinities of wild-type or mutant MST2 with RAPL were determined by BLI experiment. Biotinylated-RAPL was immobilized on SA biosensors and incubated with wild-type or mutant MST2 at a concentration of 800 nM. Curves are the experimental trace obtained from BLI experiments. (B) Purified wild-type or mutant MST2 were assayed for determining the ability of MST2 to phosphorylate MBP in the absence or presence of RAPL. Error bars represent SD of data obtained in three independent experiments.
Fig.6. MST2 and RAPL promote apoptosis in Jurkat cells. (A–H) Jurkat cells were transfected with the indicated constructs encoding Myc-tagged wild-type or mutant MST2 or/ and Flag-tagged RAPL. Cells were stained with PI and analyzed by flow cytometer. The apoptosis fractions are indicated by lines. The numbers are the percentage of apoptosis in each sample. (I–K) Jurkat cells were transfected with the indicated siRNA for 48 h. Cells were then stained with PI and cell cycle distribution was analyzed by flow cytometer. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
G. Liu et al. / Journal of Structural Biology 185 (2014) 366–374
did not significantly alter its interaction with RAPL. Together, these results indicate that SARAH domain of MST2 is essential for its homodimerization and interaction with RAPL. We then determined the effects of RAPL on kinase activity of MST2. Our results showed that RAPL promoted MST2 activity slightly (about 1.5-fold) (Fig. 5B). Mutation M459A disrupting the homodimerization of MST2 SARAH domain reduced its kinase activity with or without RAPL (Fig. 5B). These results suggest that SARAH domain mediated homodimerization and hetero-interaction with RAPL is required to fully activate MST2 kinase.
3.5. Complex of MST2 and RAPL promotes apoptosis To further assess functional importance of MST2 SARAH domain mediated homodimerization or hetero-interaction with RAPL, we examined the apoptotic effects of wild-type and mutant MST2 with or without RAPL. Our flow cytometry assay in Jurkat T cells showed that transfection of MST2 dramatically promoted apoptosis as expected (Fig. 6A and B); while transfection of RAPL also significantly enhanced apoptosis (Fig. 6C). Moreover, co-transfection of wildtype MST2 with RAPL further increased apoptosis (Fig. 6D); yet co-transfection of a kinase dead version of MST2 (D164A) with RAPL did not cause apoptosis (Fig. 6E). Similarly, co-transfection of RAPL and MST2 mutants M459A and L466A that partially disrupted SARAH domain mediated homodimerization and interaction with RAPL did not induce substantial apoptosis either (Fig. 6F and G). However, co-transfection of RAPL and MST2 mutant L452A, which did not influence its homodimerization, also induced significant apoptosis, although to a lesser extent probably due to disturbed conformation and impaired kinase activation of MST2 (Fig. 6H). Consistent with these observations, knockdown of MST2 or RAPL promoted cell cycle progression (Fig. 6I–K). Taken together, these results indicate that MST2 and RAPL may form a signaling complex through their SARAH domains in lymphocytes to synergistically promote apoptosis in a way dependent on the kinase activity of MST2.
3.6. Conclusion We determined the crystal structure of MST2 SARAH domain, which forms an antiparallel dimeric coiled coil. Structural comparison suggests that SARAH domains make use of a shared interface to mediate homo- or hetero- dimerization. Using pull-down and gel filtration assays, we found that MST2 directly interacts with RAPL via their SARAH domains. Mutations of the dimeric interface of MST2 SARAH domain not only impaired its homodimerization but also decreased its hetero-interaction with RAPL, suggesting that a similar set of interface residues mediate either homo- or hetero- dimerization of SARAH domains. Consistently, these mutations also abrogated MST2-RAPL induced apoptosis in Jurkat T cells. Besides RAPL, MST2 also functions by association with many other partners, such as other RASSF family members and Salvador, through their SARAH domain to transduce cell signaling (Mardin et al., 2010; Pfeifer et al., 2010; Scheel and Hofmann, 2003). We proposed that these proteins interact and function with MST2 via a common mechanism like RAPL.
4. Accession numbers The coordinate file and structure factor for the MST2 SARAH domain crystal structure was deposited in the Protein Data Bank under accession code 4HKD.
373
Acknowledgments We thank the staff at beamline BL17U of Shanghai Synchrotron Radiation Facility for help of data collection. This work was supported by the 973 program of the Ministry of Science and Technology of China (2012CB910204 and 2010CB529701), the Science and Technology Commission of Shanghai Municipality (11JC14140000, 13ZR1446400), the National Natural Science Foundation of China (30970565, 31170688, 31270808, 31300734, 31340044), the Science and Technological Fund of Anhui Province for Outstanding Youth (10040606Y15), and the ‘‘Cross and cooperation in science and technology innovation team’’ project of the Chinese Academy of Sciences. Dr. Z.Z. is a scholar of the Hundred Talents Program of the Chinese Academy of Sciences. References Abdollahpour, H., Appaswamy, G., Kotlarz, D., Diestelhorst, J., Beier, R., Schaffer, A.A., Gertz, E.M., Schambach, A., Kreipe, H.H., Pfeifer, D., Engelhardt, K.R., Rezaei, N., Grimbacher, B., Lohrmann, S., Sherkat, R., Klein, C., 2012. The phenotype of human STK4 deficiency. Blood 119, 3450–3457. Adams, P.D., Afonine, P.V., Bunkoczi, G., Chen, V.B., Davis, I.W., Echols, N., Headd, J.J., Hung, L.W., Kapral, G.J., Grosse-Kunstleve, R.W., McCoy, A.J., Moriarty, N.W., Oeffner, R., Read, R.J., Richardson, D.C., Richardson, J.S., Terwilliger, T.C., Zwart, P.H., 2010. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221. Avruch, J., Praskova, M., Ortiz-Vega, S., Liu, M., Zhang, X.F., 2006. Nore1 and RASSF1 regulation of cell proliferation and of the MST1/2 kinases. Methods Enzymol. 407, 290–310. CCP4, J., 1994. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763. Cinar, B., Fang, P.K., Lutchman, M., Di Vizio, D., Adam, R.M., Pavlova, N., Rubin, M.A., Yelick, P.C., Freeman, M.R., 2007. The pro-apoptotic kinase Mst1 and its caspase cleavage products are direct inhibitors of Akt1. EMBO J. 26, 4523– 4534. Creasy, C.L., Ambrose, D.M., Chernoff, J., 1996. The Ste20-like protein kinase, Mst1, dimerizes and contains an inhibitory domain. J. Biol. Chem. 271, 21049–21053. Deng, Y., Pang, A., Wang, J.H., 2003. Regulation of mammalian STE20-like kinase 2 (MST2) by protein phosphorylation/dephosphorylation and proteolysis. J. Biol. Chem. 278, 11760–11767. Dong, Y., Du, X., Ye, J., Han, M., Xu, T., Zhuang, Y., Tao, W., 2009. A cell-intrinsic role for Mst1 in regulating thymocyte egress. J. Immunol. 183, 3865–3872. Emsley, P., Lohkamp, B., Scott, W.G., Cowtan, K., 2010. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501. Hastie, C.J., McLauchlan, H.J., Cohen, P., 2006. Assay of protein kinases using radiolabeled ATP: a protocol. Nat. Protoc. 1, 968–971. Hwang, E., Ryu, K.S., Paakkonen, K., Guntert, P., Cheong, H.K., Lim, D.S., Lee, J.O., Jeon, Y.H., Cheong, C., 2007. Structural insight into dimeric interaction of the SARAH domains from Mst1 and RASSF family proteins in the apoptosis pathway. Proc. Natl. Acad. Sci. USA 104, 9236–9241. Ikeda, M., Kawata, A., Nishikawa, M., Tateishi, Y., Yamaguchi, M., Nakagawa, K., Hirabayashi, S., Bao, Y., Hidaka, S., Hirata, Y., Hata, Y., 2009. Hippo pathwaydependent and -independent roles of RASSF6. Sci. Signal. 2, ra59. Katagiri, K., Imamura, M., Kinashi, T., 2006. Spatiotemporal regulation of the kinase Mst1 by binding protein RAPL is critical for lymphocyte polarity and adhesion. Nat. Immunol. 7, 919–928. Katagiri, K., Katakai, T., Ebisuno, Y., Ueda, Y., Okada, T., Kinashi, T., 2009. Mst1 controls lymphocyte trafficking and interstitial motility within lymph nodes. EMBO J. 28, 1319–1331. Makbul, C., Constantinescu Aruxandei, D., Hofmann, E., Schwarz, D., Wolf, E., Herrmann, C., 2013. Structural and thermodynamic characterization of Nore1SARAH: a small, helical module important in signal transduction networks. Biochemistry 52, 1045–1054. Mardin, B.R., Lange, C., Baxter, J.E., Hardy, T., Scholz, S.R., Fry, A.M., Schiebel, E., 2010. Components of the Hippo pathway cooperate with Nek2 kinase to regulate centrosome disjunction. Nat. Cell Biol. 12, 1166–1176. Mou, F., Praskova, M., Xia, F., Van Buren, D., Hock, H., Avruch, J., Zhou, D., 2012. The Mst1 and Mst2 kinases control activation of rho family GTPases and thymic egress of mature thymocytes. J. Exp. Med. 209, 741–759. Murshudov, G.N., Skubak, P., Lebedev, A.A., Pannu, N.S., Steiner, R.A., Nicholls, R.A., Winn, M.D., Long, F., Vagin, A.A., 2011. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 67, 355–367. Ni, L., Li, S., Yu, J., Min, J., Brautigam, C.A., Tomchick, D.R., Pan, D., Luo, X., 2013. Structural basis for autoactivation of human Mst2 kinase and its regulation by RASSF5. Structure 21, 1757–1768. Oh, S., Lee, D., Kim, T., Kim, T.S., Oh, H.J., Hwang, C.Y., Kong, Y.Y., Kwon, K.S., Lim, D.S., 2009. Crucial role for Mst1 and Mst2 kinases in early embryonic development of the mouse. Mol. Cell Biol. 29, 6309–6320.
374
G. Liu et al. / Journal of Structural Biology 185 (2014) 366–374
Oka, T., Mazack, V., Sudol, M., 2008. Mst2 and Lats kinases regulate apoptotic function of Yes kinase-associated protein (YAP). J. Biol. Chem. 283, 27534– 27546. O’Neill, E., Kolch, W., 2005. Taming the Hippo: Raf-1 controls apoptosis by suppressing MST2/Hippo. Cell Cycle 4, 365–367. O’Neill, E., Rushworth, L., Baccarini, M., Kolch, W., 2004. Role of the kinase MST2 in suppression of apoptosis by the proto-oncogene product Raf-1. Science 306, 2267–2270. Otwinowski, Z., Minor, W., 1997. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326. Pfeifer, G.P., Dammann, R., Tommasi, S., 2010. RASSF proteins. Curr. Biol. 20, R344–345. Romano, D., Matallanas, D., Weitsman, G., Preisinger, C., Ng, T., Kolch, W., 2010. Proapoptotic kinase MST2 coordinates signaling crosstalk between RASSF1A, Raf-1, and Akt. Cancer Res. 70, 1195–1203. Scheel, H., Hofmann, K., 2003. A novel interaction motif, SARAH, connects three classes of tumor suppressor. Curr. Biol. 13, R899–900. Song, H., Mak, K.K., Topol, L., Yun, K., Hu, J., Garrett, L., Chen, Y., Park, O., Chang, J., Simpson, R.M., Wang, C.Y., Gao, B., Jiang, J., Yang, Y., 2010. Mammalian Mst1 and Mst2 kinases play essential roles in organ size control and tumor suppression. Proc. Natl. Acad. Sci. USA 107, 1431–1436.
Vichalkovski, A., Gresko, E., Cornils, H., Hergovich, A., Schmitz, D., Hemmings, B.A., 2008. NDR kinase is activated by RASSF1A/MST1 in response to Fas receptor stimulation and promotes apoptosis. Curr. Biol. 18, 1889–1895. Wu, S., Huang, J., Dong, J., Pan, D., 2003. Hippo encodes a Ste-20 family protein kinase that restricts cell proliferation and promotes apoptosis in conjunction with salvador and warts. Cell 114, 445–456. Yun, H.J., Yoon, J.H., Lee, J.K., Noh, K.T., Yoon, K.W., Oh, S.P., Oh, H.J., Chae, J.S., Hwang, S.G., Kim, E.H., Maul, G.G., Lim, D.S., Choi, E.J., 2011. Daxx mediates activation-induced cell death in microglia by triggering MST1 signalling. EMBO J. 30, 2465–2476. Zhao, B., Lei, Q., Guan, K.L., 2009. Mst out and HCC in. Cancer Cell 16, 363–364. Zhou, D., Conrad, C., Xia, F., Park, J.S., Payer, B., Yin, Y., Lauwers, G.Y., Thasler, W., Lee, J.T., Avruch, J., Bardeesy, N., 2009. Mst1 and Mst2 maintain hepatocyte quiescence and suppress hepatocellular carcinoma development through inactivation of the Yap1 oncogene. Cancer Cell 16, 425–438. Zhou, D., Medoff, B.D., Chen, L., Li, L., Zhang, X.F., Praskova, M., Liu, M., Landry, A., Blumberg, R.S., Boussiotis, V.A., Xavier, R., Avruch, J., 2008. The Nore1B/Mst1 complex restrains antigen receptor-induced proliferation of naive T cells. Proc. Natl. Acad. Sci. USA 105, 20321–20326.
Contents lists available at ScienceDirect
Journal of Structural Biology journal homepage: www.elsevier.com/locate/yjsbi
Structure of MST2 SARAH domain provides insights into its interaction with RAPL Guoguang Liu a,1, Zhubing Shi b,1, Shi Jiao b,1, Zhenzhen Zhang a, Wenjia Wang b, Cuicui Chen b, Qiao Hao b, Meng Zhang b, Miao Feng b, Liang Xu b, Zhen Zhang b, Zhaocai Zhou b,⇑, Min Zhang a,⇑ a b
School of Life Sciences, Anhui University, Hefei, Anhui 230039, China State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
a r t i c l e
i n f o
Article history: Received 31 August 2013 Received in revised form 13 January 2014 Accepted 20 January 2014 Available online 25 January 2014 Keywords: MST2 RAPL/RASSF5/NORE1 SARAH domain Coiled coil Apoptosis
a b s t r a c t The STE20 kinases MST1 and MST2 are key players in mammalian Hippo pathway. The SARAH domains of MST1/2 act as a platform to mediate homodimerization and hetero-interaction with a range of adaptors including RASSFs and Salvador, which also possess SARAH domains. Here, we determined the crystal structure of human MST2 SARAH domain, which forms an antiparallel homodimeric coiled coil. Structural comparison indicates that SARAH domains of different proteins may utilize a shared dimerization module to form homodimer or heterodimer. Structure-guided mutational study identified specific interface residues critical for MST2 homodimerization. MST2 mutations disrupting its homodimerization also impaired its hetero-interaction with RAPL (also named RASSF5 and NORE1), which is mediated by their SARAH domains. Further biochemical and cellular assays indicated that SARAH domain-mediated homodimerization and hetero-interaction with RAPL are required for full activation of MST2 and therefore apoptotic functions in T cells. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction The STE20 like kinases MST1 and MST2 are core components of mammalian Hippo signaling pathway, which plays a fundamental role in organ size control and tumor suppression (Avruch et al., 2006; O’Neill and Kolch, 2005; O’Neill et al., 2004; Song et al., 2010). MST1/2 in conjunction with an adaptor protein Salvador phosphorylates and activates LATS1/2 kinases, which in turn associates with adaptor protein MOB1, to phosphorylate and prevent nuclear translocation of the downstream transcription coactivator Yes-associated protein (YAP), and thereby inhibit cell proliferation and promote apoptosis (Oka et al., 2008; Wu et al., 2003). MST1 and MST2 share high sequence homology with identical N-terminal kinase domains for catalysis and distinct C-terminal SARAH (Salvador, RASSF and Hpo homology) domains for homodimerization or hetero-interaction with Salvador and RASSF (Ras association domain family) proteins (Fig. 1A) (Pfeifer et al., 2010; Scheel and Hofmann, 2003). Functional redundancy between MST1 and MST2 has been evidenced by animal studies showing ⇑ Corresponding authors. Address: State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, 320 Yueyang Road, Shanghai 200031, China. Fax: +86 21 54921291 (Z. Zhou). E-mail addresses: [email protected] (Z. Zhou), [email protected] (M. Zhang). 1 These authors contribute equally to this work. http://dx.doi.org/10.1016/j.jsb.2014.01.008 1047-8477/Ó 2014 Elsevier Inc. All rights reserved.
that MST1/2 double knockout causes embryonic lethality while single knockout of either MST1 or MST2 does not impair organ development (Oh et al., 2009; Zhou et al., 2009). Ablation of both MST1 and MST2 caused liver tumorigenesis through largely abolishing YAP phosphorylation and increasing YAP nuclear localization (Zhao et al., 2009). On the other hand, despite of high degree sequence homology and functional redundancy, MST1 and MST2 displayed differential regulatory properties in certain aspects such as susceptibility to protein phosphatases (Deng et al., 2003). The structure of MST1 SARAH domain has been determined by NMR, showing an antiparallel dimeric conformation (Hwang et al., 2007), yet the structure of MST2 SARAH domain has not been studied in detail. In addition to their critical functions in development, MST1/2 kinases have been implicated in immune regulation including T cell proliferation, activation, and homeostasis, lymphocyte chemotaxis and trafficking, as well as inflammatory responses (Dong et al., 2009; Katagiri et al., 2009; Mou et al., 2012; Yun et al., 2011; Zhou et al., 2008). Crosstalk of MST1/2 with Akt signaling may antagonize Akt1 activity and therefore modulate T cell costimulation and NFjB target gene transcription (Cinar et al., 2007). In MST1 and MST2 double knockout mice, mature T cells could not efficiently migrate from thymus to the circulation and secondary lymphoid organs, indicating that MST1/2 may control lymphocyte trafficking
G. Liu et al. / Journal of Structural Biology 185 (2014) 366–374
and migration, which is important for efficient immunosurveillance and effective immune responses (Katagiri et al., 2009; Mou et al., 2012). Consistently, mice lacking MST1 exhibit a variety of T cell abnormalities (Mou et al., 2012); while patients lacking MST1 displayed primary immunodeficiency like features (Abdollahpour et al., 2012). Recent studies suggest that a group of RASSF proteins including RASSF1-6 may interact with MST1/2 to differentially regulate Hippo signaling (Hwang et al., 2007; Ikeda et al., 2009; Pfeifer et al., 2010; Romano et al., 2010; Vichalkovski et al., 2008). The specific mechanism of RASSF-mediated differential regulation of MST1/2, and its functional role in immune regulation remains largely unknown. The regulator for cell adhesion and polarization enriched in lymphoid tissues (RAPL), also named RASSF5 and NORE1, has been found to associate with MST1 to induce cell polarity and adhesion of lymphocytes (Katagiri et al., 2006). RAPL is an effector for small GTPase Rap1 that is essential for lymphocyte trafficking. During TCR stimulated induction of polarized morphology and clustering of the integrin LFA-1 in T cells, RAPL can associate with MST1 to alter its cellular localization and kinase activity (Katagiri et al., 2009). Meanwhile, animal study suggested that RAPL-MST1 complex negatively regulate naïve T cell proliferation and that MST1 is required for the maintenance of RAPL level in lymphoid cells (Zhou et al., 2008). It is generally understood that RAPL may associate with MST1/2 through their SARAH domains. Here, we performed structural and biochemical studies of MST2-RAPL signaling. Our crystallographic analysis of MST2 SARAH domain revealed an antiparallel conformation similar to that of MST1 with local variations. SARAH domains are sufficient for MST2-RAPL stable association. Mutations of the SARAH domain interface impaired MST2 homodimerization and interaction with RAPL, preventing full activation of MST2. RAPL stimulates the kinase activity of MST2 in vitro, and promotes its apoptotic function in vivo. Mutation disrupting MST2 association with RAPL abrogated their cooperative function in T cell apoptosis.
367
2.2. Crystallization, structure determination and refinement The native or Se-Met-substituted MST2 SARAH domain was concentrated to 16 mg/ml and crystallized by sitting drop vapor diffusion method at 16 °C, in a drop with 1 ll protein solution and 1 ll reservoir solution. The MST2 SARAH domain was crystallized in 0.4 M calcium chloride dihydrate, 0.1 M sodium acetate trihydrate pH 4.6, 5% v/v 2-propanol. Diffraction date were collected from a flash cooled crystal at 100 K on beamline BL17U of Shanghai Synchrotron Radiation facility (SSRF), China, and processed in HKL2000 (Otwinowski and Minor, 1997). The structure of MST2 SARAH domain was solved using single-wavelength anomalous diffraction method from a Se-Met derivatized with program AutoSol in Phenix (Adams et al., 2010). The model building was performed in Coot (Emsley et al., 2010) and the structure was refined using REFMAC5 (CCP4, 1994; Murshudov et al., 2011). 2.3. Immunoprecipitation HEK293T cells were maintained in DMEM medium with 5% (vol/vol) fetal calf serum, 2 mM L-glutamine, penicillin (100 units/ ml), and streptomycin (100 lg/ml) in 5% CO2/95% humidified air at 37 °C at a density of 106 cells per ml. Cells were then transiently with expression vectors encoding Flag-tagged wild-type MST2 and Myc-tagged MST2 mutants. Cell extracts were prepared in lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 10% glycerol, 0.1% Triton X-100, 0.5 mg/ml BSA and protease inhibitor cocktail), and incubated with anti-Flag antibody (sigma) for 4 h at 4 °C followed by overnight incubation with Protein A/G agarose (Santa Cruz). The immunoprecipitates were washed three times in the lysis buffer before loading onto SDS–PAGE gels. The samples separated by SDS–PAGE is blotted and developed with anti-Myc antibodies. 2.4. Cross-linking experiment
2. Materials and methods 2.1. Cloning, protein expression and purification Human MST2 SARAH domain (amino acids 436–484) were cloned into HT-pET-28a and expressed in Escherichia coli (E. coli) BL21(DE3) codon plus cells. The expression of proteins was induced by 0.5 mM isopropyl b-D-thiogalactopyranoside in LuriaBertani medium. After cultured for 12 h at 16 °C, bacterial cells were harvested by centrifugation and suspended with lysis buffer (20 mM Hepes, 500 mM NaCl, 5% glycerol, 1 mM DTT and 20 mM imidazole, pH 7.5), and then lysed. The soluble cell lysate was fractionated after centrifuged at 18,000g for 40 min. The protein was purified with affinity column pre-charged with Ni2+. The proteins was eluted with elution buffer (20 mM Hepes, 500 mM NaCl, 5% glycerol, 1 mM DTT and 300 mM imidazole, pH 7.5) and digested by TEV protease to remove the N-terminal His-tag. Then the proteins were concentrated and loaded on HiLoad 16/60 Superdex 200 column in 20 mM Hepes, 100 mM NaCl, 1 mM DTT, pH 7.5. The purity of proteins was monitored by SDS–PAGE. Selenomethionine (Se-Met)-substituted MST2 SARAH domain was purified as described above, except that E. coli cells were cultured in M9 minimal medium containing amino-acid supplement (lysine, phenylalanine, threonine to final concentration of 100 mg/ l, isoleucine, leucine, valine to 50 mg/l, and L-Se-Met to 60 mg/l). Other MST2 mutants and mouse RAPL fragments were constructed and purified by similar procedures described above. For cell-based assays, Flag-tagged MST2, Myc-tagged MST2 and Flagtagged human RAPL were subcloned into BamHI/XhoI sites of pcDNA3.1.
Purified 1 mg/ml wild-type or mutant MST2 was incubated with 0 and 0.005% (vol/vol) glutaraldehyde (GA) in conjugation buffer (20 mM Hepes, 100 mM NaCl, 1 mM DTT, pH 7.5) at room temperature for 1 h. After the reaction was quenched with 50 mM Tris–Cl pH 8.0 for 30 min, the cross-linked samples were analyzed on a 8% SDS–PAGE. 2.5. Kinase assay The activity of purified wild-type or mutant MST2 was assayed by using myelin basic protein (MBP) as substrate. Kinase activity of wild-type or mutant MST2 was measured in a total assay volume of 40 ll consisting of 50 mM Tris–HCl pH 7.5, 0.1 mM EGTA, 1 mM DTT, 10 mM magnesium acetate, 0.1 mM [c-32P]ATP (200 c.p.m./pmol) and 1 mg/ml MBP, with 18.5 lM MST2 and 92.5 lM RAPL. The assays were carried out at 30 °C and were terminated after 20 min by spotting the 40 ll reaction mixture onto P81 membranes. The membranes were washed in phosphoric acid for 3 times, and the incorporated radioactivity was measured by scintillation counting (Beckman LS6500) (Hastie et al., 2006). 2.6. GST pulldown assay GST-tagged MST2 and its SARAH domain protein coupled on glutathione-Sepharose beads were mixed with RAPL and its SARAH domain respectively at 4 °C for 1 h in 20 mM Hepes pH7.5, 100 mM NaCl, 1 mM DTT, and washed three times. The input and output samples were boiled and loaded on 12% SDS–PAGE followed by Coomassie blue staining.
368
G. Liu et al. / Journal of Structural Biology 185 (2014) 366–374
2.7. Kinetic assay
Table 1 Data collection and refinement statistics.
Interaction analysis was performed using an Octet Red 96 instrument (ForteBio). RAPL was labeled by biotin in 20 mM Hepes pH 7.5, 100 mM NaCl, 1 mM DTT, and biotinylated proteins were immobilized on streptavidin (SA) biosensors and incubated with 800 nM wild-type or mutant MST2 in 1 kinetics buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4, 0.01% BSA, and 0.002% Tween 20). All binding experiment were performed in 1 kinetics buffer at 25 °C. The experiments comprised 5 steps: (1) Baseline acquisition; (2) Biotinylated proteins loading onto SA biosensor; (3) Second baseline acquisition; (4) Association of interacting protein for kon measurement; (5) Dissociation of interacting protein for koff measurement. Data were analyzed using Octet Data Analysis Software 7.0 (ForteBio).
MST2 SARAH Native
SeMet
Data collection Wavelength (Å) Space group
0.97930 C2221
0.97915 C2221
Cell dimensions a, b, c (Å) a, b, c (°) Resolution (Å) Rmerge I/rI Completeness (%) Redundancy
42.7, 45.3, 208.8 90.0, 90.0, 90.0 50.00–2.00 (2.00–2.03)a 0.145 (0.600) 14.9 (4.5) 96.9 (99.9) 4.5 (5.6)
42.2, 44.6, 209.2 90.0, 90.0, 90.0 50.00–1.50 (1.53–1.50) 0.125 (0.660) 17.1 (3.8) 92.3 (85.7) 5.3 (5.7)
Refinement Resolution (Å) No. reflections Rwork/Rfree No. atoms Protein Ligand/ion Water
2.8. Flow cytometry Jurkat cells were cultured in RPMI 1640 medium and then transfected with different constructs encoding Myc-tagged MST2 and Flag-tagged RAPL. The cells were incubated with 50 lg/ml propidium iodide (PI) in PBS with 0.2 mg/ml RNase A and 0.1% Triton X-100, and analyzed by flow cytometer. siRNA of MST2 and RAPL were bought from Santa Cruz. 3. Results and discussion 3.1. MST2 SARAH domain forms an antiparallel dimeric coiled coil a
34.86–1.50 29481 0.19/0.23 1751 – 239
B-factors Protein Ligand/ion Water
16.9 – 23.9
R.m.s. deviations Bond lengths (Å) Bond angles (°)
0.007 1.179
Values in parentheses are for highest-resolution shell.
Human MST2 comprises a kinase domain (amino acids 1–297), an inhibitory domain (amino acids 327–392), and a C-terminal
Fig.1. Structure of human MST2 SARAH domain. (A) Schematic illustration of domain organization for human MST2. (B) MST2 SARAH domain forms an antiparallel dimeric coiled coil. Two chains (monomers) of MST2 SARAH domain are shown in cartoon and colored slate and green, respectively. The interaction between helices 2 of the two chains is mainly mediated by hydrophobic interactions. (C) Helix 1 of one MST2 monomer interacts with helix 2 of the other via hydrophobic interactions. (D) The heptad repeats of MST2 SARAH domain coiled coil. Two tetrad transitions are colored red.
G. Liu et al. / Journal of Structural Biology 185 (2014) 366–374
SARAH domain (amino acids 436–491) (Creasy et al., 1996) (Fig. 1A). We purified and crystallized MST2 SARAH domain (amino acids 436–484). The structure was solved by single-wavelength anomalous diffraction method from a Se-Met derivative crystal with statistics summarized in Table 1. There are four copies of MST2 SARAH domain in the asymmetric unit of the crystals, forming two symmetric dimeric coiled coil. Each monomer is composed of two helices, helix 1 (amino acids 436–442) and helix 2 (amino acids 445–484) (Fig. 1B and C). These two dimers are very similar except the head of helix 1. The r.m.s.d
369
values of superimposition for all atoms and Ca only are 1.118 Å and 0.995 Å, respectively. Two chains of MST2 SARAH domains form an antiparallel dimeric coiled coil (Fig. 1B). A classical coiled coil usually adopts a heptad repeat in its sequence, consisting a–g positions. Amino acid residues at positions a and d often mediate core packing or interlocking of the alpha helical chains. However, unlike the ideal coiled coil conformation, the MST2 SARAH domain has two transitions in periodicity from heptad (a–g) to tetrad (d–g, Met459-Glu462 and Ile477-Ala479) (Fig. 1D), which are punctuated by two prolines
Fig.2. Structural comparison of SARAH domains. (A and B) The structures of MST2 SARAH domain colored green (A) and slate (B, PDB code 4L0N), respectively. (C) Comparison of two structures of the MST2 SARAH domain. (D) The structure of MST1 SARAH domain (PDB code 2JO8) colored yellow. (E) The structure of RAPL SARAH domain (PDB code 2YMY) colored cyan. (F) The heterodimeric structure of MST2 and RAPL SARAH domains (PDB code 4LGD) colored magenta. (G) Structural comparison of SARAH domains. (H) Sequence alignment of SARAH domains of MST1/2, RASSF1–6 and Salvador SARAH domains. The heptad repeats are labeled. Resides in a and d positions are colored red.
370
G. Liu et al. / Journal of Structural Biology 185 (2014) 366–374
(Pro457 and Pro476) (Fig. 1B). The two tetrads result in a decreased degree of supercoiling. The coiled coil formed by MST2 SARAH domain buries a total of 1,439 Å2 surface area. Hydrophobic interactions dominates the association between helices 2 of two chains, involving residues Leu445, Leu448, Leu452, Leu455, Met459, Ile463, Leu466, Tyr470, Lys473, Ile477, Met481 and Lys484 in helix 2 (Fig. 1B). The homodimer is further stabilized by hydrogen bonding between residues Glu462 and Tyr470, and salt bridges formed by residues Asp456, Glu460, Arg467 and Arg474. Helix 1 of one chain folds back to contact helix 2 of the other chain (Fig. 1C). Residues Leu440 and Phe437 of helix 1 interact with residues Pro476, Ile477, Ala480, and Ala483 from helix 2 via hydrophobic interaction. 3.2. Structural comparison of SARAH domains During the preparation of this manuscript, Structural Genomics Consortium also determined the structure of MST2 SARAH domain (PDB code 4L0N). The helices 2 have similar conformations with a root-mean-square deviation (r.m.s.d) value of 1.071 Å for Ca superimposition of structure 4L0N and the structure determined in this work (Fig. 2A–C). However, positions of the helix 1 shows derivation relative to helix 2, resulting in an r.m.s.d value of 1.940 Å for Ca superimposition of the coiled coil. This is partially due to the flexible loop between the helix 1 and the helix 2. Relative position between two chains in two structures also changes, although the hydrophobic interface of coiled coil, as well as
residues in the interface, is the same as that in our structure. These differences between the two structures indicate the flexibility of SARAH domain. The structure of MST1 SARAH domain, which was previously determined by nuclear magnetic resonance, folds into an antiparallel dimer (Hwang et al., 2007). The monomeric structure of MST2 SARAH domain resembles that of MST1, with r.m.s.d values of 1.853 Å and 1.300 Å for overlapping Ca atoms of the whole SARAH domain and only helix 2, respectively (Fig. 2A and D). Like differences between two structures of the MST2 SARAH domain, the conformation of MST1 and MST2 homodimers varies. When helices 2 of MST1 and MST2 SARAH domains are superimposed, the position of helix 1 is altered relative to the helix 2, and one chain shows a bend with different degree relative to the other chain, although sequences, especially residues involved in core packing of coiled coil, are highly conserved and the interfaces are very similar (Fig. 2G). The conformational differences of MST1 and MST2 SARAH domains further indicate that structures of SARAH domains are dynamic. Recently, structures of RAPL SARAH domain alone and its complex with MST2 were determined (Makbul et al., 2013; Ni et al., 2013). Although there are certain degrees of conformational variations, the overall structure of MST2-RAPL SARAH domain heterodimer is very similar to that of MST2 or RAPL SARAH domain homodimer (Fig. 2E–G). Residues at a and d positions of the coiled coils are highly conserved in these SARAH domains (Fig. 2H). Transitions in periodicity from heptad to tetrad are also present in MST1 and RAPL SARAH domains. Besides MST1/2 and RAPL, other
Fig.3. Interface mutations impair MST2 SARAH domain-mediated homodimerization and its kinase activity. (A) The indicated constructs of Myc-tagged MST2 and Flagtagged MST2 were co-transfected into HEK293T cells. Cells were lysed, and lysate were immunoprecipitated (IP) with anti-Flag antibody and subjected to immunoblot (IB) analysis. (B) The relative association was calculated by ratio of Myc and Flag gray level in IBs and the association between wild-type Myc-tagged MST2 and Flag-tagged MST2 was set to 1.0. (C) Cross-linking experiment of wild-type or mutant MST2. 1 mg/ml wild-type or mutant MST2 was incubated with 0% and 0.005% (vol/vol) glutaraldehyde (GA) at room temperature for 1 h. The cross-linked samples were analyzed on 8% SDS–PAGE. MST2 oligomer was indicated by asterisk. (D) Purified wild-type and mutant MST2 were assayed for determining the ability of MST2 to phosphorylate MBP. Error bars represent SD of data obtained in three independent experiments.
G. Liu et al. / Journal of Structural Biology 185 (2014) 366–374
SARAH domain-containing proteins, such as RASSF1–4, RASSF6 and Salvador, also possess these sequence features (Fig. 2H). Together, these observations suggest that SARAH domains of MST1/2 and their adaptor proteins share a common dimerization module with conserved overall conformation for the formation of either homodimer or heterodimer. 3.3. Kinase activity of MST2 is regulated by SARAH domain-mediated homodimerization Following our structural analysis of MST2 SARAH domain, we performed biochemical and cellular assays to identify specific amino acids most critical for MST2 SARAH domain-mediated homodimerization. Mutations M459A and L466A, but not L452A, partially disrupted MST2 homodimerization as shown by immunoprecipitation (IP) assay in HEK293T cells (Fig. 3A and B). Moreover, we perform cross-linking experiment using purified recombinant proteins of wild-type and mutant MST2. MST2 M452A mutant formed oligomer as does wild-type. However, 0.005% glutaraldehyde didn’t induce band shift of mutant M459A, consistent with IP results (Fig. 3C). In the structure of MST2 SARAH domain, residues Met459 and Leu466 locate in the center of SARAH domain, suggesting that they would play a key role in homodimerization of MST2. These binding assays further confirmed this notion. To further assess the function of MST2 SARAH domain, we used purified proteins to measure the kinase activity of wild-type and mutant MST2 towards MBP, a general kinase substrate. Mutations M459A and L466A, which disrupted SARAH domain-mediated MST2 homodimerization in vitro and in vivo, impaired the kinase activity of MST2 (Fig. 3D). Residue Leu452 is in the distal end of MST2 SARAH domain and may contribute less to its association. However, mutation of L452A also impaired MST2 kinase activity (Fig. 3D). This could be explained, in part, by mutation-induced local conformational change even though the overall association remain unbroken. As a negative control, the kinase dead mutant of
371
MST2 (D164A) did not show activity (Fig. 3D). These results indicated that hydrophobic interactions mediated by Met459 and Leu466 are critical for SARAH domain-mediated homodimerization and full activation of MST2. 3.4. SARAH domains mediates hetero-interaction between MST2 and RAPL As mentioned, RAPL functions as an adaptor protein for the small GTPase RAP1, and is required for efficient immune cell trafficking. It has been reported that MST1 may act as a key participant in the control of cell polarization and adhesion ‘downstream’ of RAP1 and RAPL activation triggered by chemokine and antigen stimulation (Katagiri et al., 2006). To verify and map the interacting domains between MST2 and RAPL, we performed pulldown and gel filtration assays using purified proteins of MST2 and RAPL. In addition to the full-length MST2 and its SARAH domain, two fragments of RAPL proteins were used, corresponding to its SARAH domain (amino acids 365–413) and Ras association (RA) domain plus SARAH domain (amino acids 200–413), respectively. As shown in Fig. 4A, RAPL and its SARAH domain could be pulled down by GST-tagged MST2 and MST2 SARAH domain. Moreover, mixtures of full-length MST2 or MST2 SARAH domain and RAPL or RAPL SARAH domain were eluted in one peak in gel filtration assay (Fig. 4B–E), indicating that MST2 and RAPL directly interact with each other through their SARAH domains. These results are consistent with a recent structural study of MST2 in complex with RAPL SARAH domain (Ni et al., 2013). To further dissect the interaction between MST2 and RAPL, purified RAPL and wild-type or mutant MST2 were used to detect their binding affinities using bio-layer interferometry (BLI). RAPL binds to wild-type MST2 with an equilibrium dissociation constant (Kd) of 6.54 nM. Single mutation of MST2 M459A or L466A, which impaired its homodimerization, substantially weakened its interaction with RAPL (Fig. 5A). In addition, mutation L452A of MST2
Fig.4. MST2 interacts with RAPL through their SARAH domains. (A) The indicated proteins of GST-tagged MST2 were coupled on glutathione Sepharose beads, and then mixed with RAPL and its SARAH domain, respectively. The input and output samples were loaded on SDS–PAGE followed by Coomassie blue staining. RAPL was indicated by asterisk. (B–E) MST2 or MST2 SARAH domain and RAPL or RAPL SARAH domain were mixed and analyzed by gel filtration using Superdex 200 10/300 GL column. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
372
G. Liu et al. / Journal of Structural Biology 185 (2014) 366–374
Fig.5. Disruption of MST2 SARAH domain homodimerization affects its interaction with RAPL. (A) Sensorgrams for wild-type or mutant MST2 on dimerization interface binding to RAPL. Binding affinities of wild-type or mutant MST2 with RAPL were determined by BLI experiment. Biotinylated-RAPL was immobilized on SA biosensors and incubated with wild-type or mutant MST2 at a concentration of 800 nM. Curves are the experimental trace obtained from BLI experiments. (B) Purified wild-type or mutant MST2 were assayed for determining the ability of MST2 to phosphorylate MBP in the absence or presence of RAPL. Error bars represent SD of data obtained in three independent experiments.
Fig.6. MST2 and RAPL promote apoptosis in Jurkat cells. (A–H) Jurkat cells were transfected with the indicated constructs encoding Myc-tagged wild-type or mutant MST2 or/ and Flag-tagged RAPL. Cells were stained with PI and analyzed by flow cytometer. The apoptosis fractions are indicated by lines. The numbers are the percentage of apoptosis in each sample. (I–K) Jurkat cells were transfected with the indicated siRNA for 48 h. Cells were then stained with PI and cell cycle distribution was analyzed by flow cytometer. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
G. Liu et al. / Journal of Structural Biology 185 (2014) 366–374
did not significantly alter its interaction with RAPL. Together, these results indicate that SARAH domain of MST2 is essential for its homodimerization and interaction with RAPL. We then determined the effects of RAPL on kinase activity of MST2. Our results showed that RAPL promoted MST2 activity slightly (about 1.5-fold) (Fig. 5B). Mutation M459A disrupting the homodimerization of MST2 SARAH domain reduced its kinase activity with or without RAPL (Fig. 5B). These results suggest that SARAH domain mediated homodimerization and hetero-interaction with RAPL is required to fully activate MST2 kinase.
3.5. Complex of MST2 and RAPL promotes apoptosis To further assess functional importance of MST2 SARAH domain mediated homodimerization or hetero-interaction with RAPL, we examined the apoptotic effects of wild-type and mutant MST2 with or without RAPL. Our flow cytometry assay in Jurkat T cells showed that transfection of MST2 dramatically promoted apoptosis as expected (Fig. 6A and B); while transfection of RAPL also significantly enhanced apoptosis (Fig. 6C). Moreover, co-transfection of wildtype MST2 with RAPL further increased apoptosis (Fig. 6D); yet co-transfection of a kinase dead version of MST2 (D164A) with RAPL did not cause apoptosis (Fig. 6E). Similarly, co-transfection of RAPL and MST2 mutants M459A and L466A that partially disrupted SARAH domain mediated homodimerization and interaction with RAPL did not induce substantial apoptosis either (Fig. 6F and G). However, co-transfection of RAPL and MST2 mutant L452A, which did not influence its homodimerization, also induced significant apoptosis, although to a lesser extent probably due to disturbed conformation and impaired kinase activation of MST2 (Fig. 6H). Consistent with these observations, knockdown of MST2 or RAPL promoted cell cycle progression (Fig. 6I–K). Taken together, these results indicate that MST2 and RAPL may form a signaling complex through their SARAH domains in lymphocytes to synergistically promote apoptosis in a way dependent on the kinase activity of MST2.
3.6. Conclusion We determined the crystal structure of MST2 SARAH domain, which forms an antiparallel dimeric coiled coil. Structural comparison suggests that SARAH domains make use of a shared interface to mediate homo- or hetero- dimerization. Using pull-down and gel filtration assays, we found that MST2 directly interacts with RAPL via their SARAH domains. Mutations of the dimeric interface of MST2 SARAH domain not only impaired its homodimerization but also decreased its hetero-interaction with RAPL, suggesting that a similar set of interface residues mediate either homo- or hetero- dimerization of SARAH domains. Consistently, these mutations also abrogated MST2-RAPL induced apoptosis in Jurkat T cells. Besides RAPL, MST2 also functions by association with many other partners, such as other RASSF family members and Salvador, through their SARAH domain to transduce cell signaling (Mardin et al., 2010; Pfeifer et al., 2010; Scheel and Hofmann, 2003). We proposed that these proteins interact and function with MST2 via a common mechanism like RAPL.
4. Accession numbers The coordinate file and structure factor for the MST2 SARAH domain crystal structure was deposited in the Protein Data Bank under accession code 4HKD.
373
Acknowledgments We thank the staff at beamline BL17U of Shanghai Synchrotron Radiation Facility for help of data collection. This work was supported by the 973 program of the Ministry of Science and Technology of China (2012CB910204 and 2010CB529701), the Science and Technology Commission of Shanghai Municipality (11JC14140000, 13ZR1446400), the National Natural Science Foundation of China (30970565, 31170688, 31270808, 31300734, 31340044), the Science and Technological Fund of Anhui Province for Outstanding Youth (10040606Y15), and the ‘‘Cross and cooperation in science and technology innovation team’’ project of the Chinese Academy of Sciences. Dr. Z.Z. is a scholar of the Hundred Talents Program of the Chinese Academy of Sciences. References Abdollahpour, H., Appaswamy, G., Kotlarz, D., Diestelhorst, J., Beier, R., Schaffer, A.A., Gertz, E.M., Schambach, A., Kreipe, H.H., Pfeifer, D., Engelhardt, K.R., Rezaei, N., Grimbacher, B., Lohrmann, S., Sherkat, R., Klein, C., 2012. The phenotype of human STK4 deficiency. Blood 119, 3450–3457. Adams, P.D., Afonine, P.V., Bunkoczi, G., Chen, V.B., Davis, I.W., Echols, N., Headd, J.J., Hung, L.W., Kapral, G.J., Grosse-Kunstleve, R.W., McCoy, A.J., Moriarty, N.W., Oeffner, R., Read, R.J., Richardson, D.C., Richardson, J.S., Terwilliger, T.C., Zwart, P.H., 2010. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221. Avruch, J., Praskova, M., Ortiz-Vega, S., Liu, M., Zhang, X.F., 2006. Nore1 and RASSF1 regulation of cell proliferation and of the MST1/2 kinases. Methods Enzymol. 407, 290–310. CCP4, J., 1994. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763. Cinar, B., Fang, P.K., Lutchman, M., Di Vizio, D., Adam, R.M., Pavlova, N., Rubin, M.A., Yelick, P.C., Freeman, M.R., 2007. The pro-apoptotic kinase Mst1 and its caspase cleavage products are direct inhibitors of Akt1. EMBO J. 26, 4523– 4534. Creasy, C.L., Ambrose, D.M., Chernoff, J., 1996. The Ste20-like protein kinase, Mst1, dimerizes and contains an inhibitory domain. J. Biol. Chem. 271, 21049–21053. Deng, Y., Pang, A., Wang, J.H., 2003. Regulation of mammalian STE20-like kinase 2 (MST2) by protein phosphorylation/dephosphorylation and proteolysis. J. Biol. Chem. 278, 11760–11767. Dong, Y., Du, X., Ye, J., Han, M., Xu, T., Zhuang, Y., Tao, W., 2009. A cell-intrinsic role for Mst1 in regulating thymocyte egress. J. Immunol. 183, 3865–3872. Emsley, P., Lohkamp, B., Scott, W.G., Cowtan, K., 2010. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501. Hastie, C.J., McLauchlan, H.J., Cohen, P., 2006. Assay of protein kinases using radiolabeled ATP: a protocol. Nat. Protoc. 1, 968–971. Hwang, E., Ryu, K.S., Paakkonen, K., Guntert, P., Cheong, H.K., Lim, D.S., Lee, J.O., Jeon, Y.H., Cheong, C., 2007. Structural insight into dimeric interaction of the SARAH domains from Mst1 and RASSF family proteins in the apoptosis pathway. Proc. Natl. Acad. Sci. USA 104, 9236–9241. Ikeda, M., Kawata, A., Nishikawa, M., Tateishi, Y., Yamaguchi, M., Nakagawa, K., Hirabayashi, S., Bao, Y., Hidaka, S., Hirata, Y., Hata, Y., 2009. Hippo pathwaydependent and -independent roles of RASSF6. Sci. Signal. 2, ra59. Katagiri, K., Imamura, M., Kinashi, T., 2006. Spatiotemporal regulation of the kinase Mst1 by binding protein RAPL is critical for lymphocyte polarity and adhesion. Nat. Immunol. 7, 919–928. Katagiri, K., Katakai, T., Ebisuno, Y., Ueda, Y., Okada, T., Kinashi, T., 2009. Mst1 controls lymphocyte trafficking and interstitial motility within lymph nodes. EMBO J. 28, 1319–1331. Makbul, C., Constantinescu Aruxandei, D., Hofmann, E., Schwarz, D., Wolf, E., Herrmann, C., 2013. Structural and thermodynamic characterization of Nore1SARAH: a small, helical module important in signal transduction networks. Biochemistry 52, 1045–1054. Mardin, B.R., Lange, C., Baxter, J.E., Hardy, T., Scholz, S.R., Fry, A.M., Schiebel, E., 2010. Components of the Hippo pathway cooperate with Nek2 kinase to regulate centrosome disjunction. Nat. Cell Biol. 12, 1166–1176. Mou, F., Praskova, M., Xia, F., Van Buren, D., Hock, H., Avruch, J., Zhou, D., 2012. The Mst1 and Mst2 kinases control activation of rho family GTPases and thymic egress of mature thymocytes. J. Exp. Med. 209, 741–759. Murshudov, G.N., Skubak, P., Lebedev, A.A., Pannu, N.S., Steiner, R.A., Nicholls, R.A., Winn, M.D., Long, F., Vagin, A.A., 2011. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 67, 355–367. Ni, L., Li, S., Yu, J., Min, J., Brautigam, C.A., Tomchick, D.R., Pan, D., Luo, X., 2013. Structural basis for autoactivation of human Mst2 kinase and its regulation by RASSF5. Structure 21, 1757–1768. Oh, S., Lee, D., Kim, T., Kim, T.S., Oh, H.J., Hwang, C.Y., Kong, Y.Y., Kwon, K.S., Lim, D.S., 2009. Crucial role for Mst1 and Mst2 kinases in early embryonic development of the mouse. Mol. Cell Biol. 29, 6309–6320.
374
G. Liu et al. / Journal of Structural Biology 185 (2014) 366–374
Oka, T., Mazack, V., Sudol, M., 2008. Mst2 and Lats kinases regulate apoptotic function of Yes kinase-associated protein (YAP). J. Biol. Chem. 283, 27534– 27546. O’Neill, E., Kolch, W., 2005. Taming the Hippo: Raf-1 controls apoptosis by suppressing MST2/Hippo. Cell Cycle 4, 365–367. O’Neill, E., Rushworth, L., Baccarini, M., Kolch, W., 2004. Role of the kinase MST2 in suppression of apoptosis by the proto-oncogene product Raf-1. Science 306, 2267–2270. Otwinowski, Z., Minor, W., 1997. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326. Pfeifer, G.P., Dammann, R., Tommasi, S., 2010. RASSF proteins. Curr. Biol. 20, R344–345. Romano, D., Matallanas, D., Weitsman, G., Preisinger, C., Ng, T., Kolch, W., 2010. Proapoptotic kinase MST2 coordinates signaling crosstalk between RASSF1A, Raf-1, and Akt. Cancer Res. 70, 1195–1203. Scheel, H., Hofmann, K., 2003. A novel interaction motif, SARAH, connects three classes of tumor suppressor. Curr. Biol. 13, R899–900. Song, H., Mak, K.K., Topol, L., Yun, K., Hu, J., Garrett, L., Chen, Y., Park, O., Chang, J., Simpson, R.M., Wang, C.Y., Gao, B., Jiang, J., Yang, Y., 2010. Mammalian Mst1 and Mst2 kinases play essential roles in organ size control and tumor suppression. Proc. Natl. Acad. Sci. USA 107, 1431–1436.
Vichalkovski, A., Gresko, E., Cornils, H., Hergovich, A., Schmitz, D., Hemmings, B.A., 2008. NDR kinase is activated by RASSF1A/MST1 in response to Fas receptor stimulation and promotes apoptosis. Curr. Biol. 18, 1889–1895. Wu, S., Huang, J., Dong, J., Pan, D., 2003. Hippo encodes a Ste-20 family protein kinase that restricts cell proliferation and promotes apoptosis in conjunction with salvador and warts. Cell 114, 445–456. Yun, H.J., Yoon, J.H., Lee, J.K., Noh, K.T., Yoon, K.W., Oh, S.P., Oh, H.J., Chae, J.S., Hwang, S.G., Kim, E.H., Maul, G.G., Lim, D.S., Choi, E.J., 2011. Daxx mediates activation-induced cell death in microglia by triggering MST1 signalling. EMBO J. 30, 2465–2476. Zhao, B., Lei, Q., Guan, K.L., 2009. Mst out and HCC in. Cancer Cell 16, 363–364. Zhou, D., Conrad, C., Xia, F., Park, J.S., Payer, B., Yin, Y., Lauwers, G.Y., Thasler, W., Lee, J.T., Avruch, J., Bardeesy, N., 2009. Mst1 and Mst2 maintain hepatocyte quiescence and suppress hepatocellular carcinoma development through inactivation of the Yap1 oncogene. Cancer Cell 16, 425–438. Zhou, D., Medoff, B.D., Chen, L., Li, L., Zhang, X.F., Praskova, M., Liu, M., Landry, A., Blumberg, R.S., Boussiotis, V.A., Xavier, R., Avruch, J., 2008. The Nore1B/Mst1 complex restrains antigen receptor-induced proliferation of naive T cells. Proc. Natl. Acad. Sci. USA 105, 20321–20326.
