CCM3/PDCD10 Heterodimerizes with Germinal Center Kinase III (GCKIII) Proteins Using a Mechanism Analogous to CCM3 Homodimerization

CCM3 mutations give rise to cerebral cavernous malformations (CCMs) of the vasculature through a mechanism that remains unclear. Interaction of CCM3 with the germinal center kinase III (GCKIII) subfamily of Sterile 20 protein kinases, MST4, STK24, and STK25, has been implicated in cardiovascular development in the zebrafish, raising the possibility that dysregulated GCKIII function may contribute to the etiology of CCM disease. Here, we show that the amino-terminal region of CCM3 is necessary and sufficient to bind directly to the C-terminal tail region of GCKIII proteins. This same region of CCM3 was shown previously to mediate homodimerization through the formation of an interdigitated α-helical domain. Sequence conservation and binding studies suggest that CCM3 may preferentially heterodimerize with GCKIII proteins through a manner structurally analogous to that employed for CCM3 homodimerization. CCM3 mutations give rise to cerebral cavernous malformations (CCMs) of the vasculature through a mechanism that remains unclear. Interaction of CCM3 with the germinal center kinase III (GCKIII) subfamily of Sterile 20 protein kinases, MST4, STK24, and STK25, has been implicated in cardiovascular development in the zebrafish, raising the possibility that dysregulated GCKIII function may contribute to the etiology of CCM disease. Here, we show that the amino-terminal region of CCM3 is necessary and sufficient to bind directly to the C-terminal tail region of GCKIII proteins. This same region of CCM3 was shown previously to mediate homodimerization through the formation of an interdigitated α-helical domain. Sequence conservation and binding studies suggest that CCM3 may preferentially heterodimerize with GCKIII proteins through a manner structurally analogous to that employed for CCM3 homodimerization. IntroductionCerebral cavernous malformations (CCMs) 5The abbreviations used are: CCMcerebral cavernous malformationGCKIIIgerminal center kinase group IIISTRIPAKstriatin-interacting phosphatase and kinaseTEVtobacco etch virusSEC-MALSsize exclusion chromatography-multiangle light scatteringFATfocal adhesion targetingPP4Ccatalytic subunit of the serine/threonine protein phosphatase 4mAUmilli absorbance units. are vascular abnormalities in the brain characterized by focal dilations of cranial vasculature that can progress to hemorrhages and stroke (OMIM ID 116860). Mutations have been identified in three distinct genes, denoted CCM1–3, that are causative for the formation of most familial CCM lesions (1Labauge P. Denier C. Bergametti F. Tournier-Lasserve E. Lancet Neurol. 2007; 6: 237-244Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 2Revencu N. Vikkula M. J. Med. Genet. 2006; 43: 716-721Crossref PubMed Scopus (147) Google Scholar). CCM3, also named PDCD10, is a 212 amino acid protein conserved among metazoans (3Bergametti F. Denier C. Labauge P. Arnoult M. Boetto S. Clanet M. Coubes P. Echenne B. Ibrahim R. Irthum B. Jacquet G. Lonjon M. Moreau J.J. Neau J.P. Parker F. Tremoulet M. Tournier-Lasserve E. Am. J. Hum. Genet. 2005; 76: 42-51Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar, 4Guclu B. Ozturk A.K. Pricola K.L. Bilguvar K. Shin D. O'Roak B.J. Gunel M. Neurosurgery. 2005; 57: 1008-1013Crossref PubMed Scopus (56) Google Scholar). Knockdown of CCM3 in zebrafish causes an enlarged heart phenotype (5Zheng X. Xu C. Di Lorenzo A. Kleaveland B. Zou Z. Seiler C. Chen M. Cheng L. Xiao J. He J. Pack M.A. Sessa W.C. Kahn M.L. J. Clin. Invest. 2010; 120: 2795-2804Crossref PubMed Scopus (116) Google Scholar), whereas targeted deletion of CCM3 in the mouse results in defects of early angiogenesis and early embryonic lethality, a phenotype also observed following tissue-specific deletion in the vascular endothelium (6He Y. Zhang H. Yu L. Gunel M. Boggon T.J. Chen H. Min W. Sci. Signal. 2010; 3: ra26Crossref PubMed Scopus (141) Google Scholar). A non-cell autonomous role for CCM3 in neuroglia on the vasculature has also been uncovered in mouse recently, indicating that CCMs may arise in the central nervous system by the loss of CCM3 signaling in neural as well as endothelial populations (7Louvi A. Chen L. Two A.M. Zhang H. Min W. Günel M. Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 3737-3742Crossref PubMed Scopus (78) Google Scholar).CCM3 has been detected in complex with CCM1 and CCM2 proteins, suggesting that the three proteins may share a common biochemical function (8Hilder T.L. Malone M.H. Bencharit S. Colicelli J. Haystead T.A. Johnson G.L. Wu C.C. J. Proteome Res. 2007; 6: 4343-4355Crossref PubMed Scopus (119) Google Scholar, 9Voss K. Stahl S. Schleider E. Ullrich S. Nickel J. Mueller T.D. Felbor U. Neurogenetics. 2007; 8: 249-256Crossref PubMed Scopus (136) Google Scholar, 10Stahl S. Gaetzner S. Voss K. Brackertz B. Schleider E. Sürücü O. Kunze E. Netzer C. Korenke C. Finckh U. Habek M. Poljakovic Z. Elbracht M. Rudnik-Schöneborn S. Bertalanffy H. Sure U. Felbor U. Hum. Mut. 2008; 29: 709-717Crossref PubMed Scopus (92) Google Scholar). Yeast two-hybrid, co-immunoprecipitation, and GST pulldown experiments from cell lysates demonstrated that CCM3 also readily interacts with MST4, STK24, and STK25, a grouping of protein kinases termed the germinal center kinase class III (GCKIII) family (10Stahl S. Gaetzner S. Voss K. Brackertz B. Schleider E. Sürücü O. Kunze E. Netzer C. Korenke C. Finckh U. Habek M. Poljakovic Z. Elbracht M. Rudnik-Schöneborn S. Bertalanffy H. Sure U. Felbor U. Hum. Mut. 2008; 29: 709-717Crossref PubMed Scopus (92) Google Scholar, 11Rual J.F. Venkatesan K. Hao T. Hirozane-Kishikawa T. Dricot A. Li N. Berriz G.F. Gibbons F.D. Dreze M. Ayivi-Guedehoussou N. Klitgord N. Simon C. Boxem M. Milstein S. Rosenberg J. Goldberg D.S. Zhang L.V. Wong S.L. Franklin G. Li S. Albala J.S. Lim J. Fraughton C. Llamosas E. Cevik S. Bex C. Lamesch P. Sikorski R.S. Vandenhaute J. Zoghbi H.Y. Smolyar A. Bosak S. Sequerra R. Doucette-Stamm L. Cusick M.E. Hill D.E. Roth F.P. Vidal M. Nature. 2005; 437: 1173-1178Crossref PubMed Scopus (2259) Google Scholar, 12Ma X. Zhao H. Shan J. Long F. Chen Y. Chen Y. Zhang Y. Han X. Ma D. Mol. Biol. Cell. 2007; 18: 1965-1978Crossref PubMed Scopus (123) Google Scholar, 13Fidalgo M. Fraile M. Pires A. Force T. Pombo C. Zalvide J. J. Cell Sci. 2010; 123: 1274-1284Crossref PubMed Scopus (92) Google Scholar). CCM3 and the GCKIII proteins have also been detected as part of a large multiprotein complex termed STRIPAK (striatin-interacting phosphatase and kinase; see Refs. 14Goudreault M. D'Ambrosio L.M. Kean M.J. Mullin M.J. Larsen B.G. Sanchez A. Chaudhry S. Chen G.I. Sicheri F. Nesvizhskii A.I. Aebersold R. Raught B. Gingras A.C. Mol. Cell Proteomics. 2009; 8: 157-171Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 15Glatter T. Wepf A. Aebersold R. Gstaiger M. Mol. Syst. Biol. 2009; 5: 237Crossref PubMed Scopus (211) Google Scholar). The knockdown of GCKIII proteins in zebrafish gives rise to the same cardiovascular defects as CCM3 knockdown, suggesting the CCM3-GCKIII protein interaction is important for proper CCM3 function (5Zheng X. Xu C. Di Lorenzo A. Kleaveland B. Zou Z. Seiler C. Chen M. Cheng L. Xiao J. He J. Pack M.A. Sessa W.C. Kahn M.L. J. Clin. Invest. 2010; 120: 2795-2804Crossref PubMed Scopus (116) Google Scholar). 6B. Yoruk, B. S. Gillers, N. C. Chi, and Ian C. Scott, submitted for publication. The GCKIII proteins (STK24, STK25, and MST4) are members of the larger Sterile 20 kinase family and are characterized by highly conserved catalytic domains and a 100–120 residue carboxyl-terminal tail, whose function is not currently known. The closely related GCKII proteins MST1 and MST2 possess completely distinct C-terminal tails that mediate homotypic and heterotypic interactions (16Hwang E. Ryu K.S. Pääkkönen K. Güntert P. Cheong H.K. Lim D.S. Lee J.O. Jeon Y.H. Cheong C. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 9236-9241Crossref PubMed Scopus (105) Google Scholar), raising the possibility that an analogous function might be served by the tail region of GCKIII proteins, albeit through an unrelated structural mechanism.Crystal structures of the CCM3 protein revealed an architecture consisting of two distinct structural domains (17Li X. Zhang R. Zhang H. He Y. Ji W. Min W. Boggon T.J. J. Biol. Chem. 2010; 285: 24099-24107Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 18Ding J. Wang X. Li D.F. Hu Y. Zhang Y. Wang D.C. Biochem. Biophys. Res. Commun. 2010; 399: 587-592Crossref PubMed Scopus (17) Google Scholar). The N-terminal helical domain of CCM3 mediates homodimerization. The C-terminal four-helix bundle, termed the focal adhesion targeting (FAT) homology domain (17Li X. Zhang R. Zhang H. He Y. Ji W. Min W. Boggon T.J. J. Biol. Chem. 2010; 285: 24099-24107Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), functions as a linear peptide binding module that mediates direct interactions with CCM2, paxillin, and the striatin component of STRIPAK (17Li X. Zhang R. Zhang H. He Y. Ji W. Min W. Boggon T.J. J. Biol. Chem. 2010; 285: 24099-24107Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). 7M. J. Kean, D. F. Ceccarelli, M. Goudreault, S. Tate, B. Larsen, M. Sanches, L. C. D. Gibson, W. B. Derry, I. C. Scott, L. Pelletier, G. S. Baillie, F. Sicheri, and A.-C. Gingras, submitted for publication. Of note, the N-terminal region of CCM3 has also been implicated in the interaction with GCKIII proteins in cells and model organisms (5Zheng X. Xu C. Di Lorenzo A. Kleaveland B. Zou Z. Seiler C. Chen M. Cheng L. Xiao J. He J. Pack M.A. Sessa W.C. Kahn M.L. J. Clin. Invest. 2010; 120: 2795-2804Crossref PubMed Scopus (116) Google Scholar, 13Fidalgo M. Fraile M. Pires A. Force T. Pombo C. Zalvide J. J. Cell Sci. 2010; 123: 1274-1284Crossref PubMed Scopus (92) Google Scholar, 19Voss K. Stahl S. Hogan B.M. Reinders J. Schleider E. Schulte-Merker S. Felbor U. Hum. Mut. 2009; 30: 1003-1011Crossref PubMed Scopus (63) Google Scholar). Given the critical role for CCM3 and GCKIII proteins in maintaining vascular integrity, we have probed the basis for their interaction in close detail. The results presented here demonstrate that the amino terminus of CCM3 interacts directly with the C-terminal regions of GCKIII proteins. Based on sequence similarity between the interacting regions of CCM3 and GCKIII proteins, we propose that heterodimerization of the two proteins is achieved through an analogous structural mechanism to that reported for the homodimerization for CCM3 and present data indicating that heterodimerization may be favored over homodimerization.DISCUSSIONIn this study, we mapped the determinants of a direct interaction between CCM3 and GCKIII proteins to the N-terminal region of CCM3 and the C-terminal tail region of GCKIII proteins. These elements of both protein families are highly related in amino acid sequence, suggesting a common folded structure and binding function (Fig. 2A). Our data lead us to propose that CCM3-MST4 complex formation is achieved through the adoption of a heterodimeric helical structure analogous to that revealed by the CCM3 homodimer crystal structure (17Li X. Zhang R. Zhang H. He Y. Ji W. Min W. Boggon T.J. J. Biol. Chem. 2010; 285: 24099-24107Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). We also confirmed the existence of CCM3 homodimers in solution and that mutations within the N-terminal region of CCM3 disrupt both homodimerization and heterodimerization with purified GCKIII proteins (Fig. 4). Dimerization of STE20 family kinases mediated by conserved auxiliary domains has now been observed for GCKIII proteins (this study), the GCKII proteins (16Hwang E. Ryu K.S. Pääkkönen K. Güntert P. Cheong H.K. Lim D.S. Lee J.O. Jeon Y.H. Cheong C. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 9236-9241Crossref PubMed Scopus (105) Google Scholar), and the p21 activated kinases (23Lei M. Lu W. Meng W. Parrini M.C. Eck M.J. Mayer B.J. Harrison S.C. Cell. 2000; 102: 387-397Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar, 24Parrini M.C. Lei M. Harrison S.C. Mayer B.J. Molecular cell. 2002; 9: 73-83Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). We reason that conserved regions flanking the kinase domains of other STE20 family kinases might serve analogous interaction functions albeit through the adoption of distinct structures.The uncovered binding mode between CCM3 and GCKIII proteins helps to explain the following biological observations. Depletion of CCM3 led to the destabilization of STK25/SOK1 in cells (13Fidalgo M. Fraile M. Pires A. Force T. Pombo C. Zalvide J. J. Cell Sci. 2010; 123: 1274-1284Crossref PubMed Scopus (92) Google Scholar) demonstrating an interdependence of protein function. A mutant in exon 5 of CCM3 that results in deletion of residues 33–50 within the N-terminal region (3Bergametti F. Denier C. Labauge P. Arnoult M. Boetto S. Clanet M. Coubes P. Echenne B. Ibrahim R. Irthum B. Jacquet G. Lonjon M. Moreau J.J. Neau J.P. Parker F. Tremoulet M. Tournier-Lasserve E. Am. J. Hum. Genet. 2005; 76: 42-51Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar) failed to bind the GCKIII proteins MST4, STK24, and STK25 (5Zheng X. Xu C. Di Lorenzo A. Kleaveland B. Zou Z. Seiler C. Chen M. Cheng L. Xiao J. He J. Pack M.A. Sessa W.C. Kahn M.L. J. Clin. Invest. 2010; 120: 2795-2804Crossref PubMed Scopus (116) Google Scholar, 19Voss K. Stahl S. Hogan B.M. Reinders J. Schleider E. Schulte-Merker S. Felbor U. Hum. Mut. 2009; 30: 1003-1011Crossref PubMed Scopus (63) Google Scholar). This observation is consistent with our finding that point mutations within the N-terminal region of CCM3 disrupt binding to GCKIII proteins both in vitro and in cells (Fig. 4). Exon 5-deleted CCM3 also failed to rescue the cardiac phenotype of zebrafish, further demonstrating the biological importance of the CCM3-GCKIII protein interaction (5Zheng X. Xu C. Di Lorenzo A. Kleaveland B. Zou Z. Seiler C. Chen M. Cheng L. Xiao J. He J. Pack M.A. Sessa W.C. Kahn M.L. J. Clin. Invest. 2010; 120: 2795-2804Crossref PubMed Scopus (116) Google Scholar, 19Voss K. Stahl S. Hogan B.M. Reinders J. Schleider E. Schulte-Merker S. Felbor U. Hum. Mut. 2009; 30: 1003-1011Crossref PubMed Scopus (63) Google Scholar). Because these regions of CCM3 and GCKIII proteins mediate both hetero- and homotypic dimerization, the relative importance of each state in the etiology of CCM disease needs to be explored further.The interacting tail regions of GCKIII proteins and CCM3 are similar in sequence across the three α-helices that mediate CCM3 homodimerization (Fig. 2) (19Voss K. Stahl S. Hogan B.M. Reinders J. Schleider E. Schulte-Merker S. Felbor U. Hum. Mut. 2009; 30: 1003-1011Crossref PubMed Scopus (63) Google Scholar). Although highly similar, the observed differences (29 of 39 contact residues are not identical) likely account for the tendency of CCM3 and MST4 kinases to preferentially heterodimerize versus homodimerize. Because the dimerization-mediating tails of the two other GCKIII proteins, STK24 and STK25, are more similar to MST4 than to CCM3 (Fig. 2A), we predict that they too will preferentially heterodimerize with CCM3. This, however, remains to be tested region of GCKIII proteins is unrelated in sequence to the C-terminal domain of GCKII kinase an that not with CCM3 (Fig. with the of CCM3 and GCKIII proteins not other proteins in the that might with CCM3 GCKIII through a related structural the regions of CCM3 and GCKIII proteins are well conserved in more as and the the most related of to (Fig. N. M.J. S.L. A. M. J. S. U. Y. I. M. D. N. A. R. W. M. D. D. Li W. A. S. A. P. Lim G. W. H. R. D. Nature. 2008; PubMed Scopus Google Scholar). This conserved of CCM3 and GCKIII proteins further the of their observed A kinase in the however, a CCM3 has not been identified in this suggesting that GCKIII proteins may functions of a CCM3 IntroductionCerebral cavernous malformations (CCMs) 5The abbreviations used are: CCMcerebral cavernous malformationGCKIIIgerminal center kinase group IIISTRIPAKstriatin-interacting phosphatase and kinaseTEVtobacco etch virusSEC-MALSsize exclusion chromatography-multiangle light scatteringFATfocal adhesion targetingPP4Ccatalytic subunit of the serine/threonine protein phosphatase 4mAUmilli absorbance units. are vascular abnormalities in the brain characterized by focal dilations of cranial vasculature that can progress to hemorrhages and stroke (OMIM ID 116860). Mutations have been identified in three distinct genes, denoted CCM1–3, that are causative for the formation of most familial CCM lesions (1Labauge P. Denier C. Bergametti F. Tournier-Lasserve E. Lancet Neurol. 2007; 6: 237-244Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 2Revencu N. Vikkula M. J. Med. Genet. 2006; 43: 716-721Crossref PubMed Scopus (147) Google Scholar). CCM3, also named PDCD10, is a 212 amino acid protein conserved among metazoans (3Bergametti F. Denier C. Labauge P. Arnoult M. Boetto S. Clanet M. Coubes P. Echenne B. Ibrahim R. Irthum B. Jacquet G. Lonjon M. Moreau J.J. Neau J.P. Parker F. Tremoulet M. Tournier-Lasserve E. Am. J. Hum. Genet. 2005; 76: 42-51Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar, 4Guclu B. Ozturk A.K. Pricola K.L. Bilguvar K. Shin D. O'Roak B.J. Gunel M. Neurosurgery. 2005; 57: 1008-1013Crossref PubMed Scopus (56) Google Scholar). Knockdown of CCM3 in zebrafish causes an enlarged heart phenotype (5Zheng X. Xu C. Di Lorenzo A. Kleaveland B. Zou Z. Seiler C. Chen M. Cheng L. Xiao J. He J. Pack M.A. Sessa W.C. Kahn M.L. J. Clin. Invest. 2010; 120: 2795-2804Crossref PubMed Scopus (116) Google Scholar), whereas targeted deletion of CCM3 in the mouse results in defects of early angiogenesis and early embryonic lethality, a phenotype also observed following tissue-specific deletion in the vascular endothelium (6He Y. Zhang H. Yu L. Gunel M. Boggon T.J. Chen H. Min W. Sci. Signal. 2010; 3: ra26Crossref PubMed Scopus (141) Google Scholar). A non-cell autonomous role for CCM3 in neuroglia on the vasculature has also been uncovered in mouse recently, indicating that CCMs may arise in the central nervous system by the loss of CCM3 signaling in neural as well as endothelial populations (7Louvi A. Chen L. Two A.M. Zhang H. Min W. Günel M. Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 3737-3742Crossref PubMed Scopus (78) Google Scholar).CCM3 has been detected in complex with CCM1 and CCM2 proteins, suggesting that the three proteins may share a common biochemical function (8Hilder T.L. Malone M.H. Bencharit S. Colicelli J. Haystead T.A. Johnson G.L. Wu C.C. J. Proteome Res. 2007; 6: 4343-4355Crossref PubMed Scopus (119) Google Scholar, 9Voss K. Stahl S. Schleider E. Ullrich S. Nickel J. Mueller T.D. Felbor U. Neurogenetics. 2007; 8: 249-256Crossref PubMed Scopus (136) Google Scholar, 10Stahl S. Gaetzner S. Voss K. Brackertz B. Schleider E. Sürücü O. Kunze E. Netzer C. Korenke C. Finckh U. Habek M. Poljakovic Z. Elbracht M. Rudnik-Schöneborn S. Bertalanffy H. Sure U. Felbor U. Hum. Mut. 2008; 29: 709-717Crossref PubMed Scopus (92) Google Scholar). Yeast two-hybrid, co-immunoprecipitation, and GST pulldown experiments from cell lysates demonstrated that CCM3 also readily interacts with MST4, STK24, and STK25, a grouping of protein kinases termed the germinal center kinase class III (GCKIII) family (10Stahl S. Gaetzner S. Voss K. Brackertz B. Schleider E. Sürücü O. Kunze E. Netzer C. Korenke C. Finckh U. Habek M. Poljakovic Z. Elbracht M. Rudnik-Schöneborn S. Bertalanffy H. Sure U. Felbor U. Hum. Mut. 2008; 29: 709-717Crossref PubMed Scopus (92) Google Scholar, 11Rual J.F. Venkatesan K. Hao T. Hirozane-Kishikawa T. Dricot A. Li N. Berriz G.F. Gibbons F.D. Dreze M. Ayivi-Guedehoussou N. Klitgord N. Simon C. Boxem M. Milstein S. Rosenberg J. Goldberg D.S. Zhang L.V. Wong S.L. Franklin G. Li S. Albala J.S. Lim J. Fraughton C. Llamosas E. Cevik S. Bex C. Lamesch P. Sikorski R.S. Vandenhaute J. Zoghbi H.Y. Smolyar A. Bosak S. Sequerra R. Doucette-Stamm L. Cusick M.E. Hill D.E. Roth F.P. Vidal M. Nature. 2005; 437: 1173-1178Crossref PubMed Scopus (2259) Google Scholar, 12Ma X. Zhao H. Shan J. Long F. Chen Y. Chen Y. Zhang Y. Han X. Ma D. Mol. Biol. Cell. 2007; 18: 1965-1978Crossref PubMed Scopus (123) Google Scholar, 13Fidalgo M. Fraile M. Pires A. Force T. Pombo C. Zalvide J. J. Cell Sci. 2010; 123: 1274-1284Crossref PubMed Scopus (92) Google Scholar). CCM3 and the GCKIII proteins have also been detected as part of a large multiprotein complex termed STRIPAK (striatin-interacting phosphatase and kinase; see Refs. 14Goudreault M. D'Ambrosio L.M. Kean M.J. Mullin M.J. Larsen B.G. Sanchez A. Chaudhry S. Chen G.I. Sicheri F. Nesvizhskii A.I. Aebersold R. Raught B. Gingras A.C. Mol. Cell Proteomics. 2009; 8: 157-171Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 15Glatter T. Wepf A. Aebersold R. Gstaiger M. Mol. Syst. Biol. 2009; 5: 237Crossref PubMed Scopus (211) Google Scholar). The knockdown of GCKIII proteins in zebrafish gives rise to the same cardiovascular defects as CCM3 knockdown, suggesting the CCM3-GCKIII protein interaction is important for proper CCM3 function (5Zheng X. Xu C. Di Lorenzo A. Kleaveland B. Zou Z. Seiler C. Chen M. Cheng L. Xiao J. He J. Pack M.A. Sessa W.C. Kahn M.L. J. Clin. Invest. 2010; 120: 2795-2804Crossref PubMed Scopus (116) Google Scholar). 6B. Yoruk, B. S. Gillers, N. C. Chi, and Ian C. Scott, submitted for publication. The GCKIII proteins (STK24, STK25, and MST4) are members of the larger Sterile 20 kinase family and are characterized by highly conserved catalytic domains and a 100–120 residue carboxyl-terminal tail, whose function is not currently known. The closely related GCKII proteins MST1 and MST2 possess completely distinct C-terminal tails that mediate homotypic and heterotypic interactions (16Hwang E. Ryu K.S. Pääkkönen K. Güntert P. Cheong H.K. Lim D.S. Lee J.O. Jeon Y.H. Cheong C. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 9236-9241Crossref PubMed Scopus (105) Google Scholar), raising the possibility that an analogous function might be served by the tail region of GCKIII proteins, albeit through an unrelated structural mechanism.Crystal structures of the CCM3 protein revealed an architecture consisting of two distinct structural domains (17Li X. Zhang R. Zhang H. He Y. Ji W. Min W. Boggon T.J. J. Biol. Chem. 2010; 285: 24099-24107Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 18Ding J. Wang X. Li D.F. Hu Y. Zhang Y. Wang D.C. Biochem. Biophys. Res. Commun. 2010; 399: 587-592Crossref PubMed Scopus (17) Google Scholar). The N-terminal helical domain of CCM3 mediates homodimerization. The C-terminal four-helix bundle, termed the focal adhesion targeting (FAT) homology domain (17Li X. Zhang R. Zhang H. He Y. Ji W. Min W. Boggon T.J. J. Biol. Chem. 2010; 285: 24099-24107Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), functions as a linear peptide binding module that mediates direct interactions with CCM2, paxillin, and the striatin component of STRIPAK (17Li X. Zhang R. Zhang H. He Y. Ji W. Min W. Boggon T.J. J. Biol. Chem. 2010; 285: 24099-24107Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). 7M. J. Kean, D. F. Ceccarelli, M. Goudreault, S. Tate, B. Larsen, M. Sanches, L. C. D. Gibson, W. B. Derry, I. C. Scott, L. Pelletier, G. S. Baillie, F. Sicheri, and A.-C. Gingras, submitted for publication. Of note, the N-terminal region of CCM3 has also been implicated in the interaction with GCKIII proteins in cells and model organisms (5Zheng X. Xu C. Di Lorenzo A. Kleaveland B. Zou Z. Seiler C. Chen M. Cheng L. Xiao J. He J. Pack M.A. Sessa W.C. Kahn M.L. J. Clin. Invest. 2010; 120: 2795-2804Crossref PubMed Scopus (116) Google Scholar, 13Fidalgo M. Fraile M. Pires A. Force T. Pombo C. Zalvide J. J. Cell Sci. 2010; 123: 1274-1284Crossref PubMed Scopus (92) Google Scholar, 19Voss K. Stahl S. Hogan B.M. Reinders J. Schleider E. Schulte-Merker S. Felbor U. Hum. Mut. 2009; 30: 1003-1011Crossref PubMed Scopus (63) Google Scholar). Given the critical role for CCM3 and GCKIII proteins in maintaining vascular integrity, we have probed the basis for their interaction in close detail. The results presented here demonstrate that the amino terminus of CCM3 interacts directly with the C-terminal regions of GCKIII proteins. Based on sequence similarity between the interacting regions of CCM3 and GCKIII proteins, we propose that heterodimerization of the two proteins is achieved through an analogous structural mechanism to that reported for the homodimerization for CCM3 and present data indicating that heterodimerization may be favored over homodimerization.

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