title: CCM2-CCM3 interaction stabilizes their protein expression and permits endothelial network formation.
abstract: Mutations in the essential adaptor proteins CCM2 or CCM3 lead to cerebral cavernous malformations (CCM), vascular lesions that most frequently occur in the brain and are strongly associated with hemorrhagic stroke, seizures, and other neurological disorders. CCM2 binds CCM3, but the molecular basis of this interaction, and its functional significance, have not been elucidated. Here, we used x-ray crystallography and structure-guided mutagenesis to show that an α-helical LD-like motif within CCM2 binds the highly conserved "HP1" pocket of the CCM3 focal adhesion targeting (FAT) homology domain. By knocking down CCM2 or CCM3 and rescuing with binding-deficient mutants, we establish that CCM2-CCM3 interactions protect CCM2 and CCM3 proteins from proteasomal degradation and show that both CCM2 and CCM3 are required for normal endothelial cell network formation. However, CCM3 expression in the absence of CCM2 is sufficient to support normal cell growth, revealing complex-independent roles for CCM3.
authors.affiliation: Department of Pharmacology and Department of Cell Biology, Yale University, New Haven, CT 06520.
authors.forename: Kyle M
authors.initials: KM
authors.lastname: Draheim
authors.affiliation: Department of Pharmacology and Department of Cell Biology, Yale University, New Haven, CT 06520.
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Li
authors.affiliation: Department of Pharmacology and Department of Cell Biology, Yale University, New Haven, CT 06520.
authors.forename: Rong
authors.initials: R
authors.lastname: Zhang
authors.affiliation: Department of Pharmacology and Department of Cell Biology, Yale University, New Haven, CT 06520.
authors.forename: Oriana S
authors.initials: OS
authors.lastname: Fisher
authors.affiliation: Department of Pharmacology and Department of Cell Biology, Yale University, New Haven, CT 06520.
authors.forename: Giulia
authors.initials: G
authors.lastname: Villari
authors.affiliation: Department of Pharmacology and Department of Cell Biology, Yale University, New Haven, CT 06520 titus.boggon@yale.edu david.calderwood@yale.edu.
authors.forename: Titus J
authors.initials: TJ
authors.lastname: Boggon
authors.affiliation: Department of Pharmacology and Department of Cell Biology, Yale University, New Haven, CT 06520 Department of Pharmacology and Department of Cell Biology, Yale University, New Haven, CT 06520 titus.boggon@yale.edu david.calderwood@yale.edu.
authors.forename: David A
authors.initials: DA
authors.lastname: Calderwood
mesh_terms: D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D002460:Cell Line; D049109:Cell Proliferation / Q000235:genetics*; D002490:Central Nervous System / Q000098:blood supply*; D018360:Crystallography, X-Ray; D015870:Gene Expression; D020786:Hemangioma, Cavernous, Central Nervous System / Q000235:genetics; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D016296:Mutagenesis; D018919:Neovascularization, Physiologic / Q000235:genetics*; D051419:Paxillin / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D034622:RNA Interference; D034741:RNA, Small Interfering; D016415:Sequence Alignment
chemical_list: D051017:Apoptosis Regulatory Proteins; C502544:CCM2 protein, human; D002352:Carrier Proteins; D008565:Membrane Proteins; C495390:PDCD10 protein, human; D051419:Paxillin; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering
references.citation: Angiogenesis. 2001;4(2):91-102
references.pmid: 11806248
references.citation: Mol Cell Biol. 2003 Mar;23(5):1750-63
references.pmid: 12588993
references.citation: Structure. 2003 Oct;11(10):1207-17
references.pmid: 14527389
references.citation: Am J Hum Genet. 2003 Dec;73(6):1459-64
references.pmid: 14624391
references.citation: Indian J Cancer. 2004 Jan-Mar;41(1):47
references.pmid: 15105580
references.citation: Nucleic Acids Res. 2004 Jul 1;32(Web Server issue):W615-9
references.pmid: 15215462
references.citation: Acta Crystallogr D Biol Crystallogr. 1994 Sep 1;50(Pt 5):760-3
references.pmid: 15299374
references.citation: Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
references.pmid: 15572765
references.citation: Mol Cell. 2006 Feb 3;21(3):337-47
references.pmid: 16455489
references.citation: Neurogenetics. 2007 Nov;8(4):249-56
references.pmid: 17657516
references.citation: J Mol Biol. 2007 Sep 21;372(3):774-97
references.pmid: 17681537
references.citation: Nucleic Acids Res. 2008 May;36(9):e54
references.pmid: 18424799
references.citation: Hum Mol Genet. 2008 Aug 15;17(16):2424-32
references.pmid: 18469344
references.citation: Mol Cell Proteomics. 2009 Jan;8(1):157-71
references.pmid: 18782753
references.citation: Biochem Biophys Res Commun. 2009 Jun 5;383(3):347-52
references.pmid: 19358827
references.citation: Acta Crystallogr D Biol Crystallogr. 2009 May;65(Pt 5):510-2
references.pmid: 19390156
references.citation: J Appl Crystallogr. 2007 Aug 1;40(Pt 4):658-674
references.pmid: 19461840
references.citation: FEBS J. 2010 Mar;277(5):1076-83
references.pmid: 20096037
references.citation: Acta Crystallogr D Biol Crystallogr. 2010 Feb;66(Pt 2):213-21
references.pmid: 20124702
references.citation: J Biol Chem. 2010 Apr 16;285(16):11760-4
references.pmid: 20181950
references.citation: J Biol Chem. 2010 Jul 30;285(31):24099-107
references.pmid: 20489202
references.citation: Proc Natl Acad Sci U S A. 2010 Jul 13;107(28):12640-5
references.pmid: 20616044
references.citation: Acta Crystallogr D Biol Crystallogr. 2011 Apr;67(Pt 4):355-67
references.pmid: 21460454
references.citation: Acta Crystallogr D Biol Crystallogr. 2011 Apr;67(Pt 4):386-94
references.pmid: 21460457
references.citation: J Biol Chem. 2011 Jul 15;286(28):25065-75
references.pmid: 21561862
references.citation: J Biol Chem. 2011 Jul 22;286(29):26138-47
references.pmid: 21632544
references.citation: BMC Biochem. 2011 Oct 10;12:54
references.pmid: 21985334
references.citation: J Biol Chem. 2012 Mar 30;287(14):11556-65
references.pmid: 22291017
references.citation: Dev Cell. 2012 Aug 14;23(2):342-55
references.pmid: 22898778
references.citation: FEBS Lett. 2013 Jan 31;587(3):272-7
references.pmid: 23266514
references.citation: Mol Syndromol. 2013 Apr;4(4):165-72
references.pmid: 23801932
references.citation: Biochem J. 2013 Aug 15;454(1):13-30
references.pmid: 23889253
references.citation: J Cell Biol. 2013 Aug 5;202(3):545-61
references.pmid: 23918940
references.citation: Cell Mol Life Sci. 2014 May;71(10):1881-92
references.pmid: 24287896
references.citation: J Cell Sci. 2014 Feb 15;127(Pt 4):701-7
references.pmid: 24481819
references.citation: Biochem J. 2014 Jun 15;460(3):317-29
references.pmid: 24870021
references.citation: J Biol Chem. 2015 Jan 30;290(5):2842-53
references.pmid: 25525273
references.citation: Methods Enzymol. 1997;276:307-26
references.pmid: 27754618
references.citation: Biochem Biophys Res Commun. 1998 Aug 10;249(1):118-23
references.pmid: 9705842