CCM2-CCM3 interaction stabilizes their protein expression and permits endothelial network formation.
Bottom Line: 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.However, CCM3 expression in the absence of CCM2 is sufficient to support normal cell growth, revealing complex-independent roles for CCM3.
Affiliation: Department of Pharmacology and Department of Cell Biology, Yale University, New Haven, CT 06520.Show MeSH
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Mentions: To validate the crystallographically observed interfaces, we introduced point mutations that we expected would interrupt the interactions between CCM3 and CCM2. We then probed the impact of these mutations using our pull-down assay followed by Western blot analysis. We began by generating a quadruple lysine–to–glutamic acid mutation in the FAT-H domain of CCM3 (K132E, K139E, K172E, K179E), termed CCM34KE (Fig. S1, C and D). We previously showed that charge reversal for these four exquisitely conserved lysines interrupts CCM3 interactions with both paxillin (Li et al., 2011) and CCM2 (Li et al., 2010). To confirm this using the current system, we compared CCM3 and CCM34KE binding to GST fusions of CCM2FL-, CCM2PTB-LL-, CCM2PTB-SL-, or CCM2LD-bound to beads. We found that in all cases the amount of CCM34KE binding to beads approximated to background, clearly indicating that mutants of these conserved lysines interrupt CCM3’s interaction with CCM2 (Fig. 3, A and B).
Affiliation: Department of Pharmacology and Department of Cell Biology, Yale University, New Haven, CT 06520.