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CCM2-CCM3 interaction stabilizes their protein expression and permits endothelial network formation.

Draheim KM, Li X, Zhang R, Fisher OS, Villari G, Boggon TJ, Calderwood DA - J. Cell Biol. (2015)

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.

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Affiliation: Department of Pharmacology and Department of Cell Biology, Yale University, New Haven, CT 06520.

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Biochemical analysis of the interaction between CCM3 and CCM2. (A and B) Pull-down of 6×His-CCM3 or 6×His-CCM34KE by GST fusion CCM2 constructs. Pull-down was probed by immunoblotting (A) and quantified (mean ± SEM, error bars; n = 3) as a percentage of CCM3 that binds to each CCM2 construct (B). (C and D) Pull-down of 6×His-CCM3 by mutated GST-CCM2LD. Pull-down was probed by immunoblotting (C) and quantified (mean ± SEM, error bars; n = 4) as a percentage of CCM3 that binds to wild-type CCM2LD construct (D). (E and F) Pull-down of 6×His-CCM3 by mutated GST fusion CCM2 constructs. Pull-down was probed by Western blotting (E) and quantified (mean ± SEM, error bars; n = 4) as a percentage of CCM3 that binds to each wild-type CCM2 construct (F). (G) Pull-down of heterologously expressed GFP-CCM2 by purified GST-CCM3. GFP-fusions of either full-length CCM2 or the CCM2 LD–like motif expressed in HEK 293T cells were pulled down by either GST or GST-CCM3 immobilized on beads. 3.6% input is shown. (H) Quantification of pull-downs of CCM2 LD–like motif constructs shown in G (mean ± SEM, error bars; n = 3) as a percentage of GFP-CCM2LD that binds to GST-CCM3. (I) Quantification of pull-downs of full-length CCM2 constructs shown in G (mean ± SEM, error bars; n = 3) as a percentage of GFP-CCM2FL that binds to GST-CCM3.
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fig3: Biochemical analysis of the interaction between CCM3 and CCM2. (A and B) Pull-down of 6×His-CCM3 or 6×His-CCM34KE by GST fusion CCM2 constructs. Pull-down was probed by immunoblotting (A) and quantified (mean ± SEM, error bars; n = 3) as a percentage of CCM3 that binds to each CCM2 construct (B). (C and D) Pull-down of 6×His-CCM3 by mutated GST-CCM2LD. Pull-down was probed by immunoblotting (C) and quantified (mean ± SEM, error bars; n = 4) as a percentage of CCM3 that binds to wild-type CCM2LD construct (D). (E and F) Pull-down of 6×His-CCM3 by mutated GST fusion CCM2 constructs. Pull-down was probed by Western blotting (E) and quantified (mean ± SEM, error bars; n = 4) as a percentage of CCM3 that binds to each wild-type CCM2 construct (F). (G) Pull-down of heterologously expressed GFP-CCM2 by purified GST-CCM3. GFP-fusions of either full-length CCM2 or the CCM2 LD–like motif expressed in HEK 293T cells were pulled down by either GST or GST-CCM3 immobilized on beads. 3.6% input is shown. (H) Quantification of pull-downs of CCM2 LD–like motif constructs shown in G (mean ± SEM, error bars; n = 3) as a percentage of GFP-CCM2LD that binds to GST-CCM3. (I) Quantification of pull-downs of full-length CCM2 constructs shown in G (mean ± SEM, error bars; n = 3) as a percentage of GFP-CCM2FL that binds to GST-CCM3.

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).


CCM2-CCM3 interaction stabilizes their protein expression and permits endothelial network formation.

Draheim KM, Li X, Zhang R, Fisher OS, Villari G, Boggon TJ, Calderwood DA - J. Cell Biol. (2015)

Biochemical analysis of the interaction between CCM3 and CCM2. (A and B) Pull-down of 6×His-CCM3 or 6×His-CCM34KE by GST fusion CCM2 constructs. Pull-down was probed by immunoblotting (A) and quantified (mean ± SEM, error bars; n = 3) as a percentage of CCM3 that binds to each CCM2 construct (B). (C and D) Pull-down of 6×His-CCM3 by mutated GST-CCM2LD. Pull-down was probed by immunoblotting (C) and quantified (mean ± SEM, error bars; n = 4) as a percentage of CCM3 that binds to wild-type CCM2LD construct (D). (E and F) Pull-down of 6×His-CCM3 by mutated GST fusion CCM2 constructs. Pull-down was probed by Western blotting (E) and quantified (mean ± SEM, error bars; n = 4) as a percentage of CCM3 that binds to each wild-type CCM2 construct (F). (G) Pull-down of heterologously expressed GFP-CCM2 by purified GST-CCM3. GFP-fusions of either full-length CCM2 or the CCM2 LD–like motif expressed in HEK 293T cells were pulled down by either GST or GST-CCM3 immobilized on beads. 3.6% input is shown. (H) Quantification of pull-downs of CCM2 LD–like motif constructs shown in G (mean ± SEM, error bars; n = 3) as a percentage of GFP-CCM2LD that binds to GST-CCM3. (I) Quantification of pull-downs of full-length CCM2 constructs shown in G (mean ± SEM, error bars; n = 3) as a percentage of GFP-CCM2FL that binds to GST-CCM3.
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fig3: Biochemical analysis of the interaction between CCM3 and CCM2. (A and B) Pull-down of 6×His-CCM3 or 6×His-CCM34KE by GST fusion CCM2 constructs. Pull-down was probed by immunoblotting (A) and quantified (mean ± SEM, error bars; n = 3) as a percentage of CCM3 that binds to each CCM2 construct (B). (C and D) Pull-down of 6×His-CCM3 by mutated GST-CCM2LD. Pull-down was probed by immunoblotting (C) and quantified (mean ± SEM, error bars; n = 4) as a percentage of CCM3 that binds to wild-type CCM2LD construct (D). (E and F) Pull-down of 6×His-CCM3 by mutated GST fusion CCM2 constructs. Pull-down was probed by Western blotting (E) and quantified (mean ± SEM, error bars; n = 4) as a percentage of CCM3 that binds to each wild-type CCM2 construct (F). (G) Pull-down of heterologously expressed GFP-CCM2 by purified GST-CCM3. GFP-fusions of either full-length CCM2 or the CCM2 LD–like motif expressed in HEK 293T cells were pulled down by either GST or GST-CCM3 immobilized on beads. 3.6% input is shown. (H) Quantification of pull-downs of CCM2 LD–like motif constructs shown in G (mean ± SEM, error bars; n = 3) as a percentage of GFP-CCM2LD that binds to GST-CCM3. (I) Quantification of pull-downs of full-length CCM2 constructs shown in G (mean ± SEM, error bars; n = 3) as a percentage of GFP-CCM2FL that binds to GST-CCM3.
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).

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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology and Department of Cell Biology, Yale University, New Haven, CT 06520.

Show MeSH
Related in: MedlinePlus