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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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CCM3 is required for cell growth. (A) Stable CCM2 or CCM3 knockdown cells were evaluated for 5 d in a cell growth assay and compared with uninfected/parental cells (Ø) and cells infected with a virus expressing a scrambled hairpin (shSCR). Bar graphs represents the mean cell number ± SEM (error bars) from at least nine experiments. (B) The effects of expressing GFP, or wild-type or mutant GFP-CCM2 and GFP-CCM3 in shSCR, shCCM2 #1, or shCCM3 #2 EA.hy926 cells were evaluated in a 5-d cell growth assay. Bar graphs represent the mean cell number ± SEM (error bars) from at least nine experiments. (C and D) Immunoblotting was used to verify the expression of the exogenous and endogenous CCM2 and CCM3 proteins in CCM2 knockdown (C) and CCM3 knockdown (D) cells.
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fig5: CCM3 is required for cell growth. (A) Stable CCM2 or CCM3 knockdown cells were evaluated for 5 d in a cell growth assay and compared with uninfected/parental cells (Ø) and cells infected with a virus expressing a scrambled hairpin (shSCR). Bar graphs represents the mean cell number ± SEM (error bars) from at least nine experiments. (B) The effects of expressing GFP, or wild-type or mutant GFP-CCM2 and GFP-CCM3 in shSCR, shCCM2 #1, or shCCM3 #2 EA.hy926 cells were evaluated in a 5-d cell growth assay. Bar graphs represent the mean cell number ± SEM (error bars) from at least nine experiments. (C and D) Immunoblotting was used to verify the expression of the exogenous and endogenous CCM2 and CCM3 proteins in CCM2 knockdown (C) and CCM3 knockdown (D) cells.

Mentions: When generating the CCM2 and CCM3 knockdown cells used in Fig. 4, we observed that knockdown cultures expanded more slowly than parental EA.hy926 cells or control shRNA–expressing cells. Careful assessment over 5 d of culture clearly established that knockdown of CCM2 or CCM3 strongly impaired the increase in cell number (Fig. 5 A). This effect was evident for both knockdown constructs of each gene and appeared to be strongest with the more efficient knockdown constructs. Re-expression of wild-type but not mutant CCM2 or CCM3 restored cell numbers in CCM2 and CCM3 knockdown cells, respectively (Fig. 5 B). These data suggested that a CCM2–CCM3 complex is important for regulating cell number, either through effects on cell proliferation or on cell survival. However, whether formation of a CCM2–CCM3 complex directly contributes to the process, or is important only because it stabilizes protein expression, was not clear. We therefore performed additional GFP-CCM expression experiments in knockdown cells. Although CCM2–CCM3 complex formation is clearly normally important in regulating protein levels, partner binding is not absolutely essential for protein expression because overexpressed binding-defective GFP-CCM mutants can be detected (Fig. 4 F). We therefore expressed GFP-CCM2 in CCM3 knockdown cells and assessed cell numbers. CCM3 knockdown cells have very little CCM3 and exhibit reduced levels of CCM2. Reconstitution with GFP-CCM2 increased total CCM2 levels without rescuing endogenous CCM3 levels (Fig. 5 C) but failed to increase cell growth (Fig. 5 B). Indeed, overexpressing CCM2 in the absence of CCM3 further reduced cell numbers below those seen in CCM3 knockdown cells (Fig. 5 B). Thus, CCM2 expression in the absence of CCM3 was not sufficient to permit normal expansion of cell number. However, when we overexpressed GFP-CCM3 in CCM2 knockdown cells, it was sufficient to largely rescue the cell number phenotype (Fig. 5 B), without rescuing the expression of endogenous CCM2 (Fig. 5 D). Consistent with the inability of GFP-CCM34KE to rescue defects in CCM3 knockdown cells (Fig. 5 B), expressing GFP-CCM34KE also failed to rescue cell numbers in CCM2 knockdown cells (Fig. S3 A). These data clearly implicate CCM3 in the control of cell numbers. Expression of GFP-CCM2LI/RR in CCM2 knockdown cells is presumably unable to rescue cell numbers because it cannot restore CCM3 expression levels (Fig. 4 F). We suggest that the inability of GFP-CCM34KE to restore growth of CCM3 knockdown cells is not due to its inability to bind and stabilize CCM2, but instead is because it disrupts CCM3 binding to other partner proteins, such as striatin or paxillin (Kean et al., 2011; Li et al., 2011). Consistent with this suggestion, paxillin knockdown EA.hy926 cells exhibit growth inhibition comparable to that seen in CCM3 knockdown cells, whereas striatin 1 knockdown cells show an even more profound growth defect (Fig. S3, B and C). CCM3 expression is not required to maintain paxillin or striatin expression levels, and CCM3 levels are normal in paxillin and striatin knockdown cells (Fig. S3, B and D). Future work will address the question of whether the CCM3–paxillin and CCM3–striatin interactions contribute to their effects on cell number. Nonetheless, we conclude that loss of CCM3 results in cell proliferation defects and that, even in the absence of CCM2, forced CCM3 expression restores cell numbers.


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)

CCM3 is required for cell growth. (A) Stable CCM2 or CCM3 knockdown cells were evaluated for 5 d in a cell growth assay and compared with uninfected/parental cells (Ø) and cells infected with a virus expressing a scrambled hairpin (shSCR). Bar graphs represents the mean cell number ± SEM (error bars) from at least nine experiments. (B) The effects of expressing GFP, or wild-type or mutant GFP-CCM2 and GFP-CCM3 in shSCR, shCCM2 #1, or shCCM3 #2 EA.hy926 cells were evaluated in a 5-d cell growth assay. Bar graphs represent the mean cell number ± SEM (error bars) from at least nine experiments. (C and D) Immunoblotting was used to verify the expression of the exogenous and endogenous CCM2 and CCM3 proteins in CCM2 knockdown (C) and CCM3 knockdown (D) cells.
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fig5: CCM3 is required for cell growth. (A) Stable CCM2 or CCM3 knockdown cells were evaluated for 5 d in a cell growth assay and compared with uninfected/parental cells (Ø) and cells infected with a virus expressing a scrambled hairpin (shSCR). Bar graphs represents the mean cell number ± SEM (error bars) from at least nine experiments. (B) The effects of expressing GFP, or wild-type or mutant GFP-CCM2 and GFP-CCM3 in shSCR, shCCM2 #1, or shCCM3 #2 EA.hy926 cells were evaluated in a 5-d cell growth assay. Bar graphs represent the mean cell number ± SEM (error bars) from at least nine experiments. (C and D) Immunoblotting was used to verify the expression of the exogenous and endogenous CCM2 and CCM3 proteins in CCM2 knockdown (C) and CCM3 knockdown (D) cells.
Mentions: When generating the CCM2 and CCM3 knockdown cells used in Fig. 4, we observed that knockdown cultures expanded more slowly than parental EA.hy926 cells or control shRNA–expressing cells. Careful assessment over 5 d of culture clearly established that knockdown of CCM2 or CCM3 strongly impaired the increase in cell number (Fig. 5 A). This effect was evident for both knockdown constructs of each gene and appeared to be strongest with the more efficient knockdown constructs. Re-expression of wild-type but not mutant CCM2 or CCM3 restored cell numbers in CCM2 and CCM3 knockdown cells, respectively (Fig. 5 B). These data suggested that a CCM2–CCM3 complex is important for regulating cell number, either through effects on cell proliferation or on cell survival. However, whether formation of a CCM2–CCM3 complex directly contributes to the process, or is important only because it stabilizes protein expression, was not clear. We therefore performed additional GFP-CCM expression experiments in knockdown cells. Although CCM2–CCM3 complex formation is clearly normally important in regulating protein levels, partner binding is not absolutely essential for protein expression because overexpressed binding-defective GFP-CCM mutants can be detected (Fig. 4 F). We therefore expressed GFP-CCM2 in CCM3 knockdown cells and assessed cell numbers. CCM3 knockdown cells have very little CCM3 and exhibit reduced levels of CCM2. Reconstitution with GFP-CCM2 increased total CCM2 levels without rescuing endogenous CCM3 levels (Fig. 5 C) but failed to increase cell growth (Fig. 5 B). Indeed, overexpressing CCM2 in the absence of CCM3 further reduced cell numbers below those seen in CCM3 knockdown cells (Fig. 5 B). Thus, CCM2 expression in the absence of CCM3 was not sufficient to permit normal expansion of cell number. However, when we overexpressed GFP-CCM3 in CCM2 knockdown cells, it was sufficient to largely rescue the cell number phenotype (Fig. 5 B), without rescuing the expression of endogenous CCM2 (Fig. 5 D). Consistent with the inability of GFP-CCM34KE to rescue defects in CCM3 knockdown cells (Fig. 5 B), expressing GFP-CCM34KE also failed to rescue cell numbers in CCM2 knockdown cells (Fig. S3 A). These data clearly implicate CCM3 in the control of cell numbers. Expression of GFP-CCM2LI/RR in CCM2 knockdown cells is presumably unable to rescue cell numbers because it cannot restore CCM3 expression levels (Fig. 4 F). We suggest that the inability of GFP-CCM34KE to restore growth of CCM3 knockdown cells is not due to its inability to bind and stabilize CCM2, but instead is because it disrupts CCM3 binding to other partner proteins, such as striatin or paxillin (Kean et al., 2011; Li et al., 2011). Consistent with this suggestion, paxillin knockdown EA.hy926 cells exhibit growth inhibition comparable to that seen in CCM3 knockdown cells, whereas striatin 1 knockdown cells show an even more profound growth defect (Fig. S3, B and C). CCM3 expression is not required to maintain paxillin or striatin expression levels, and CCM3 levels are normal in paxillin and striatin knockdown cells (Fig. S3, B and D). Future work will address the question of whether the CCM3–paxillin and CCM3–striatin interactions contribute to their effects on cell number. Nonetheless, we conclude that loss of CCM3 results in cell proliferation defects and that, even in the absence of CCM2, forced CCM3 expression restores cell numbers.

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