CCM1 and CCM2 protein interactions in cell signaling: implications for cerebral cavernous malformations pathogenesis

Zawistowski, Jon S.; Stalheim, Lisa; Uhlik, Mark; Abell, Amy N.; Ancrile, Brooke B.; Johnson, Gary L.; Marchuk, Douglas A.

doi:10.1093/hmg/ddi256

Cited by 234 publications

(291 citation statements)

References 29 publications

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“…CCM2 Localizes Smurf1 by Binding the HECT Domain-We have shown that CCM2 regulates localization of the MEKK3-MKK3-p38 signaling module and localization of KRIT1 (1,13). Therefore, we hypothesized that CCM2 regulates Smurf1 localization.…”

Section: Ccm2 Is Not a Smurf1 Substrate Nor Does It Affect Smurf1mentioning

confidence: 97%

Cerebral Cavernous Malformation 2 Protein Promotes Smad Ubiquitin Regulatory Factor 1-mediated RhoA Degradation in Endothelial Cells

Crose

Hilder

Sciaky

et al. 2009

Journal of Biological Chemistry

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Mutation of CCM2 predisposes individuals to cerebral cavernous malformations, vascular abnormalities that cause seizures and hemorrhagic stroke. CCM2 has been proposed to regulate the activity of RhoA for maintenance of vascular integrity. Herein, we define a novel mechanism where the CCM2 phosphotyrosine binding (PTB) domain binds the ubiquitin ligase (E3) Smurf1, controlling RhoA degradation. Brain endothelial cells with knockdown of CCM2 have increased RhoA protein and display impaired directed cell migration. CCM2 binding of Smurf1 increases Smurf1-mediated degradation of RhoA. CCM2 does not significantly alter the catalytic activity of Smurf1, nor is CCM2 a Smurf1 substrate. Rather the CCM2-Smurf1 interaction functions to localize Smurf1 for RhoA degradation. These findings provide a molecular mechanism for the pathogenesis of cerebral cavernous malformations (CCM) resulting from loss of CCM2-mediated localization of Smurf1, which controls RhoA degradation required for maintenance of normal endothelial cell physiology.We previously characterized a scaffold-like protein named osmosensing scaffold for MEKK3 (OSM) for its ability to bind actin and localize to Rac-containing membrane ruffles and its obligate requirement for p38 activation in response to hyperosmotic stress (1). Subsequently, the gene encoding OSM, CCM2, was found to be mutated in the human disease cerebral cavernous malformations (CCM) 2 (2). Cerebral cavernous malformations are vascular lesions of the central nervous system characterized as clusters of dilated, thin walled blood vessels. CCM lesions are fragile and prone to vascular leakiness and rupture, leading to hemorrhages that cause seizure and stroke (3, 4).Recently, CCM2 knockdown endothelial cells were shown to have increased activation of RhoA (5), although the mechanism was not defined. Herein, we demonstrate a molecular mechanism for activation of this pathway. Through a novel CCM2 PTB domain interaction with the Smurf1 homologous to the E6-AP C terminus (HECT) domain, we now show that CCM2 binds the E3 ligase Smurf1 for the control of RhoA degradation. EXPERIMENTAL PROCEDURESRNAi-bEND.3 cells were purchased from ATCC. pLKO.1 and pLKO.1 CCM2 shRNA plasmids were purchased from Open Biosystems. Lentiviral infection was done according to the RNAi consortium protocol.In Vitro Ubiquitination Assay-The in vitro ubiquitination assay was done as described (6).In Vitro Wound Healing Assay-bEND.3 cells stably expressing pLKO.1 or CCM2 shRNAs were transduced with pLL5.0 for expression of green fluorescent protein. Cells were grown in 24-well dishes to a monolayer and imaged on the BD Biosciences Pathway high content imager. Wounds were imaged every 15 min for 16 h on a ϫ10 objective. The percentage of wound healing was determined as the area of the wound remaining after 16 h divided by the initial area of the wound (ImageJ). RESULTS AND DISCUSSIONCCM2 Binds Smurf1-We have previously demonstrated that MEKK3, a Ste11-like MAP3K, regulates the p38 pathway in mammalian cells in response to hypero...

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Section: Ccm2 Is Not a Smurf1 Substrate Nor Does It Affect Smurf1mentioning

confidence: 97%

Cerebral Cavernous Malformation 2 Protein Promotes Smad Ubiquitin Regulatory Factor 1-mediated RhoA Degradation in Endothelial Cells

Crose

Hilder

Sciaky

et al. 2009

Journal of Biological Chemistry

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“…In humans, the genetic disorder CCM is characterized by vascular lesions localized to the CNS. The most common form of CCM (CCM1) is due to mutations in KRIT1 (35), which interacts with CCM2 (the gene mutated in CCM2), with both proteins hypothesized to play a role in endothelial cell signal transduction via the p38 MAPK pathway (36). Because PAK2 has been implicated in the activation of the p38 signaling cascade (37), defects in the Pak2/␤-pix pathway could account for additional, unexplained cases of CCM or could function as modifier genes in these disorders or in clinical settings such as CNS hemorrhage in premature infants.…”

Section: Discussionmentioning

confidence: 99%

pak2a mutations cause cerebral hemorrhage in redhead zebrafish

Buchner

Su²,

Yamaoka

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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The zebrafish is a powerful model for studying vascular development, demonstrating remarkable conservation of this process with mammals. Here, we identify a zebrafish mutant, redhead (rhd mi149 ), that exhibits embryonic CNS hemorrhage with intact gross development of the vasculature and normal hemostatic function. We show that the rhd phenotype is caused by a hypomorphic mutation in p21-activated kinase 2a (pak2a). PAK2 is a kinase that acts downstream of the Rho-family GTPases CDC42 and RAC and has been implicated in angiogenesis, regulation of cytoskeletal structure, and endothelial cell migration and contractility among other functions. Correction of the Pak2a-deficient phenotype by Pak2a overexpression depends on kinase activity, implicating Pak2 signaling in the maintenance of vascular integrity. Rescue by an endothelial-specific transgene further suggests that the hemorrhage seen in Pak2a deficiency is the result of an autonomous endothelial cell defect. Reduced expression of another PAK2 ortholog, pak2b, in Pak2a-deficient embryos results in a more severe hemorrhagic phenotype, consistent with partially overlapping functions for these two orthologs. These data provide in vivo evidence for a critical function of Pak2 in vascular integrity and demonstrate a severe disease phenotype resulting from loss of Pak2 function.␤-pix ͉ CNS ͉ endothelial cell ͉ p21-activated kinase ͉ vasculature

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“…To date, it has been established that the three CCM proteins can form a complex. [39][40][41] How and whether disruption of this complex of CCM1, CCM2, and CCM3 is involved in the pathogenesis of CCM is still highly unknown.…”

Section: 32mentioning

confidence: 99%