Proteomic Identification of the Cerebral Cavernous Malformation Signaling Complex

Abstract

Cerebral cavernous malformations (CCM) are sporadic or inherited vascular lesions of the central nervous system characterized by dilated, thin-walled, leaky vessels. Linkage studies have mapped autosomal dominant mutations to three loci: ccm1 (KRIT1), ccm2 (OSM), and ccm3 (PDCD10). All three proteins appear to be scaffolds or adaptor proteins, as no enzymatic function can be attributed to them. Our previous results demonstrated that OSM is a scaffold for the assembly of the GTPase Rac and the MAPK kinase kinase MEKK3, for the hyperosmotic stress-dependent activation of p38 MAPK. Herein, we show that the three CCM proteins are members of a larger signaling complex. To define this complex, epitope-tagged wild type OSM or OSM harboring the mutation of F217-->A, which renders the OSM phosphotyrosine binding (PTB) domain unable to bind KRIT1, were stably introduced into RAW264.7 mouse macrophages. FLAG-OSM or FLAG-OSMF217A and the associated complex members were purified by immunoprecipitation using anti-FLAG antibody. OSM binding partners were identified by gel-based methods combined with electrospray ionization-MS or by multidimensional protein identification technology (MudPIT). Previously identified proteins that associate with OSM including KRIT1, MEKK3, Rac, and the KRIT1-binding protein ICAP-1 were found in the immunoprecipitates. In addition, we show for the first time that PDCD10 binds to OSM and is found in cellular CCM complexes. Other prominent proteins that bound the CCM complex include EF1A1, RIN2, and tubulin, with each interaction disrupted with the OSMF217A mutant protein. We further show that PDCD10 binds phosphatidylinositol di- and triphosphates and OSM binds phosphatidylinositol monophosphates. The findings define the targeting of the CCM complex to membranes and to proteins regulating trafficking and the cytoskeleton.

Structure-Function Analysis of Core STRIPAK Proteins

“…Finally, although our data point to the STRIPAK complex as the major interactor for epitope-tagged or endogenous CCM3 protein in HEK293 cells (4), HeLa cells, C2C12 myoblasts, and myotubes and in bovine endothelial aortic cells (data not shown) CCM3 is also capable of interacting with CCM2 (48) and paxillin (30). Because these interactions are apparently mediated via the same surface as the striatin binding site on CCM3, we propose here that they may be mutually exclusive.…”

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

“…Therefore, we asked whether Smurf1 was found in the CCM protein complex (8), possibly by binding to the PY motif of MEKK3. Cells stably expressing FLAG-tagged wild type CCM2 or a CCM2 PTB domain mutant (CCM2-F217A) that is unable to bind NPXY motifs in target proteins were used in co-immunoprecipitation assays to define endogenous proteins that bind to the CCM protein complex (8). Fig.…”

“…MEKK3 binds CCM2 independent of the CCM2 PTB domain (8), suggesting that Smurf1 association with the CCM complex is independent of MEKK3. However, we found that Smurf1 was also able to associate with MEKK3 by co-immunoprecipitation (supplemental Fig.…”

Cerebral cavernous malformation protein CCM1 inhibits sprouting angiogenesis by activating DELTA-NOTCH signaling

“…AKT is phosphorylated after interaction with phosphatidylinositol trisphosphates (PIP3) at the cell membrane. PIP3 is generated from PIP2 by phosphatidylinositol-3-kinase (PI3K), and CCM proteins can interact with PIP2 or PIP3 molecules (9,26). The PI3K inhibitors Wortmannin and LY294002 almost completely abolished CCM1-induced AKT phosphorylation (Fig.…”

“…The CCM1 protein is part of a large protein complex together with CCM2 and CCM3, components of the cytoskeleton and cell junctions, as well as components of signal transduction pathways and lipids (9). The interaction with RAP1 is essential for stabilizing endothelial cell-cell contacts (5).…”