Abstract
The serine/threonine protein phosphatases are targeted to specific subcellular locations and substrates in part via interactions with a wide variety of regulatory proteins. Understanding these interactions is thus critical to understanding phosphatase function. Using an iterative affinity purification/mass spectrometry approach, we generated a high density interaction map surrounding the protein phosphatase 2A catalytic subunit. This approach recapitulated the assembly of the PP2A catalytic subunit into many different trimeric complexes but also revealed several new protein-protein interactions. Here we define a novel large multiprotein assembly, referred to as the striatin-interacting phosphatase and kinase (STRIPAK) complex. STRIPAK contains the PP2A catalytic (PP2Ac) and scaffolding (PP2A A) subunits, the striatins (PP2A regulatory B''' subunits), the striatin-associated protein Mob3, the novel proteins STRIP1 and STRIP2 (formerly FAM40A and FAM40B), the cerebral cavernous malformation 3 (CCM3) protein, and members of the germinal center kinase III family of Ste20 kinases. Although the function of the CCM3 protein is unknown, the CCM3 gene is mutated in familial cerebral cavernous malformations, a condition associated with seizures and strokes. Our proteomics survey indicates that a large portion of the CCM3 protein resides within the STRIPAK complex, opening the way for further studies of CCM3 biology. The STRIPAK assembly establishes mutually exclusive interactions with either the CTTNBP2 proteins (which interact with the cytoskeletal protein cortactin) or a second subcomplex consisting of the sarcolemmal membrane-associated protein (SLMAP) and the related coiled-coil proteins suppressor of IKKepsilon (SIKE) and FGFR1OP2. We have thus identified several novel PP2A-containing protein complexes, including a large assembly linking kinases and phosphatases to a gene mutated in human disease.
Highlights
The serine/threonine protein phosphatases are targeted to specific subcellular locations and substrates in part via interactions with a wide variety of regulatory proteins
cerebral cavernous malformation 3 (CCM3) and Its GCK-III Kinase Subfamily Partners Are Physically Associated with phosphatase 2A (PP2A) Complexes—Here we report the identification of a large molecular assembly (STRIPAK) that links the PDCD10 gene product CCM3 and associated GCK-III kinases to a PP2A1⁄7striatin1⁄7Mob31⁄7STRIP complex
Mutations in the CCM1, CCM2, and CCM3 genes account for most cases of familial cerebral cavernous malformations [68]
Summary
CDNA and Stable Cell Lines—Constructs used in this study are described in supplemental Table S2; all inserts were sequenced in their entirety. The ammonium bicarbonate was evaporated, and the samples were resuspended in HPLC buffer A2 (2% acetonitrile, 0.1% formic acid) and directly loaded onto capillary columns packed in-house with Magic 5 m, 100 Å, C18AQ. Immunoprecipitation/Western Blot—Lysates from stable cell lines (prepared as above) were incubated with FLAG M2-agarose beads for 1.5 h, washed as above, and directly eluted by boiling in Laemmli or Criterion XT sample buffer. Lysates from untransfected HEK293 cells were incubated with protein G-Sepharose (GE Healthcare) and antibodies to endogenous striatin (5 l), striatin (2.5 l), MST4 (2.5 l), PP4R2 (5 l), or the FLAG epitope (2.5 l) for 2 h and washed three times in lysis buffer and once in PBS prior to elution in 25 l of 2ϫ Laemmli sample buffer. Precipitated proteins were separated via SDS-PAGE, transferred to nitrocellulose, and detected with antibodies directed against the FLAG or HA epitopes or endogenous proteins
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