title: SETA/CIN85/Ruk and its binding partner AIP1 associate with diverse cytoskeletal elements, including FAKs, and modulate cell adhesion.
abstract: The adaptor protein SETA/CIN85/Ruk is involved in regulating diverse signal transduction pathways, including the internalization of tyrosine kinase receptors via the Cbl ubiquitin ligases, and attenuating PI3K activity by interaction with its regulatory subunit. Here we present evidence for a new aspect of SETA function, based on the initial observation that it co-localizes with actin in microfilaments and at focal adhesions, and with microtubules. Although there was no evidence for direct molecular interactions between SETA and cytoskeletal proteins, the SETA-interacting protein AIP1, which is a rat ortholog of the Xenopus src substrate Xp95, strongly interacted with structural proteins of the cytoskeleton, including actin and tubulins. Both SETA and AIP1 interacted with focal adhesion kinase (FAK) and proline rich tyrosine kinase 2 (PYK-2), and c-Cbl interacted with PYK-2. AIP1, which interacted more strongly than either SETA or c-Cbl, required an intact consensus tyrosine kinase phosphorylation sequence at Y319 to bind to focal adhesion kinases, which suggests that phosphorylation is an important mediator of this complex. SETA, which interacted as a dimer with focal adhesion kinases, promoted the interaction between PYK-2 and AIP1. Direct analysis of the impact of these proteins on cell adhesion, by use of an electrical cell-substrate impedance sensor (ECIS), showed that SETA promoted cell adhesion while AIP1 and c-Cbl reduced it. Furthermore, the ability of AIP1 and AIP1 mutants to decrease cell adhesion in ECIS analysis correlated with their presence in PYK-2 complexes, providing a direct link between AIP1-mediated molecular interactions and cellular behavior. Transfection of AIP1 also reduced the level of phosphorylation of endogenous PYK-2 and FAK, suggesting that this protein may directly regulate focal adhesion kinases, and thereby cell adhesion. These data are the first to implicate the adaptor protein SETA and its binding partner AIP1 as being involved with the cytoskeleton and in the regulation of cell adhesion, and suggest that they may be part of the focal adhesion kinase regulatory complex.
authors.affiliation: William and Karen Davidson Laboratory of Brain Tumor Biology, Hermelin Brain Tumor Center, Department of Neurosurgery, Henry Ford Hospital, 2799 West Grand Blvd, Detroit, MI 48202, USA.
authors.forename: Mirko H H
authors.initials: MHH
authors.lastname: Schmidt
authors.forename: Baihua
authors.initials: B
authors.lastname: Chen
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Randazzo
authors.forename: Oliver
authors.initials: O
authors.lastname: Bogler
mesh_terms: D000199:Actins / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001253:Astrocytes / Q000378:metabolism / Q000473:pathology; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002448:Cell Adhesion; D002460:Cell Line; D003599:Cytoskeleton / Q000378:metabolism*; D019281:Dimerization; D017097:Electric Impedance; D004594:Electrophysiology; D051417:Focal Adhesion Kinase 1; D051418:Focal Adhesion Kinase 2; D051416:Focal Adhesion Protein-Tyrosine Kinases; D005910:Glioma / Q000473:pathology; D006801:Humans; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D008870:Microtubules / Q000378:metabolism; D009154:Mutation; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D014404:Tubulin / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism
chemical_list: D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; C433810:SH3KBP1 protein, human; C411889:Sh3kbp1 protein, rat; D014404:Tubulin; D025801:Ubiquitin; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051418:Focal Adhesion Kinase 2; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; C496052:Ptk2 protein, rat; C496054:Ptk2b protein, rat; D002118:Calcium