title: The integrin beta1 subunit transmembrane domain regulates phosphatidylinositol 3-kinase-dependent tyrosine phosphorylation of Crk-associated substrate.
abstract: Our previous studies on the transmembrane domain of human integrin subunits have shown that a conserved basic amino acid in both subunits of integrin heterodimers is positioned in the plasma membrane in the absence of interacting proteins. To investigate the possible functional role of the lipid-embedded lysine in the mouse integrin beta1 subunit, this amino acid was replaced with leucine, and the mutated beta1 subunit (beta1A(K756L)) was stably expressed in beta1-deficient GD25 cells. The extracellular domain of beta1A(K756L) integrins possesses a competent conformation for ligand binding as determined by the ability to mediate cell adhesion, and by the presence of the monoclonal antibody 9EG7 epitope. However, the spreading of GD25-beta1A(K756L) cells on fibronectin and laminin-1 was impaired, and the rate of migration of GD25-beta1A(K756L) cells on fibronectin was reduced compared with GD25-beta1A cells. Phosphorylation of tyrosines in focal adhesion kinase (FAK) and the Y416 in c-Src in response to beta1A(K756L)-mediated adhesion was similar to that induced by wild-type beta1. The tyrosine phosphorylation level of paxillin, a downstream target of FAK/Src, was unaffected by the beta1 mutation, whereas tyrosine phosphorylation of CAS was strongly reduced. The results demonstrate that CAS is a target for phosphorylation both by FAK-dependent and -independent pathways after integrin ligation. The latter pathway was inhibited by wortmannin and LY294002, implicating that it required an active phosphatidylinositol 3-kinase. Furthermore, the K756L mutation in the beta1 subunit was found to interfere with beta1-induced activation of Akt. The results from this study identify phosphatidylinositol 3-kinase as an early component of a FAK-independent integrin signaling pathway triggered by the membrane proximal part of the beta1 subunit.
authors.affiliation: Department of Medical Biochemistry and Microbiology, The Biomedical Center, Uppsala University, SE-751 23, Uppsala, Sweden. annika.armulik@cmb.ki.se
authors.forename: Annika
authors.initials: A
authors.lastname: Armulik
authors.forename: Teet
authors.initials: T
authors.lastname: Velling
authors.forename: Staffan
authors.initials: S
authors.lastname: Johansson
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002448:Cell Adhesion; D002460:Cell Line; D002462:Cell Membrane; D002465:Cell Movement; D050719:Crk-Associated Substrate Protein; D004789:Enzyme Activation; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D019012:Integrin beta1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007930:Leucine / Q000235:genetics; D008239:Lysine / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D050717:Retinoblastoma-Like Protein p130; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: C493842:Bcar1 protein, mouse; D050719:Crk-Associated Substrate Protein; D016326:Extracellular Matrix Proteins; D019012:Integrin beta1; D021122:Protein Subunits; D011506:Proteins; D050717:Retinoblastoma-Like Protein p130; D019000:Phosphotyrosine; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496051:Ptk2 protein, mouse; D019061:src-Family Kinases; D007930:Leucine; D008239:Lysine