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Vol. 15, Issue 6, 2558-2567, June 2004

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The Integrin 1 Subunit Transmembrane Domain Regulates Phosphatidylinositol 3-Kinase-dependent Tyrosine Phosphorylation of Crk-associated Substrate

Annika Armulik ^*  , Teet Velling ^*, and Staffan Johansson

Department of Medical Biochemistry and Microbiology, The Biomedical Center, Uppsala University, SE-751 23, Uppsala, Sweden

Submitted September 26, 2003; Revised January 30, 2004; Accepted February 26, 2004
Monitoring Editor: Anne Ridley

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 1 subunit, this amino acid was replaced with leucine, and the mutated 1 subunit (1A^K756L) was stably expressed in 1-deficient GD25 cells. The extracellular domain of 1A^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 GD25-1A^K756L cells on fibronectin and laminin-1 was impaired, and the rate of migration of GD25-1A^K756L cells on fibronectin was reduced compared with GD25-1A cells. Phosphorylation of tyrosines in focal adhesion kinase (FAK) and the Y416 in c-Src in response to 1A^K756L-mediated adhesion was similar to that induced by wild-type 1. The tyrosine phosphorylation level of paxillin, a downstream target of FAK/Src, was unaffected by the 1 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 1 subunit was found to interfere with 1-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 1 subunit.

Abbreviations used: BSA, bovine serum albumin; CAS, Crk-associated substrate; ECM, extracellular matrix; FAK, focal adhesion kinase; FN, fibronectin; GST, glutathione S-transferase; LN, laminin; PI3K, phosphatidylinositol 3-kinase; SH, Src homology; VN, vitronectin.

^* These authors contributed equally to this work.

Present address: Department of Cell and Molecular Biology, Medical Nobel Institute, Karslinska Institute, SE-171 77 Stockholm, Sweden.

Corresponding author. E-mail address: annika.armulik{at}cmb.ki.se.

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