Altered localization and cytoplasmic domain-binding properties of tyrosine-phosphorylated beta 1 integrin.

Abstract

Abstract. We describe a novel approach to study tyrosine-phosphorylated (PY) integrins in cells transformed by virally encoded tyrosine kinases. We have synthesized a peptide (PY/~ peptide) that represents a portion of the cytoplasmic domain of the/~1 integrin subunit and is phosphorylated on the tyrosine residue known to be the target of oncogenic tyrosine kinases. Antibodies prepared against the PY/3~ peptide, after removal of cross-reacting antibodies by absorption and affinity purification, recognized the PYfll peptide and the tyrosine-phosphorylated form of the intact/~ subunit, but did not bind the nonphosphorylated/~ peptide, the nonphosphorylated/~ subunit or other unrelated tyrosine-phosphorylated proteins. The anti-PY/~I antibodies labeled the podosomes of Rous sarcoma virus-transformed fibroblasts, but did not detectably stain nontransformed fibroblasts. The localization of the tyrosine phosphorylated ~ subunit appeared distinct from that of the/~1 subunit. Adhesion plaques were stained by the anti-/~l subunit antibodies in Rous sarcoma virus-transformed fibroblasts plated on fibronectin, whereas neither podosomes nor adhesion plaques were labeled on vitronectin or on uncoated plates. Anti-phosphotyrosine antibodies labeled podosomes, adhesion plaques and cell-cell boundaries regardless of the substratum. One of the SH2 domains of the p85 subunit of phosphatidylinositol-3-kinase bound to the PY~I peptide, but not to the nonphosphorylated ~1 cytoplasmic peptide. Other SH2 domains did not bind to the PY~1 peptide. These results show that the phosphorylated form of the ~ integrin subunit is detected in a different subcellular localization than the nonphosphorylated form and suggest that the phosphorylation on tyrosine of the ~ subunit cytoplasmic domain may affect cellular signaling pathways. INTEGRINS are a family of transmembrane proteins composed of an c~ and/3 subunit. Many of the integrins are receptors for extracellular matrix proteins and mediate cell adhesion to matrices (for reviews, see ' Ruoslahti, 1991;Hynes, 1992). The//~ integrin subunit combines with several c~ subunits to form receptors for extracelhlar matrix proteins such as fibronectin, laminin, and collagens. The cytoplasmic domains of the integrins are thought to bind cytoskeletal and other intraceilular components. The ~1 integrin subunit has been shown to interact with talin ) and ce-actinin (Otey et al., 1990(Otey et al., , 1993 in vitro. Furthermore, the cytoplasmic tail is necessary and sufficient for ~1 integrins to localize in adhesion plaques (Solowska et al., 1989; l_aFlamme et al., 1992). Cell transformation by vitally encoded tyrosine kinases has been shown to cause increased phosphorylation on tyrosine of the ~1 subunit cytoplasmic domain (Hirst et al., 1986). The transforming protein of Rous sarcoma virus (RSV),I p60 v~'c, is a cytoplasmic tyrosine kinase with constitutive activity . Fibroblasts transformed by RSV express high levels of p60v-s% which causes extensive protein tyrosine phosphorylation of cellular proteins ...

Role of the cytoplasmic tyrosines of β1A integrins in transformation by v-src

“…14, nontransformed ␤1A-GD25, Y783͞795F, Y783F, and Y795F cells all have a well-developed F-actin-containing cables that terminate in ␤1A-and vinculin-containing focal contacts. In v-src-transformed GD25 cells expressing wild-type or Y795F mutant ␤1A, ␤1A was present in circular podosomes, as reported previously for a variety of src-transformed cells (4,11,22,23) (Fig. 4A).…”

“…v-src-transformed fibroblasts exhibit several properties related to cell adhesion, including morphological changes, increased growth rate, loss of anchorage dependence of growth, loss of focal contacts, and decreased synthesis and deposition of extracellular matrix (3)(4)(5)(6)(7). v-src causes extensive protein tyrosine phosphorylation of cellular proteins (3,8), including tyrosines in the NPXY motifs of the cytoplasmic domain of the ␤1A integrin subunit (9)(10)(11). The role of phosphorylation of ␤1 integrins in transformation by v-src is not known.…”

Increased extracellular pressure enhances cancer cell integrin-binding affinity through phosphorylation of β1-integrin at threonine 788/789

“…Although increased ␤ 1 -integrin tyrosine phosphorylation has been observed in v-Src-transformed cells (24) and increased serine phosphorylation in parietal endoderm has been demonstrated following treatment with okadaic acid (39), the transient nature of ␤ 1 -integrin phosphorylation has limited most site-specific functional studies to using mutational analysis of conserved cytoplasmic serine-threonine residues without direct evidence of conditional phosphorylation. Increased pressure stimulates robust phosphorylation of ␤ 1 -integrin S785 and T788/9 residues and provides a unique model by which to study the role of site-specific phosphorylation events on ␤ 1 -integrin conformational activation and cell adhesion.…”

Distinct regulatory mechanisms control integrin adhesive processes during tissue morphogenesis

“…Although the effects of NPxY tyrosine phosphorylation have been studied extensively, conflicting reports demonstrate differential effects on integrin function (reviewed in Legate and Fassler, 2009). Several lines of evidence indicate that these mutations activate vertebrate integrins by enhancing binding of the adapter protein, talin (Johansson et al, 1994;Datta et al, 2002;MillonFrémillon et al, 2008;Oxley et al, 2008;Anthis et al, 2009b), which has an established role in activating integrins (reviewed in Calderwood, 2004). We tested the effects of the YY>FF mutation in flies using fluorescence recovery after photobleaching (FRAP) and found that the mobile fraction, a proven method for measuring integrin turnover (Yuan et al, 2010), was lower than WT at MTJs (Supp.…”