P130Cas-associated protein (p140Cap) as a new tyrosine-phosphorylated protein involved in cell spreading.

Integrin-mediated cell adhesion stimulates a cascade of signaling pathways that control cell proliferation, migration, and survival, mostly through tyrosine phosphorylation of signaling molecules. p130Cas, originally identified as a major substrate of v-Src, is a scaffold molecule that interacts with several proteins and mediates multiple cellular events after cell adhesion and mitogen treatment. Here, we describe a novel p130Cas-associated protein named p140Cap (Cas-associated protein) as a new tyrosine phosphorylated molecule involved in integrin- and epidermal growth factor (EGF)-dependent signaling. By affinity chromatography of human ECV304 cell extracts on a MBP-p130Cas column followed by mass spectrometry matrix-assisted laser desorption ionization/time of flight analysis, we identified p140Cap as a protein migrating at 140 kDa. We detected its expression in human, mouse, and rat cells and in different mouse tissues. Endogenous and transfected p140Cap proteins coimmunoprecipitate with p130Cas in ECV304 and in human embryonic kidney 293 cells and associate with p130Cas through their carboxy-terminal region. By immunofluorescence analysis, we demonstrated that in ECV304 cells plated on fibronectin, the endogenous p140Cap colocalizes with p130Cas in the perinuclear region as well as in lamellipodia. In addition p140Cap codistributes with cortical actin and actin stress fibers but not with focal adhesions. We also show that p140Cap is tyrosine phosphorylated within 15 min of cell adhesion to integrin ligands. p140Cap tyrosine phosphorylation is also induced in response to EGF through an EGF receptor dependent-mechanism. Interestingly expression of p140Cap in NIH3T3 and in ECV304 cells delays the onset of cell spreading in the early phases of cell adhesion to fibronectin. Therefore, p140Cap is a novel protein associated with p130Cas and actin cytoskeletal structures. Its tyrosine phosphorylation by integrin-mediated adhesion and EGF stimulation and its involvement in cell spreading on matrix proteins suggest that p140Cap plays a role in controlling actin cytoskeleton organization in response to adhesive and growth factor signaling.

Systematic analysis of the epidermal growth factor receptor by mass spectrometry reveals stimulation-dependent multisite phosphorylation.

Multisite phosphorylation of proteins is a general mechanism for modulation of protein function and molecular interactions. Definition of phosphorylation sites and elucidation of the functional interplay between multiple phosphorylated residues in proteins are, however, a major analytical challenge in current molecular cell biology and proteomic research. In the present study, we used mass spectrometry to determine the major phosphorylated residues of the human epidermal growth factor (EGF) receptor at various well defined cellular conditions. Activation of EGF receptor was achieved by several types of stimulation, i.e. by sodium pervanadate, EGF, and integrin-dependent adhesion. The contribution of cell-matrix adhesion was also determined by activating the EGF receptor by EGF in cells kept in suspension. We developed an analytical strategy that combined miniaturized sample preparation techniques and MALDI tandem mass spectrometry and determined a total of nine phosphorylation sites in the EGF receptor. We discovered one novel phosphorylation site (Ser967) and revealed constitutive phosphorylation of Thr669, Ser967, Ser1002, and Tyr1045 and stimulation-dependent differential phosphorylation of Tyr1068, Tyr1086, Ser1142, Tyr1148, and Tyr1173. The EGF receptor was purified from HeLa cells or ECV304 cells by immunoprecipitation and SDS-PAGE and then digested with trypsin. Phosphopeptides in the range of 0.8-3.7 kDa were recovered by combinations of IMAC, perfusion chromatography, and graphite powder chromatography and subsequently detected and sequenced by MALDI quadrupole time-of-flight tandem mass spectrometry. Two phosphorylation sites were detected in the peptide 1137GSHQISLDNPDYQQDFFPK1155; however, only Tyr1148 was phosphorylated upon EGF treatment; in contrast Ser1142 was only phosphorylated by integrin-dependent adhesion in the absence of EGF treatment, suggesting differential phosphorylation of this region by distinct stimuli. This MALDI MS/MS-based analytical approach demonstrates the feasibility of systematic analysis of signaling molecules by mass spectrometry and provides new insights into the dynamics of receptor signaling processes.

Integrin-induced epidermal growth factor (EGF) receptor activation requires c-Src and p130Cas and leads to phosphorylation of specific EGF receptor tyrosines.

Integrin-mediated cell adhesion cooperates with growth factor receptors in the control of cell proliferation, cell survival, and cell migration. One mechanism to explain these synergistic effects is the ability of integrins to induce phosphorylation of growth factor receptors, for instance the epidermal growth factor (EGF) receptor. Here we define some aspects of the molecular mechanisms regulating integrin-dependent EGF receptor phosphorylation. We show that in the early phases of cell adhesion integrins associate with EGF receptors on the cell membrane in a macromolecular complex including the adaptor protein p130Cas and the c-Src kinase, the latter being required for adhesion-dependent assembly of the macromolecular complex. We also show that the integrin cytoplasmic tail, c-Src kinase, and the p130Cas adaptor protein are required for phosphorylation of EGF receptor in response to integrin-mediated adhesion. We show that integrins induce phosphorylation of EGF receptor on tyrosine residues 845, 1068, 1086, and 1173, but not on residue 1148, a major site of phosphorylation in response to EGF. In addition we find that integrin-mediated adhesion increases the amount of EGF receptor expressed on the cell surface. Therefore these data indicate that integrin-mediated adhesion induces assembly of a macromolecular complex containing c-Src and p130Cas and leads to phosphorylation of specific EGF receptor tyrosine residues.