Abstract
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.
Highlights
Multisite phosphorylation of proteins is a general mechanism for modulation of protein function and molecular interactions
In subsequent experiments we systematically studied adhesion-dependent phosphorylation of the epidermal growth factor (EGF) receptor from ECV304 cells grown in suspension with stimulation by soluble EGF or under adhesion conditions with and without EGF treatment
The EGF receptor purified in this condition was analyzed by MALDI MS and MS/MS, and the results show that, in cells kept in suspension, only the residues Tyr1045, Thr669, Ser967, and Ser1002 were phosphorylated upon EGF treatment
Summary
Multisite phosphorylation of proteins is a general mechanism for modulation of protein function and molecular interactions. The EGF receptor is composed of a large extracellular domain that contains the ligand-binding site, a single hydrophobic transmembrane region, and an intracellular domain This intracellular domain contains the tyrosine kinase activity and a carboxyl-terminal region that is characterized by the presence of several important tyrosine residues. In addition to the canonical activation by its soluble ligands, EGF receptor responds to a host of signals outside the ligand family; EGF receptor transactivation has been detected by G-protein-coupled receptor agonists, phorbol esters, cytokines, estrogen, and cell stress signals [3] These data expand the traditional view of highly specific receptor-ligand interactions, suggesting a wealth of signals impinging on the EGF receptor. In addition integrin-mediated adhesion cooperates with soluble ligand EGF in activation of EGF receptor downstream signaling [8]
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