Identification of post-translationally modified proteins in proteome studies.

Proteome studies are powerful tools to solve many different problems in metabolism, signal transduction, drug discovery, and other areas of interest in life sciences. Up to now, high-sensitive methods for protein identification after two-dimensional gel electrophoresis using mass spectrometry are available. However, the identification of post-translational modifications after two-dimensional gel electrophoresis is still an unsolved problem. In this paper, we want to give several examples for the successful identification of post-translational modifications and point mutations.

Identification of major tyrosine phosphorylation sites in the human insulin receptor substrate Gab-1 by insulin receptor kinase in vitro.

Gab-1 (Grb2-associated binder-1), which appears to play a central role in cellular growth response, transformation, and apoptosis, is a member of the insulin receptor substrate (IRS) family. IRS proteins act downstream in the signaling pathways of different receptor tyrosine kinases, including the insulin receptor (IR). In this paper, we characterize the phosphorylation of recombinant human Gab-1 (hGab-1) by IR in vitro. Kinetic phosphorylation data revealed that hGab-1 is a high affinity substrate for the IR (K(M): 12.0 microM for native IR vs 23.3 microM for recombinant IR). To elucidate the IR-specific phosphorylation pattern of hGab-1, we used phosphopeptide mapping by two-dimensional HPLC analysis. Phosphorylated tyrosine residues were subsequently identified by sequencing the separated phosphopeptides by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) and Edman degradation. Our results demonstrate that hGab-1 was phosphorylated by IR at eight tyrosine residues (Y242, Y285, Y373, Y447, Y472, Y619, Y657, and Y689). Seventy-five percent of the identified radioactivity was incorporated into tyrosine residues Y447, Y472, and Y619 exhibiting features (NYVPM motif) of potential binding sites for the regulatory subunit (p85) of phosphatidylinositol (PI)-3 kinase. Accordingly, pull down assays with human HepG2 cell lysates showed that IR-specific phosphorylation of wild-type hGab-1 strongly enhanced PI-3 kinase binding. This is still the case when a single tyrosine residue in the NYVPM motif was mutated to phenylalanine. In contrast, phosphorylation-dependent binding of PI-3 kinase was completely abolished by changing a second tyrosine residue in a NYVPM motif independent from its location. Recently, we identified a similar cohort of tyrosine phosphorylation sites for the epidermal growth factor receptor (EGFR) with a predominant phosphorylation of tyrosine residue Y657 and binding of Syp [Lehr, S. et al. (1999) Biochemistry 38, 151-159]. These differences in the phosphorylation pattern of hGab-1 may contribute to signaling specificity by different tyrosine kinase receptors engaging distinct SH2 signaling molecules.

Identification of tyrosine phosphorylation sites in human Gab-1 protein by EGF receptor kinase in vitro.

Grb2-associated binder-1 (Gab-1) has been identified recently in a cDNA library of glioblastoma tumors and appears to play a central role in cellular growth response, transformation, and apoptosis. Structural and functional features indicate that Gab-1 is a multisubstrate docking protein downstream in the signaling pathways of different receptor tyrosine kinases, including the epidermal growth factor receptor (EGFR). Therefore, the aim of the study was to characterize the phosphorylation of recombinant human Gab-1 (hGab-1) protein by EGFR in vitro. Using the pGEX system to express the entire protein and different domains of hGab-1 as glutathione S-transferase proteins, kinetic data for phosphorylation of these proteins by wheat germ agglutinine-purified EGFR and the recombinant EGFR (rEGFR) receptor kinase domain were determined. Our data revealed similar affinities of hGab-1-C for both receptor preparations (KM = 2.7 microM for rEGFR vs 3.2 microM for WGA EGFR) as well as for the different recombinant hGab-1 domains. To identify the specific EGFR phosphorylation sites, hGab-1-C was sequenced by Edman degradation and mass spectrometry. The entire protein was phosphorylated by rEGFR at eight tyrosine residues (Y285, Y373, Y406, Y447, Y472, Y619, Y657, and Y689). Fifty percent of the identified radioactivity was incorporated in tyrosine Y657 as the predominant peak in HPLC analysis, a site exhibiting features of a potential Syp (PTP1D) binding site. Accordingly, GST-pull down assays with A431 and HepG2 cell lysates showed that phosphorylated intact hGab-1 was able to bind Syp. This binding appears to be specific, because it was abolished by changing the Y657 of hGab-1 to F657. These results demonstrate that hGab-1 is a high-affinity substrate for the EGFR and the major tyrosine phosphorylation site Y657 in the C terminus is a specific binding site for the tyrosine phosphatase Syp.