BIOMAT Database Logo BIOMAT Database Logo

Identification of tyrosine phosphorylation sites in human Gab-1 protein by EGF receptor kinase in vitro. 1999

S Lehr, and J Kotzka, and A Herkner, and E Klein, and C Siethoff, and B Knebel, and V Noelle, and J C Brüning, and H W Klein, and H E Meyer, and W Krone, and D Müller-Wieland
Klinik II und Poliklinik für Innere Medizin am Zentrum für Molekulare Medizin Köln, Institut für Biochemie der Universit at zu Köln, Germany.

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.

UI MeSH Term Description Entries
D007700 Kinetics The rate dynamics in chemical or physical systems.
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D010750 Phosphoproteins Phosphoprotein
D010766 Phosphorylation The introduction of a phosphoryl group into a compound through the formation of an ester bond between the compound and a phosphorus moiety. Phosphorylations
D011993 Recombinant Fusion Proteins Recombinant proteins produced by the GENETIC TRANSLATION of fused genes formed by the combination of NUCLEIC ACID REGULATORY SEQUENCES of one or more genes with the protein coding sequences of one or more genes. Fusion Proteins, Recombinant,Recombinant Chimeric Protein,Recombinant Fusion Protein,Recombinant Hybrid Protein,Chimeric Proteins, Recombinant,Hybrid Proteins, Recombinant,Recombinant Chimeric Proteins,Recombinant Hybrid Proteins,Chimeric Protein, Recombinant,Fusion Protein, Recombinant,Hybrid Protein, Recombinant,Protein, Recombinant Chimeric,Protein, Recombinant Fusion,Protein, Recombinant Hybrid,Proteins, Recombinant Chimeric,Proteins, Recombinant Fusion,Proteins, Recombinant Hybrid
D002458 Cell Fractionation Techniques to partition various components of the cell into SUBCELLULAR FRACTIONS. Cell Fractionations,Fractionation, Cell,Fractionations, Cell
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000595 Amino Acid Sequence The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION. Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D001483 Base Sequence The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence. DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D014407 Tumor Cells, Cultured Cells grown in vitro from neoplastic tissue. If they can be established as a TUMOR CELL LINE, they can be propagated in cell culture indefinitely. Cultured Tumor Cells,Neoplastic Cells, Cultured,Cultured Neoplastic Cells,Cell, Cultured Neoplastic,Cell, Cultured Tumor,Cells, Cultured Neoplastic,Cells, Cultured Tumor,Cultured Neoplastic Cell,Cultured Tumor Cell,Neoplastic Cell, Cultured,Tumor Cell, Cultured

Related Publications

S Lehr, and J Kotzka, and A Herkner, and E Klein, and C Siethoff, and B Knebel, and V Noelle, and J C Brüning, and H W Klein, and H E Meyer, and W Krone, and D Müller-Wieland
In vitro phosphorylation of insulin receptor substrate 1 by protein kinase C-zeta: functional analysis and identification of novel phosphorylation sites.
May 2004, Biochemistry,
S Lehr, and J Kotzka, and A Herkner, and E Klein, and C Siethoff, and B Knebel, and V Noelle, and J C Brüning, and H W Klein, and H E Meyer, and W Krone, and D Müller-Wieland
Modulation of human insulin receptor substrate-1 tyrosine phosphorylation by protein kinase Cdelta.
February 2004, The Biochemical journal,
S Lehr, and J Kotzka, and A Herkner, and E Klein, and C Siethoff, and B Knebel, and V Noelle, and J C Brüning, and H W Klein, and H E Meyer, and W Krone, and D Müller-Wieland
Recombinant human insulin receptor substrate-1 protein. Tyrosine phosphorylation and in vitro binding of insulin receptor kinase.
March 1995, The Journal of biological chemistry,
S Lehr, and J Kotzka, and A Herkner, and E Klein, and C Siethoff, and B Knebel, and V Noelle, and J C Brüning, and H W Klein, and H E Meyer, and W Krone, and D Müller-Wieland
The EGF receptor-kinase has multiple phosphorylation sites.
March 1982, Biochemical and biophysical research communications,
S Lehr, and J Kotzka, and A Herkner, and E Klein, and C Siethoff, and B Knebel, and V Noelle, and J C Brüning, and H W Klein, and H E Meyer, and W Krone, and D Müller-Wieland
Activation of AMP-activated protein kinase by human EGF receptor 2/EGF receptor tyrosine kinase inhibitor protects cardiac cells.
June 2007, Proceedings of the National Academy of Sciences of the United States of America,
S Lehr, and J Kotzka, and A Herkner, and E Klein, and C Siethoff, and B Knebel, and V Noelle, and J C Brüning, and H W Klein, and H E Meyer, and W Krone, and D Müller-Wieland
Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay.
May 2018, Journal of visualized experiments : JoVE,
S Lehr, and J Kotzka, and A Herkner, and E Klein, and C Siethoff, and B Knebel, and V Noelle, and J C Brüning, and H W Klein, and H E Meyer, and W Krone, and D Müller-Wieland
Identification of phosphorylation sites unique to the B form of human progesterone receptor. In vitro phosphorylation by casein kinase II.
December 1994, The Journal of biological chemistry,
S Lehr, and J Kotzka, and A Herkner, and E Klein, and C Siethoff, and B Knebel, and V Noelle, and J C Brüning, and H W Klein, and H E Meyer, and W Krone, and D Müller-Wieland
Identification of the G protein-coupled receptor kinase phosphorylation sites in the human beta2-adrenergic receptor.
June 1996, The Journal of biological chemistry,
S Lehr, and J Kotzka, and A Herkner, and E Klein, and C Siethoff, and B Knebel, and V Noelle, and J C Brüning, and H W Klein, and H E Meyer, and W Krone, and D Müller-Wieland
Identification of tyrosine phosphorylation sites on 3-phosphoinositide-dependent protein kinase-1 and their role in regulating kinase activity.
October 2001, The Journal of biological chemistry,
S Lehr, and J Kotzka, and A Herkner, and E Klein, and C Siethoff, and B Knebel, and V Noelle, and J C Brüning, and H W Klein, and H E Meyer, and W Krone, and D Müller-Wieland
Enhancement of protein kinase C-mediated EGF receptor phosphorylation by auranofin.
January 1988, Scandinavian journal of rheumatology,
Copied contents to your clipboard!