Serine 25 of oncoprotein 18 is a major cytosolic target for the mitogen-activated protein kinase.
Oncoprotein 18 (Op18) is an 18-19-kDa cytoplasmic phosphoprotein, of unknown function, that is frequently up-regulated in transformed cells. Stimulation of various cell-surface receptors results in extensive phosphorylation of Op18 and this protein has, therefore, previously been implicated in intracellular signaling. In the present study, by expression of specific Op18 cDNA mutant constructs and phosphopeptide mapping, we have identified in vivo phosphorylation sites. In conjunction with in vitro phosphorylation experiments, using purified wild-type and mutant Op18 proteins in combination with a series of kinases, these results have identified two distinct proline-directed kinase families that phosphorylate Op18 with overlapping but distinct site preference. These two kinase families, mitogen activated protein (MAP) kinases and cyclin dependent cdc2 kinases, are involved in receptor and cell cycle-regulated phosphorylation events, respectively. Therefore, Op18 may reside at a junction where receptor and cell cycle-regulated kinase families interact with the same substrate. The present study shows that the MAP kinase has a 20-fold preference for Ser25 as opposed to Ser38 of Op18, while cdc2 kinases have a 5-fold preference for the Ser38 residue. Only a minor fraction of the 4.5 x 10(6) Op18 molecules/cell in a leukemic T-cell line are normally in their Ser25 phosphorylated form. However, antigen receptor stimulation of this cell line is shown to result in a rapid conversion of 35-45% of all Op18 molecules to the Ser25 phosphorylated form. These results suggest that Ser25 of Op18 may be a major cytoplasmic target for the MAP kinase in cells with high expression of Op18.
Marklund
U
U
Department for Cell and Molecular Biology, University of Umeå, Sweden.
Brattsand
G
G
Shingler
V
V
Gullberg
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D003600:Cytosol / Q000378:metabolism*; D006801:Humans; D008868:Microtubule Proteins; D019950:Mitogen-Activated Protein Kinase 1; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*; D050777:Stathmin; D013379:Substrate Specificity; D013601:T-Lymphocytes / Q000378:metabolism; D014407:Tumor Cells, Cultured
D008868:Microtubule Proteins; D009838:Oligodeoxyribonucleotides; D010748:Phosphopeptides; D010750:Phosphoproteins; C493995:STMN1 protein, human; D050777:Stathmin; D012694:Serine; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019950:Mitogen-Activated Protein Kinase 1