Multiple signal transduction pathways induce phosphorylation of serines 16, 25, and 38 of oncoprotein 18 in T lymphocytes.
A multitude of external signals induce extensive phosphorylation of Oncoprotein 18 (Op18), which suggests a putative role for this protein in signal transduction. We have recently identified two distinct proline-directed kinase families that phosphorylates 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. In the present study, site-specific phosphorylation of Op18 in response to stimulation of the antigen receptor-associated CD3 complex was analyzed in the Jurkat T cell-line. The results show that CD3-induced phosphorylation of Ser-25 of Op18, which is the primary MAP kinase phosphorylation site, can be induced by an apparently protein kinase C (PKC)-independent signal transduction pathway. We also demonstrate that Ser-16 of Op18 is specifically phosphorylated in response to the Ca2+ signal generated by CD3 stimulation or by the Ca2+ ionophore ionomycin. Ser-16 phosphorylation occurs independently of both PKC and MAP kinase activation. Using site-specific Op18 mutants and tryptic phosphopeptide mapping, we show that phosphorylation of Ser-16 of Op18 together with Ser-25, or Ser-25 and Ser-38, generates two Op18 phosphoisomers showing a dramatic electrophoretic retardation. In conclusion, site-mapping studies of Op18 reveal that CD3 stimulation results in an apparently PKC-independent activation of both the MAP kinase and a Ca(2+)-regulated kinase pathway, which results in phosphorylation of distinct sites of Op18. The data also pinpoints the specific phosphorylation events that result in electrophoretic retardation of Op18.
Marklund
U
U
Department of Cell and Molecular Biology, University of Umeå, Sweden.
Brattsand
G
G
Osterman
O
O
Ohlsson
P I
PI
Gullberg
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D017252:CD3 Complex / Q000378:metabolism; D016203:CDC2 Protein Kinase / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D008868:Microtubule Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000302:isolation & purification; D010750:Phosphoproteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*; D015398:Signal Transduction; D050777:Stathmin; D013601:T-Lymphocytes / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D017252:CD3 Complex; D008868:Microtubule Proteins; D009838:Oligodeoxyribonucleotides; D010748:Phosphopeptides; D010750:Phosphoproteins; C493995:STMN1 protein, human; D050777:Stathmin; D012694:Serine; D004247:DNA; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D016203:CDC2 Protein Kinase