Cell-cycle-regulated phosphorylation of oncoprotein 18 on Ser16, Ser25 and Ser38.
Oncoprotein 18 (Op18) has been independently identified due to its increased phosphorylation in response to external signals and its up-regulated expression in acute leukemia. We have identified two serine residues of Op18 that are phosphorylated after triggering by the T cell antigen receptor. One of these residues, Ser25, was shown to be a likely substrate for the mitogen-activated protein (MAP) kinase, while the other residue, Ser16, was shown to be phosphorylated in response to increased intracellular calcium. Our previous site-mapping studies of Op18 also revealed that basal phosphorylation of Op18 is mainly located on Ser38, which was found to be the primary in vitro phosphorylation site of p13suc1-precipitated cdc2 kinase activities. These findings raised the possibility that Op18 may be a substrate for both receptor-regulated calcium-induced protein kinases and the MAP kinase family, as well as being a substrate for the cell-cycle-regulated cdc2 kinase family. In the present report we have performed site-mapping studies of cell-cycle-regulated fluctuations of Op18 phosphorylation. The results reveal that S-phase progression of a synchronised leukemic T cell line is associated with increased phosphorylation of both the Ser25 and Ser38 residues. Moreover, during mitosis, a burst of phosphorylation was observed and at this stage of the cell cycle a major fraction of Op18 was phosphorylated at multiple sites. Phosphorylation of Op18 during mitosis was located primarily on Ser38 and to lesser extent on Ser25, Ser16 and at an unidentified C-terminal residue. In vitro phosphorylation experiments, employing two distinct members of the cdc2 kinase family, were consistent with involvement of both p34-cdc2 and p33-cdk2 in cell-cycle-regulated phosphorylation of Ser25 and Ser38 of Op18. Most importantly, the ratio of Ser25/Ser38 phosphorylation observed in vitro, using either p34-cdc2 or p33-cdk2, was found to be the same as the ratio observed in intact cells during all phases of the cell cycle. These findings suggest that Op18 may be a physiological substrate for several members of the cdc2 kinase family during both the S-phase and the mitotic phase of the cell cycle.
Brattsand
G
G
Department for Cell and Molecular Biology, University of Umeå, Sweden.
Marklund
U
U
Nylander
K
K
Roos
G
G
Gullberg
M
M
D000595:Amino Acid Sequence; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002453:Cell Cycle / Q000502:physiology*; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D015458:Leukemia, T-Cell; D008868:Microtubule Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine; D012694:Serine / Q000378:metabolism*; D050777:Stathmin; D014407:Tumor Cells, Cultured
D008868:Microtubule Proteins; D010446:Peptide Fragments; D010748:Phosphopeptides; D010750:Phosphoproteins; C493995:STMN1 protein, human; D050777:Stathmin; D010768:Phosphoserine; D012694:Serine; D016203:CDC2 Protein Kinase