Cell-cycle-regulated Phosphorylation of Oncoprotein 18 on Ser16, Ser25 and Ser38

Eur J Biochem. 1994 Mar 1;220(2):359-68. doi: 10.1111/j.1432-1033.1994.tb18632.x.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • CDC2 Protein Kinase / metabolism*
  • Cell Cycle / physiology*
  • Cell Line
  • Electrophoresis, Polyacrylamide Gel
  • HeLa Cells
  • Humans
  • Leukemia, T-Cell
  • Microtubule Proteins*
  • Molecular Sequence Data
  • Molecular Weight
  • Peptide Fragments / chemistry
  • Peptide Fragments / isolation & purification
  • Phosphopeptides / chemistry
  • Phosphopeptides / isolation & purification
  • Phosphoproteins / chemistry
  • Phosphoproteins / isolation & purification
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Phosphoserine
  • Serine / metabolism*
  • Stathmin
  • Tumor Cells, Cultured

Substances

  • Microtubule Proteins
  • Peptide Fragments
  • Phosphopeptides
  • Phosphoproteins
  • STMN1 protein, human
  • Stathmin
  • Phosphoserine
  • Serine
  • CDC2 Protein Kinase