Mitosis-specific phosphorylation of polyomavirus middle-sized tumor antigen and its role during cell transformation.
Transformation of cells in culture by polyomavirus is mediated by one of its early gene products, middle-sized tumor antigen (MTAg). This protein forms multiple complexes with cellular enzymes such as tyrosine kinases (pp60c-src), a phosphatidylinositol 3-kinase, and phosphatase 2A. Association with MTAg leads to the activation of pp60c-src through interference with phosphorylation at Tyr-527, a site negatively regulating src kinase activity. MTAg abrogates mitosis-specific activation of pp60c-src, resulting in constitutive high kinase activity of the enzyme throughout all phases of the cell cycle. Here we report that MTAg is transiently modified during mitosis, resulting in an increase in its apparent molecular size on SDS/acrylamide gels. Similarly, MTAg isolated from interphase cells and phosphorylated by the cell cycle-regulated serine/threonine kinase p34cdc2 in vitro has increased molecular mass. The large molecular mass form of the protein can be converted to the authentic 56-kDa form upon dephosphorylation by potato acid phosphatase. Two putative phosphorylation sites for a cdc2-like kinase were identified as Thr-160 and -291, respectively. Conversion of Thr-160 to Ala resulted in a transformation-defective mutant protein that was still capable of associating with pp60c-src, phosphatidylinositol 3-kinase, and phosphatase 2A, while the corresponding mutant in position 291 was wild type with respect to all parameters measured so far. These data suggest that phosphorylation by p34cdc2 or a related cell cycle-regulated kinase modulates the interaction of MTAg with cellular targets that are crucial for cell transformation.
Pérez
L
L
Friedrich Miescher-Institute, Basel, Switzerland.
Paasinen
A
A
Schnierle
B
B
Käch
S
S
Senften
M
M
Ballmer-Hofer
K
K
D016475:3T3 Cells; D000135:Acid Phosphatase / Q000378:metabolism; D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002455:Cell Division; D002471:Cell Transformation, Neoplastic; D051379:Mice; D008938:Mitosis; D008979:Moloney murine leukemia virus / Q000235:genetics; D019869:Phosphatidylinositol 3-Kinases; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism; D011120:Polyomavirus / Q000235:genetics* / Q000378:metabolism; D054648:Protein Phosphatase 2; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D013539:Simian virus 40 / Q000235:genetics*; D014162:Transfection
D010770:Phosphotransferases; D019869:Phosphatidylinositol 3-Kinases; D016392:Proto-Oncogene Proteins pp60(c-src); D016203:CDC2 Protein Kinase; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D000135:Acid Phosphatase