Simian Virus 40 Gene A Regulates the Association Between a Highly Phosphorylated Protein and Chromatin and Ribosomes in Simian Virus 40-Transformed Cells

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Abstract
The species of proteins associated with chromatin and ribosomes of SV-40-transformed and untransformed monkey, mouse and rat cells were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after phosphorylation of these proteins in vivo or in vitro. In vitro phosphorylation was carried out by protein kinase associated with these organelles and [.gamma.-32 P]ATP as the phosphoryl donor. The reaction products contained phosphoserine and phosphothreonine in approximately equal amounts. The electrophoretic analysis of the phosphorylated proteins revealed that the highly phosphorylated protein with a MW of about 90,000 (90K protein) was associated with chromatin and ribosomes from transformed cells but not from untransformed cells. The 90K protein could be extracted from chromatin and ribosomes with 0.5-1.0 M NaCl or KCl. The 90K protein was still associated with the runoff ribosomes prepared by the puromycin reaction of the post-mitochondrial supernatant in the protein-synthesizing system. In vitro phosphorylation of chromatin and ribosomes from SV-40 tsA-transformed mouse and rat cells indicated that the amounts of 90K protein associated with these organelles decreased greatly when the cells were cultivated at the restrictive temperature. A similar temperature-dependent decrease in the amount of 32P-labeled 90K protein was observed in nonhistone chromosomal and ribosome-associated protein fractions prepared from SV-40 tsA-transformed cells labeled with [3H]leucine and [32P]Pi in vivo. In vitro phosphorylated 90K protein in nonhistone chromosomal and ribosome-associated proteins extracted with high salt was not immunoprecipitated with anti-SV-40 T [tumor antigen] sera.