Protein kinase activities in immune complexes of simian virus 40 large T-antigen and transformation-associated cellular p53 protein.

Abstract

Immune complex kinase assays in the simian virus 40 system were performed by incubation of immunoprecipitates containing tumor antigens with [-y-32P]ATP, followed by analysis of any phosphoacceptor proteins. These assays yielded mainly the viral large T-antigen and, in particular, the associated cellular p53 as endogenous substrates. The nature of these substrates was confirmed by proteolysis techniques. Under specific conditions, casein could be used as an exogenous substrate as well. The kinase reactions showed preference for ATP and MgCl2 instead of GTP or MnCl2. Both phosphoserine and phosphothreonine, but in no case phosphotyrosine, were detected after an immune complex kinase reaction. Apparently, several in vivo phosphorylation sites were recognized in vitro in both large T-antigen and p53, but the presence of some artifactual sites could not be completely excluded. Although contaminating kinases were detectable in the immune complexes, at least the p53 molecules were phosphorylated in vitro in a more specific way. This followed from several characteristics of the immune complex kinase reactions and especially from the strong inhibition of p53 phosphorylation by two anti-large-T monoclonal antibodies. It was shown that large T-antigen showed associated kinase activity, although none of our results could unambiguously demonstrate an intrinsic kinase activity of this protein. Finally, anti-p53 monoclonal antibodies only slightly affected in vitro phosphorylation reactions, whereas a p53 molecule from a simian virus 40-free, chemically transformed human cell line was not phosphorylated in vitro under any condition tested. Thus, it is highly unlikely that the p53 molecule per se carries intrinsic or even associated kinase activities.

Viral Aspects of Protein Phosphorylation

“…This protein kinase, however, appears to be a non-specific contaminant (Walser & Deppert, 1986), the properties (ability to phosphorylate seryl or threonyl residues on casein using either ATP or GTP) of which suggest that it may be cellular casein kinase II (Van Roy et al, 1984). This conclusion is consistent with the assignment to large T of different functional properties involving interaction with DNA.…”

The kinase activity of SV40 large T antigen is mediated by a cellular kinase.

“…However, the kinase activity exhibited by large T in in vitro assays, both by autophosphorylation as well as towards exogenous substrates, like casein and phosvitin, is relatively low (Van Roy et al, 1984). Furthermore, , as well as Giacherio and Hager (1979) were able to separate the kinase activity from large T during extensive biochemical purification of large T. These authors, therefore, conclude that the in vitro kinase activity of large T resulted from an association of a cellular kinase to this protein during cell extraction.…”

“…the possible inactivation of the large T-associated kinase activity or the possible loss of a kinase-positive subclass of large T during extensive purification steps), we analyzed large T obtained by immunopurification. Such preparations, so far, unequivocally had been described as kinase positive (Griffin et al, 1979;Van Roy et al, 1984). In order to prevent an unspecific association of a cellular kinase(s) during extraction of large T, we prepared individual cellular subclasses of large T by sequentially fractionating SV40-transformed cells as described previously Deppert, 1983, 1984), and compared their kinase activities with the kinase activity of large T obtained from wholecell lysates.…”

Phosphorylation of p53 in normal and simian virus 40-transformed NIH 3T3 cells.

“…The protein also forms stable complexes with the Elb tumor antigen of adenovirus (33) and the large tumor antigen (large T) of simian virus 40 (SV40; [17][18][19] and may have a direct involvement in SV40-mediated transformation (24). p53 is a phosphoprotein (5,26,31) which lacks intrinsic protein kinase activity (34). Two phosphorylated amino acids, serines 312 and 389, have been identified in mouse p53 by using Edman sequencing (32); additional phosphorylation sites have been tentatively mapped to the amino terminus between amino acid residues 28 and 98.…”