PURIFICATION AND CHARACTERIZATION OF A PROTEIN-KINASE WHICH IS ACTIVATED BY SV40 LARGE T-ANTIGEN AND PHOSPHORYLATES THE TUMOR-SUPPRESSOR PROTEIN P53

In SV40-transformed or infected rat cells phosphorylation of the tumor suppressor protein p53 is enhanced due to activation of kinases. At l east three different kinases can be co-precipitated with p53-large T ( LT) immune complexes, casein kinase II representing the major activity , a cyclin dependent kinase (Cdk), and a kinase which appears to be sp ecifically activated by LT (E Muller, B Boldyreff and KH Scheidtmann, Oncogene 8: 2193-2205, 1993). In this paper we describe the purificati on and identification of the LT-activated kinase that phosphorylates a site adjacent to the Cdk site in rat p53. To monitor the activity a s ynthetic peptide was used containing glutamic acid at the position of Ser-313, thus mimicking a phosphorylated Cdk site, With a combination of Mono Q chromatography and subsequent affinity chromatographies with p13(suc1) and a p53-fragment as ligands a 42 kDa protein kinase was p urified to near homogeneity from SV40-transformed rat cells, This kina se phosphorylated both the peptide substrate and the native rat p53, I nterestingly, phosphorylation of the specific site seemed to depend on prior phosphorylation of the Cdk site. On the other hand, the kinase seemed to be activated by LT, as the activity towards the peptide subs trate was significantly higher in extracts from wildtype LT-transforme d cells than from normal or mutant LT-transformed cells, This activati on was not restricted to rat cells but occurred in SV40-transformed mo use and infected monkey cells as well, Phosphorylation of the specific site by LT-activated kinase was not dependent on the presence of LT i n vitro suggesting that activation of the LT-activated kinase is proba bly indirect rather than through direct interaction with LT. Cell cycl e studies revealed that the LT-activated kinase is cell cycle regulate d, since its activity was not detectable in M phase but increased duri ng G1 phase after which it remained relatively constant.