P53 IS PHOSPHORYLATED BY CDK7-CYCLIN-H IN A P36(MAT1)-DEPENDENT MANNER

The tumor suppressor protein p53 acts as a transcriptional activator t hat can mediate cellular responses to DNA damage by inducing apoptosis and cell cycle arrest, p53 is a nuclear phosphoprotein, and phosphory lation has been proposed to he a means by which the activity of p53 is regulated, The cyclin-dependent kinase (CDK)-activating kinase (CAK) was originally identified as a cellular kinase required for the activa tion of a CDK-cyclin complex, and CAK is comprised of three subunits: CDK7, cyclin H, and p36(MAT1). CAK is part of the transcription factor IIH multiprotein complex, which is required for RNA polymerase II tra nscription and nucleotide excision repair. Because of the similarities between p53 and CAK in their involvement in the cell cycle, transcrip tion, and repair, we investigated whether p53 could act as a substrate for phosphorylation by CAK. While CDK7-cyclin H is sufficient for pho sphorylation of CDK2, we show that p36(MAT1) is required for efficient phosphorylation of p53 by CDK7-cyclin H, suggesting that p35(MAT1) ca n act as a substrate specificity-determining factor for CDK7-cyclin H. We have mapped a major site of phosphorylation by CAK to Ser-33 of p5 3 and have demonstrated as well that p53 is phosphorylated at this sit e in vivo. Both wild-type and tumor-derived mutant p53 proteins are ef ficiently phosphorylated by CAK. Furthermore, we show that p36 and p53 can interact both in vitro and in vivo. These studies reveal a potent ial mechanism for coupling the regulation of p53 with DNA repair and t he basal transcriptional machinery.