Hodeify R, Tarcsafalvi A, Megyesi J, Safirstein RL, Price PM. Cdk2-dependent phosphorylation of p21 regulates the role of Cdk2 in cisplatin cytotoxicity. Am J Physiol Renal Physiol 300: F1171-F1179, 2011. First published February 16, 2011 doi:10.1152/ajprenal.00507.2010.-Cisplatin cytotoxicity is dependent on cyclin-dependent kinase 2 (Cdk2) activity in vivo and in vitro. We found that an 18-kDa protein identified by mass spectrometry as p21 WAF1/Cip1 was phosphorylated by Cdk2 starting 12 h after cisplatin exposure. The analysis showed it was phosphorylated at serine 78, a site not previously identified. The adenoviral transduction of p21 before cisplatin exposure protects from cytotoxicity by inhibiting Cdk2. Although cisplatin causes induction of endogenous p21, the protection is inefficient. We hypothesized that phosphorylation of p21 at serine 78 could affect its role as a Cdk inhibitor, and thereby lessen its ability to protect from cisplatin cytotoxicity. To investigate the effect of serine 78 phosphorylation on p21 activity, we replaced serine 78 with aspartic acid, creating the phosphomimic p21 S78D . Mutant p21 S78D was an inefficient inhibitor of Cdk2 and was inefficient at protecting TKPTS cells from cisplatininduced cell death. We conclude that phosphorylation of p21 by Cdk2 limits the effectiveness of p21 to inhibit Cdk2, which is the mechanism for continued cisplatin cytotoxicity even after the induction of a protective protein.cell death CISPLATIN IS A CHEMOTHERAPEUTIC drug used as a first-line component in the treatment of several solid tumors, including testicular, head and neck, and ovarian and cervical cancers (14). Its dosage and efficacy are limited by its side effects, especially nephrotoxicity (39). Cisplatin cytotoxicity in kidney cells is dependent on cyclin-dependent kinase 2 (Cdk2) activity in vivo and in vitro (42,43). This dependence was shown by Cdk2 inhibitory drugs, dominant-negative Cdk2 transduction, and expression of the cyclin-dependent kinase inhibitor (CDKI), p21, all of which protected from cisplatininduced cell death. Cdk2 is a serine/threonine protein kinase, whose main described function is the phosphorylation of substrates necessary for cell cycle progression (37). The Cdk2 pathways involved in cisplatin-induced cell death are not yet understood, and one substrate in these pathways is described below.Cdk2 activity is regulated by several mechanisms, including binding to cyclins, positive and negative phosphorylation, and binding of CDKIs (38). Apart from its role in cell cycle progression, various studies showed increased Cdk2 activity associated with programmed cell death (apoptosis) (21,36,43,45,54,65,66), and inhibition of Cdk2 activity in vitro has been shown to protect cultured cells from apoptosis (40,42,43,61).In the present study, we identified a Cdk2 substrate induced after cisplatin exposure. An 18-kDa protein accumulated and was phosphorylated by Cdk2 starting 12 h after cisplatin exposure, which coincided with the time when Cdk2 inhibition no longer protec...