The Atr protein kinase controls UV-dependent upregulation of p16 INK4A through inhibition of Skp2-related polyubiquitination/ degradation

Abstract

The tumor suppressor p16INK4A, a phosphoprotein that exists in human cells under both phosphorylated and nonphosphorylated forms, plays crucial roles during the cellular response to UV light. However, it is still unclear how this protein is activated in response to this carcinogenic agent.We have shown here that UVC upregulates p16INK4A and the phosphorylated form of the protein at the 4 serine sites; Ser-7, Ser-8, Ser-140, and Ser-152. This accumulation of p16INK4A occurred through increasing the stability of both forms of the protein. Importantly, phospho-p16INK4A showed much higher stability, and UV treatment strongly increased its level in absence of de novo protein synthesis. Furthermore, we have shown that the UV-dependent upregulation of both forms of p16INK4A is under the control of the protein kinase Atr, which suppresses their UVC-dependent proteasomal degradation. Interestingly, although this degradation is ubiquitin-related for p16INK4A through the Skp2 ubiquitin ligase protein, it is ubiquitin-independent for the phosphorylated form. In addition, we present clear evidence that Skp2 is upregulated in ATR-deficient cells, leading to the downregulation of the p27Kip1 protein in response to UVC light. Moreover, we have shown a preferential association of endogeneous phospho-p16INK4A with Cdk4. This association increased following UV-treatment mainly for p16INK4A phosphorylated at Ser-140 and Ser-152. Besides, we have shown that Atr regulates UV-related p16/Cdk4-dependent and -independent phosphorylation of pRB and G1 cell cycle delay. Together, these results indicate that p16INK4A and p27Kip1 are key targets in the Atr-dependent signaling pathway in response to UV damage. ©2011 AACR.