Proteolysis of Xenopus Cip-type CDK inhibitor, p16Xic2, is regulated by PCNA binding and CDK2 phosphorylation

Xi-Ning Zhu,Varija N Budhavarapu,P Renee Yew,Herbert B Rosenbaum,Dong Hyun Kim,Horng-Ru Lin,Paul R Mueller

doi:10.1186/1747-1028-8-5

Abstract

BackgroundCell division is positively regulated by cyclin-dependent kinases (CDKs) partnered with cyclins and negatively regulated by CDK inhibitors. In the frog, Xenopus laevis, three types of CDK inhibitors have been described: p27Xic1 (Xic1) which shares sequence homology with both p21Cip1 and p27Kip1 from mammals, p16Xic2 (Xic2) which shares sequence homology with p21Cip1, and p17Xic3 (Xic3) which shares sequence homology with p27Kip1. While past studies have demonstrated that during DNA polymerase switching, Xic1 is targeted for protein turnover dependent upon DNA, Proliferating Cell Nuclear Antigen (PCNA), and the ubiquitin ligase CRL4Cdt2, little is known about the processes that regulate Xic2 or Xic3.MethodsWe used the Xenopus interphase egg extract as a model system to examine the regulation of Xic2 by proteolysis and phosphorylation.ResultsOur studies indicated that following primer synthesis during the initiation of DNA replication, Xic2 is targeted for DNA- and PCNA-dependent ubiquitin-mediated proteolysis and that Cdt2 can promote Xic2 turnover. Additionally, during interphase, Xic2 is phosphorylated by CDK2 at Ser-98 and Ser-131 in a DNA-independent manner, inhibiting Xic2 turnover. In the presence of double-stranded DNA ends, Xic2 is also phosphorylated at Ser-78 and Ser-81 by a caffeine-sensitive kinase, but this phosphorylation does not alter Xic2 turnover. Conversely, in the presence or absence of DNA, Xic3 was stable in the Xenopus interphase egg extract and did not exhibit a shift indicative of phosphorylation.ConclusionsDuring interphase, Xic2 is targeted for DNA- and PCNA-dependent proteolysis that is negatively regulated by CDK2 phosphorylation. During a response to DNA damage, Xic2 may be alternatively regulated by phosphorylation by a caffeine-sensitive kinase. Our studies suggest that the three types of Xenopus CDK inhibitors, Xic1, Xic2, and Xic3 appear to be uniquely regulated which may reflect their specialized roles during cell division or early development in the frog.

Highlights

Cell division is positively regulated by cyclin-dependent kinases (CDKs) partnered with cyclins and negatively regulated by Cyclin-dependent kinase (CDK) inhibitors
We have dissected the molecular mechanism of Xic1 turnover and have found that Xic1 is degraded in the egg extract during DNA polymerase switching in a DNA, Proliferating Cell Nuclear Antigen (PCNA), and CRL4Cdt2dependent manner [19,20,21,22,23]
Using the interphase egg extract, we found that Xic3 was completely stable in the egg extract, Xic2 was partially degraded and partially modified in a manner resembling ubiquitination and/or phosphorylation (Figure 1A), and Xic1 was readily degraded as shown in previous studies [21]

Summary

Introduction

Cell division is positively regulated by cyclin-dependent kinases (CDKs) partnered with cyclins and negatively regulated by CDK inhibitors. Cip-type CDK inhibitors bind to and negatively regulate the replication protein, Proliferating Cell Nuclear Antigen (PCNA) [3]. Developmental studies suggest that Xic is the only CDK inhibitor that is expressed in the early embryo and studies indicate that Xic is required for both the differentiation of nerve and muscles cells [11,12,13,14,15,16,17,18]. The expression of both Xic and Xic appears to be more tissue-specific in nature with Xic found in somites, the tail bud, lens, and the cement gland while Xic is expressed primarily in the central nervous system [11]. While overexpression of Xic and Xic in the developing embryo results in an arrest in cell division due to an inhibition of CDK2 activity, little is known about the possible regulatory pathways that may control the activities of Xic or Xic3 [11]

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Conclusion