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Open Access Research

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

Xi-Ning Zhu3, Dong Hyun Kim16, Horng-Ru Lin4, Varija N Budhavarapu5, Herbert B Rosenbaum1, Paul R Mueller2 and P Renee Yew16*

Author Affiliations

1 Department of Molecular Medicine, Institute of Biotechnology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA

2 Department of Biology, The University of Texas at San Antonio, San Antonio, TX, 78249, USA

3 Current address: DiaCarta Inc., P.O. Box 360772, Milpitas, CA, 95036, USA

4 Current address: Department of Infectious Diseases, Novartis Institute for Biomedical Research, 4560 Horton Street MS 4.3, Emeryville, CA, 94608-2916, USA

5 Current address: MD Anderson Cancer, Department of Biochemistry and Molecular Biology, University of Texas, Houston, TX, 77030, USA

6 Department of Molecular Medicine, Institute of Biotechnology, Mail Code 8257, South Texas Research Facility, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA

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Cell Division 2013, 8:5  doi:10.1186/1747-1028-8-5

Published: 22 April 2013

Abstract

Background

Cell 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.

Methods

We used the Xenopus interphase egg extract as a model system to examine the regulation of Xic2 by proteolysis and phosphorylation.

Results

Our 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.

Conclusions

During 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.

Keywords:
Xic2; Xenopus; PCNA; Phosphorylation; Proteolysis; CDK inhibitor