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Pirh2 Promotes Ubiquitin-Dependent Degradation of the Cyclin-Dependent Kinase Inhibitor p27^Kip1

¹ Department of Biochemistry 1 and ² Second Department of Surgery, Hamamatsu University School of Medicine, Higashi-ku, Hamamatsu, Japan

Requests for reprints: Masatoshi Kitagawa, Department of Biochemistry 1, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu 431-3192, Japan. Phone: 81-53-435-2322; Fax: 81-53-435-2322; E-mail: kitamasa{at}hama-med.ac.jp.

The cyclin-dependent kinase inhibitor p27^Kip1 is degraded in late G₁ phase by the ubiquitin-proteasome pathway, allowing cells to enter S phase. Due to accelerated degradation of p27^Kip1, various human cancers express low levels of p27^Kip1 associated with poor prognosis. S-phase kinase–associated protein 2, the F-box protein component of an SCF ubiquitin ligase complex, is implicated in degradation of p27^Kip1 during S-G₂ phases. Recently, Kip1 ubiquitination–promoting complex has been reported as another ubiquitin ligase that targets cytoplasmic p27^Kip1 exported from the nucleus in G₀-G₁ phases. Here, we identified a RING-H2–type ubiquitin ligase, Pirh2, as a p27^Kip1-interacting protein. Endogenous Pirh2 physically interacted with endogenous p27^Kip1 in mammalian cells. Pirh2 directly ubiquitinated p27^Kip1 in an intact RING finger domain-dependent manner in vivo, as well as in vitro. Ablation of endogenous Pirh2 by small interfering RNA increased the steady-state level of p27^Kip1 and decelerated p27^Kip1 turnover. Depletion of Pirh2 induced accumulation of p27^Kip1 in both the nucleus and cytoplasm. Pirh2 expression was induced from late G₁-S phase, whereas p27^Kip1 was decreased in synchronization with accumulation of Pirh2. Furthermore, reduction of Pirh2 resulted in an impairment of p27^Kip1 degradation and an inhibition of cell cycle progression at G₁-S transition in a p53-independent manner. Overall, the results indicate that Pirh2 acts as a negative regulator of p27^Kip1 function by promoting ubiquitin-dependent proteasomal degradation. [Cancer Res 2007;67(22):10789–95]