Pirh2 promotes ubiquitin-dependent degradation of the cyclin-dependent kinase inhibitor p27Kip1.
The cyclin-dependent kinase inhibitor p27(Kip1) is degraded in late G(1) 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-G(2) phases. Recently, Kip1 ubiquitination-promoting complex has been reported as another ubiquitin ligase that targets cytoplasmic p27(Kip1) exported from the nucleus in G(0)-G(1) 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 G(1)-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 G(1)-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.
Hattori
Takayuki
T
Department of Biochemistry 1, Hamamatsu University School of Medicine, Higashi-ku, Hamamatsu, Japan.
Isobe
Tomoyasu
T
Abe
Kenji
K
Kikuchi
Hirotoshi
H
Kitagawa
Kyoko
K
Oda
Toshiaki
T
Uchida
Chiharu
C
Kitagawa
Masatoshi
M
D000818:Animals; D002453:Cell Cycle; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D011485:Protein Binding; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D034741:RNA, Small Interfering / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism / Q000502:physiology*
C436291:CDKN1B protein, human; D047908:Intracellular Signaling Peptides and Proteins; D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D025801:Ubiquitin; D050760:Cyclin-Dependent Kinase Inhibitor p27; C485369:RCHY1 protein, human; D044767:Ubiquitin-Protein Ligases