Phosphorylation of p27  on Serine 10 Is Required for Its Binding to CRM1 and Nuclear Export

Ishida, Noriko; Hara, Taichi; Kamura, Takumi; Yoshida, Minoru; Nakayama, Keiko; Nakayama, Keiichi I.

doi:10.1074/jbc.c100762200

Cited by 241 publications

(228 citation statements)

References 31 publications

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“…Proteolysis follows a series of steps, starting with phosphorylation of p27 at Thr 187 by the cdk2/cyclin E complex, 28,29 followed by phosphorylation of p27 kip1 on Ser, 10 , which is required for p27 binding to CRM1 and nuclear export. 30 Association of p27 with the jab1 adaptor protein 31 has also been reported to play a role in transportation from the nucleus to the cytoplasm, followed by association of the jab1/p27 complex with grb2 in the cytoplasm, ubiquitination of p27 by the ubiquitin ligase SCF/skp2 complex [32][33][34] and finally proteolysis by the 26S proteasome. How might Mirk mediate downregulation of p27 kip1 , cyclin D1 and p21 cip1 ?…”

Section: Discussionmentioning

confidence: 99%

Rapid turnover of cell‐cycle regulators found in Mirk/dyrk1B transfectants

Ewton

Lee

Deng

et al. 2002

Intl Journal of Cancer

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Mirk/dyrk1B is an arginine-directed protein kinase, which functions as a transcriptional activator and mediates serumfree growth of colon carcinoma cells by an unknown mechanism. We now report that turnover of the cdk inhibitor p27 kip1 and the G 1 -phase cyclin cyclin D1 is enhanced in each of 4 Mirk stable transfectants compared to vector control transfectants and Mirk kinase-inactive mutant transfectants. This enhanced turnover is proteasome-dependent and leads to lower protein levels of both p27 kip1 and cyclin D1. Lower protein levels of the cdk inhibitor p21 cip1 were also observed in the 4 Mirk stable transfectants. Mirk did not alter the activity of a p27 kip1 promoter construct or p27 kip1 mRNA levels by stable expression, indicating that the decrease in p27 kip1 protein levels was due to a posttranscriptional mechanism. These data are consistent with mirk enhancing the expression of some component common to the proteolysis of both p27 kip1 and cyclin D1.

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Section: Discussionmentioning

confidence: 99%

Rapid turnover of cell‐cycle regulators found in Mirk/dyrk1B transfectants

Ewton

Lee

Deng

et al. 2002

Intl Journal of Cancer

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“…Because p27 export from G0 nuclei is less efficient than from nuclei in early G1, the timing of LMB addition may be important when interpreting the effects of LMB on p27 export. Using p27 immunofluorescence, others have shown that LMB blocks the transient cytoplasmic accumulation of p27 that occurs when cells are released from quiescence (Rodier et al, 2001;Ishida et al, 2002). Since treatment with LMB in quiescence blocks G0 to G1 progression, the early G1 activation of p27 export would be compromised.…”

Section: Discussionmentioning

confidence: 99%

“…Not only does p27 phosphorylation differ between the nucleus and cytoplasm (M. Connor and J.M. Slingerland, unpublished data), we and others have shown that the phosphorylation status of serine 10 (S10) critically regulates p27 export (Rodier et al, 2001;Ishida et al, 2002;Boehm et al, 2002). CRM1 mediates nuclear export by binding to a leucinerich NES motif in the export substrate.…”

Section: Discussionmentioning

confidence: 99%

CRM1/Ran-Mediated Nuclear Export of p27^Kip1Involves a Nuclear Export Signal and Links p27 Export and Proteolysis

Connor

Kotchetkov

Cariou

et al. 2003

MBoC

174

169

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We show that p27 localization is cell cycle regulated and we suggest that active CRM1/RanGTPmediated nuclear export of p27 may be linked to cytoplasmic p27 proteolysis in early G1. p27 is nuclear in G0 and early G1 and appears transiently in the cytoplasm at the G1/S transition. Association of p27 with the exportin CRM1 was minimal in G0 and increased markedly during G1-to-S phase progression. Proteasome inhibition in mid-G1 did not impair nuclear import of p27, but led to accumulation of p27 in the cytoplasm, suggesting that export precedes degradation for at least part of the cellular p27 pool. p27-CRM1 binding and nuclear export were inhibited by S10A mutation but not by T187A mutation. A putative nuclear export sequence in p27 is identified whose mutation reduced p27-CRM1 interaction, nuclear export, and p27 degradation. Leptomycin B (LMB) did not inhibit p27-CRM1 binding, nor did it prevent p27 export in vitro or in heterokaryon assays. Prebinding of CRM1 to the HIV-1 Rev nuclear export sequence did not inhibit p27-CRM1 interaction, suggesting that p27 binds CRM1 at a non-LMB-sensitive motif. LMB increased total cellular p27 and may do so indirectly, through effects on other p27 regulatory proteins. These data suggest a model in which p27 undergoes active, CRM1-dependent nuclear export and cytoplasmic degradation in early G1. This would permit the incremental activation of cyclin E-Cdk2 leading to cyclin E-Cdk2-mediated T187 phosphorylation and p27 proteolysis in late G1 and S phase. INTRODUCTIONThe Cdk inhibitor p27 is an important regulator of G1 progression. It is highly expressed in G0, where it binds tightly and inhibits cyclin E-Cdk 2 Polyak et al., 1994;Slingerland et al., 1994). In mid-G1, p27 also plays a role in the assembly and nuclear import of D-type cyclinCdk complexes (LaBaer et al., 1997;Cheng et al., 1999). p27 levels are regulated by translational controls and by proteolysis, and decrease as cells progress from G1 to S phase (Hengst and Reed, 1996;Millard et al., 1997;Slingerland and Pagano, 2000). The ubiquitin-dependent proteolysis of p27 (Pagano et al., 1995) is regulated by its phosphorylation at threonine 187 (T187) by cyclin E-Cdk 2 in late G1 and S phase (Sheaff et al., 1997;Vlach et al., 1997;Montagnoli et al., 1999). T187 phosphorylation allows recognition of p27 by its SCF-type E3 ligase, comprised of Skp1, Cul1, and the F-box protein, Skp2 and Roc1 and the Cks1 cofactor Ohta et al., 1999;Sutterluty et al., 1999;Tsvetkov et al., 1999;Ganoth et al., 2001;Spruck et al., 2001). Recent evidence suggests that p27 proteolysis is regulated by at least two distinct mechanisms, with mitogenic signaling conditioning p27 for degradation in early G1 in a manner independent of T187 phosphorylation (Hara et al., 2001;Malek et al., 2001), whereas Skp2-dependent cyclin E-Cdk 2-mediated degradation occurs in S phase after T187 phosphorylation (Malek et al., 2001). Although p27 is detected in the nuclei of most normal quiescent cells (Slingerland and Pagano, 2000), the relationship between its int...

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“…Until recently, it was thought that export of p27 to the cytoplasm was a prerequisite for its degradation [18]. However, new studies show that phosphorylation of p27 on Ser-10, an event occurring primarily during G0 and G1, re-routes a fraction of p27 from the nucleus to the cytoplasm [19,68]. Phosphorylation on Ser-10 is mitogen independent and is necessary but not sufficient for the nuclear export of p27.…”

Section: Regulation Of P27 Protein Levelsmentioning

confidence: 99%