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

Abstract

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...

Proliferation and Cdk4 expression in microsatellite unstable colon cancers with TGFBR2 mutations

“…The cellular localization of p27 Kip1 is significant because p27 Kip1 requires nuclear import in order to function as a cdk inhibitor, and TGF-b signaling has been shown to regulate the cellular localization of p27 kip1 in some cell lines via FKHRL-1-mediated phosphorylation. 55,56 Interestingly, p27 Kip1 was found to be localized in the cytoplasm predominantly or to be absent in 88% of the wild-type BAT-RII tumors and in 60% of the mutant BAT-RII tumors. Thus, predominantly nuclear p27 Kip1 was found in only 10% of the cancers with wild-type BAT-RII but in 40% of colon cancers with mutant BAT-RII, although this association was not statistically significant (p 5 0.29, Mantel-Haenszel chi-square analysis).…”

Molecular Characterization of Ring Finger Protein 11

“…Cells were fractionated into their nuclear and cytoplasmic components using a digitonin-permeabilization technique (28). When nuclear (N) and cytoplasmic (C) fractions were probed with anti-FLAG antibodies, wild-type RNF11 was located predominantly in the cytoplasmic compartment (Fig.…”

A pathway in quiescent cells that controls p27Kip1 stability, subcellular localization, and tumor suppression

“…CRM1-mediated nuclear export of p27 was necessary for KPC1-KPC2-mediated degradation, but it remains unknown whether a direct modification of p27, such as phosphorylation, is also required (Kamura et al 2004). Moreover, it is unlikely that all p27 translocates into the cytosol in G1 cells (Rodier et al 2001;Boehm et al 2002;Ishida et al 2002;Connor et al 2003;McAllister et al 2003;Kamura et al 2004;Shin et al 2005). Indeed, when p27 is confined to the nucleus either by pharmacological or genetic inhibition of its nuclear export, its stability decreases (Rodier et al 2001).…”

“…Several groups reported that Ser10 phosphorylation of p27 was required for its export from the nucleus, possibly by mediating the binding of p27 to the exportin CRM1 (Rodier et al 2001;Boehm et al 2002;Ishida et al 2002;Connor et al 2003;McAllister et al 2003;Shin et al 2005). This may trigger p27 degradation in the cytoplasm of G1 cells, possibly by the KPC pathway.…”