Phosphorylation of p27 Kip1 on Serine 10 Is Required for Its Binding to CRM1 and Nuclear Export*

Phosphorylation of the cyclin-dependent kinase inhibitor p27 Kip1 has been thought to regulate its stability. Ser10 is the major phosphorylation site of p27 Kip1 , and phosphorylation of this residue affects protein stability. Phosphorylation of p27 Kip1 on Ser10 has now been shown to be required for the binding of CRM1, a carrier protein for nuclear export. The p27 Kip1 protein was translocated from the nucleus to the cytoplasm at the G0-G1transition of the cell cycle, and this export was inhibited by leptomycin B, a specific inhibitor of CRM1-dependent nuclear export. The nuclear export and subsequent degradation of p27 Kip1 at the G0-G1transition were observed in cells lacking Skp2, the F-box protein component of an SCF ubiquitin ligase complex, indicating that these early events are independent of Skp2-mediated proteolysis. Substitution of Ser10 with Ala (S10A) markedly reduced the extent of p27 Kip1 export, whereas substitution of Ser10 with Asp (S10D) or Glu (S10E) promoted export. Co-immunoprecipitation analysis showed that CRM1 preferentially interacted with S10D and S10E but not with S10A, suggesting that the phosphorylation of p27 Kip1 on Ser10is required for its binding to CRM1 and for its subsequent nuclear export.

The cell cycle of eukaryotic cells is regulated by a series of protein complexes composed of cyclins and cyclin-dependent kinases (CDKs) 1 (1), the activity of which is suppressed by a group of CDK inhibitors (CKIs) (1,2). Among the CKIs, p27 Kip1 plays a pivotal role in the control of cell proliferation (3)(4)(5)(6)(7). The amount of p27 Kip1 is high during the G 0 phase of the cell cycle in normal cells, but it rapidly decreases on reentry of cells into G 1 phase (8,9). We and others have shown that mice homozygous for deletion of the p27 Kip1 gene are larger than normal mice and that they exhibit multiple organ hyperplasia as well as a predisposition to cancer (10 -13). These observations support the notion that p27 Kip1 is a key determinant of both body size and the size of organs as a result of its role in the control of cell proliferation and that the loss of p27 Kip1 function may lead to carcinogenesis. Indeed many studies have shown that the expression of p27 Kip1 is deregulated in various human cancers (for review, see Ref. 14).
The function of p27 Kip1 is regulated by changes in its concentration as well as in its localization in the cell. The concentration of p27 Kip1 is thought to be controlled predominantly by the ubiquitin-proteasome pathway (15). Degradation of p27 Kip1 is promoted by its phosphorylation on Thr 187 by the cyclin E-CDK2 complex (16,17), and the phosphorylation of this residue is required for the binding of p27 Kip1 to Skp2, an F-box protein that is thought to function as the receptor component of an SCF ubiquitin ligase complex; such binding then results in the ubiquitination and degradation of p27 Kip1 (18 -22). We have recently shown that the degradation of p27 Kip1 at the G 0 -G 1 transition is independent of Skp2 and occurs in the cytoplasm, whereas the Skp2-and Thr 187 phosphorylation-dependent degradation of p27 Kip1 occurs in the nucleus (23). These observations suggest that the nuclear export of p27 Kip1 may be critical for its down-regulation early during reentry of quiescent cells into the cell cycle. Consistent with this notion, Jab1 promotes the translocation of p27 Kip1 from the nucleus to the cytoplasm, decreasing the amount of p27 Kip1 in the cell by accelerating its degradation (24).
We previously identified Ser 10 as a major phosphorylation site of p27 Kip1 , accounting for ϳ70% of the total phosphorylation of this protein, and the extent of phosphorylation at this site was 75-fold greater than that at Thr 187 (25). The extent of Ser 10 phosphorylation was markedly increased in cells in the G 0 -G 1 phase of the cell cycle compared with that apparent for cells in S or M phase. Mutation analysis revealed that phosphorylation of Ser 10 , like that of Thr 187 , contributes to regulation of p27 Kip1 stability. We now show that Ser 10 phosphorylation is required for the binding of p27 Kip1 to CRM1, a carrier protein for nuclear export, and that the substitution of Ser 10 with other residues affects the nuclear export of p27 Kip1 . Our data suggest that Ser 10 phosphorylation is a key event in regulation of the function of p27 Kip1 at the G 0 -G 1 transition.

EXPERIMENTAL PROCEDURES
Cell Culture and Synchronization-Mouse embryonic fibroblasts (MEFs) were prepared from 13.5-day-postcoitum Skp2 ϩ/ϩ and Skp2 Ϫ/Ϫ embryos as described previously (26). Only nonsenescent MEFs (no more than passage 2) were used for experiments. MEFs as well as NIH 3T3 and HEK293T cells were cultured as described previously (10,25). For analysis of synchronized cells, NIH 3T3 cells or MEFs were arrested at G 0 phase by subjecting them to serum deprivation with medium supplemented with 0.1% calf serum or fetal bovine serum, respectively, for 96 h; they were then cultured for the indicated times in medium containing 20% serum. Treatment with leptomycin B and MG132 was performed for the indicated times at concentrations of 5 ng/ml and 10 M, respectively.

Construction of Expression Plasmids-Complementary
DNAs encoding all human p27 Kip1 derivatives (wild type, S10A, S10D, S10E, and T187A) tagged with the FLAG epitope were subcloned into pcDNA3 (Invitrogen) for transfection experiments (25) or into pRevTRE (CLONTECH) for the generation of stable inducible cell lines. For the expression of CRM1-enhanced green fluorescent protein (EGFP) fusion protein, human CRM1 cDNA was subcloned in pEGFP-N1 vector (CLONTECH).
Doxycycline (Dox)-regulated Retroviral Expression of p27 Kip1 -NIH 3T3 cells were cotransfected with the reverse tetracycline (tet)-responsive transcriptional activator construct (pTet-On, CLONTECH) and the tet-controlled transcriptional silencer construct (pTet-tTS, CLONTECH). A clone showing high level induction by Dox was used for the expression of tet-regulated constructs delivered as retroviral particles. The pRevTRE constructs encoding p27 Kip1 derivatives were introduced by transfection into the packaging cell line PT67 (CLONTECH) for generation of retroviral particles. The supernatant of the transfected PT67 cells was used to infect the selected NIH 3T3 cell line in the presence of Polybrene (Sigma) at a concentration of 4 g/ml. Representative clones were selected from the infected NIH 3T3 cells on the basis of the level of Dox-induced protein expression as determined by immunoblot analysis.
Immunofluorescence Analysis of p27 Kip1 Expression-NIH 3T3 cells and MEFs were grown on glass coverslips, and immunofluorescence staining was performed as described previously (27). Endogenous and exogenous p27 Kip1 was stained with a monoclonal antibody to p27 Kip1 (57, Transduction Laboratories) and antibodies to the FLAG epitope (M5, Sigma), respectively, and immune complexes were detected with Alexa488-conjugated goat antibodies to mouse immunoglobulin G (green; Molecular Probes). Nuclei were stained with Hoechst 33258 dye (blue).
Subcellular Fractionation-NIH 3T3 cells were lysed in an ice-cold solution containing 0.02% digitonin, 5 mM sodium phosphate (pH 7.4), 50 mM NaCl, 150 mM sucrose, 5 mM KCl, 2 mM dithiothreitol, 1 mM MgCl 2 , 0.5 mM CaCl 2 , and 0.1 mM phenylmethylsulfonyl fluoride. The cytoplasmic fraction was collected after centrifugation of lysates at 1000 ϫ g for 10 min at 4°C. The resulting pellet was resuspended in the lysis solution without digitonin and loaded onto a cushion of a solution containing 30% (w/v) sucrose, 2.5 mM Tris-HCl (pH 7.4), and 10 mM NaCl. After centrifugation at 1000 ϫ g for 10 min at 4°C, nuclei were collected and extracted for 30 min at 4°C with an ice-cold solution containing 0.5% Triton X-100, 50 mM Tris-HCl (pH 7.5), and 300 mM NaCl. After centrifugation of the extract at 10,000 ϫ g for 10 min at 4°C, the supernatant was collected as the nuclear fraction.

RESULTS AND DISCUSSION
Nuclear Export of p27 Kip1 Is Sensitive to Leptomycin B-NIH 3T3 cells begin to enter S phase synchronously 12 h after release from serum deprivation for 96 h (data not shown). The nuclear export of p27 Kip1 was examined by immunostaining of the endogenous protein in such synchronized NIH 3T3 cells (Fig. 1A). Whereas p27 Kip1 accumulated predominantly in the nucleus of cells arrested in G 0 phase, most of the endogenous protein had translocated to the cytoplasm by 7 h after release from G 0 arrest. The p27 Kip1 signal gradually decreased thereafter and was not detected 14 h after the onset of serum stimulation (early S phase). Similar results were obtained when the nuclear export of p27 Kip1 was monitored by immunoblot analysis of nuclear and cytoplasmic fractions of the cells (Fig. 1B). The amount of p27 Kip1 in the nuclear fraction thus gradually decreased, whereas that in the cytoplasmic fraction was transiently increased at 7-10.5 h after the onset of serum stimulation. Given that the expression of Skp2 was not detected until 10.5 h after the onset of stimulation and that Skp2 is localized predominantly to the nucleus (28), Skp2 likely does not contribute to the translocation and degradation of p27 Kip1 during this time period. Treatment of cells with leptomycin B, a specific inhibitor of CRM1-dependent nuclear export (29), blocked the translocation of p27 Kip1 but did not prevent the decrease in the abundance of this protein (Fig. 1A), suggesting that the nuclear export of p27 Kip1 is not required for its degradation. Furthermore, the addition of MG132, a rapid-acting inhibitor of the proteasome, to the culture medium together with leptomycin B inhibited the degradation of p27 Kip1 in the nucleus. These data suggest the existence of two independent pathways for p27 Kip1 proteolysis, one in the nucleus and one in the cytoplasm.  ). B, NIH 3T3 cells were arrested at G 0 and restimulated with serum as in A, and at the indicated times, they were subjected to subcellular fractionation. Nuclear and cytoplasmic fractions (25 g of protein) as well as whole cell lysates (30 g of protein) were subjected to immunoblot analysis with antibodies specific for the indicated proteins. C, NIH 3T3 cells were synchronized and subjected to subcellular fractionation as in B. The amount of lysate protein analyzed for two-dimensional electrophoresis was varied from 100 to 500 g to ensure that the amounts of endogenous p27 Kip1 were similar at the different times of G 0 -G 1 transition. The positions corresponding to unphosphorylated and phosphorylated p27 Kip1 are indicated as are the amounts of each of these two forms of the protein expressed as a percentage of total p27 Kip1 . pp27, phospho-p27; NEPHGE, nonequilibrium pH gradient electrophoresis.
Our previous report demonstrated that the extent of Ser 10 phosphorylation was markedly increased in cells in the G 0 phase of the cell cycle compared with that apparent for cells in S or M phase (25). We thus examined the relative amounts of two (phosphorylated versus nonphosphorylated) forms of p27 Kip1 in the nucleus versus cytoplasm (Fig. 1C). Two-dimensional electrophoresis and immunoblot analysis with anti-p27 Kip1 in the nucleus fraction of NIH 3T3 cells revealed that ϳ50% of endogenous p27 Kip1 was phosphorylated on Ser 10 at G 0 phase, whereas the amount of this form of the protein was reduced to ϳ25% in late G 1 phase (8 h after the serum stimulation). In contrast, ϳ70% of p27 Kip1 protein was phosphorylated in the cytoplasm in the late G 1 phase. These data suggest that p27 Kip1 is phosphorylated on Ser 10 in the nucleus at G 0 phase, and the phosphorylated p27 Kip1 is translocated from nucleus to cytoplasm in late G 1 phase.
Nuclear Export and Degradation of p27 Kip1 at G 0 -G 1 Are Not Dependent on Skp2-To confirm that the nuclear export and degradation of p27 Kip1 at the G 0 -G 1 transition are not dependent on Skp2-mediated proteolysis, we studied wild-type and Skp2 Ϫ/Ϫ MEFs. MEFs begin to enter S phase synchronously 16 h after release from serum deprivation for 96 h (data not shown). Immunofluorescence analysis revealed that p27 Kip1 was translocated from the nucleus to the cytoplasm and degraded in Skp2 Ϫ/Ϫ MEFs as well as in Skp2 ϩ/ϩ MEFs ( Fig. 2A); however, as we previously demonstrated in Skp2 Ϫ/Ϫ lymphocytes (23), p27 Kip1 began to reaccumulate in the nucleus of Skp2 Ϫ/Ϫ MEFs during S phase. Immunoblot analysis of whole cell lysates confirmed that the decrease in p27 Kip1 abundance at the G 0 -G 1 transition does not require Skp2 (Fig. 2B). These data indicate that the nuclear export and degradation of p27 Kip1 at the G 0 -G 1 transition are not dependent on Skp2.
CRM1 Interacts with p27 Kip1 in a Ser 10 Phosphorylation-dependent Manner-Given that CRM1 is implicated in the nuclear export of many proteins (30) and that the nuclear export of p27 Kip1 at the G 0 -G 1 transition was inhibited by leptomycin B, we examined whether CRM1 interacts with p27 Kip1 . Immunoblot analysis revealed that endogenous CRM1 was specifi-cally present in immunoprecipitates prepared from NIH 3T3 cells with antibodies to p27 Kip1 (Fig. 3A). Endogenous Jab1 was not detected in these immunoprecipitates, suggesting that Jab1-mediated export of p27 Kip1 is accomplished by a distinct mechanism. Given that p27 Kip1 phosphorylated on Ser 10 seems to be efficiently translocated from nucleus to cytoplasm in late G 1 phase (Fig. 1C), we next investigated the interaction between recombinant CRM1 and p27 Kip1 derivatives co-expressed in HEK293T cells (Fig. 3B). Co-immunoprecipitation analysis revealed that the substitution of Ser 10 of p27 Kip1 with the acidic residues Asp (S10D) or Glu (S10E) markedly enhanced the interaction between CRM1 and p27 Kip1 , whereas replacement of Ser 10 with Ala (S10A) reduced the extent of binding. Mutation of Thr 187 of p27 Kip1 to Ala did not affect the association with CRM1.
Finally we examined the ability of the various p27 Kip1 derivatives to undergo translocation from the nucleus to the cytoplasm. Wild-type and mutant derivatives of p27 Kip1 were expressed in NIH 3T3 cells with the use of the retroviral Dox-regulated system. The expression of the p27 Kip1 derivatives was induced by Dox during serum deprivation for 96 h and was then terminated by removal of Dox from the medium at which time serum was added back to the medium to induce the translocation of p27 Kip1 . Immunofluorescence analysis revealed that wild-type, S10D, and S10A derivatives of p27 Kip1 were located in the nucleus in the absence of serum stimulation (Fig. 4A), suggesting that Ser 10 phosphorylation is not sufficient for nuclear export. This notion is consistent with our previous observation that p27 Kip1 is located in the nucleus of quiescent cells even though Ser 10 is highly phosphorylated (25). The stimulation-induced translocation of the S10A mutant of p27 Kip1 was markedly inhibited compared with that observed with the wild-type and S10D proteins. Quantitative analysis indicated that the efficiency of export was greater for the S10D mutant than for wild-type p27 Kip1 (Fig. 4B). The amount of the S10A mutant remaining in the nucleus was markedly greater than that apparent for wild-type p27 Kip1 (Fig. 4, A and B). The observations that the translocation of the S10A mutant was not affected by leptomycin B (data not shown) and that the amount of this protein in the nucleus of stimulated cells never achieved the level apparent in quiescent cells (Fig. 4B) suggest that a   FIG. 2. Skp2-independent translocation of p27 Kip1 . A, Skp2 ϩ/ϩ and Skp2 Ϫ/Ϫ MEFs synchronized at G 0 phase by serum deprivation for 96 h were restimulated to enter the cell cycle by exposure to 20% serum for the indicated times. Cells were then subjected to immunostaining for endogenous p27 Kip1 (upper panels of each set), and the resulting images were superimposed with those of nuclear staining with Hoechst 33258 (lower panels). B, Skp2 ϩ/ϩ and Skp2 Ϫ/Ϫ MEFs synchronized at G 0 phase were restimulated with serum as in A. Whole cell lysates (30 g of protein) were then subjected to immunoblot analysis with antibodies to p27 Kip1 , to cyclin A, or to ␣-tubulin. were then subjected to immunoprecipitation (IP) with antibodies to p27 Kip1 or to Myc (control), and the resulting precipitates were subjected to immunoblot analysis with antibodies to CRM1, to Jab1, or to p27 Kip1 . A portion (10%) of the input lysates was also subjected directly to immunoblot analysis with the same antibodies. IgL, immunoglobulin light chain. B, HEK293T cells were cotransfected with vectors encoding an EGFP-CRM1 fusion protein and either FLAG-tagged wild-type (WT) p27 Kip1 or its S10D, S10E, S10A, or T187A mutants. Cell lysates (0.5 mg of protein) were then subjected to immunoprecipitation with antibodies to the FLAG epitope, and the resulting precipitates were subjected to immunoblot (IB) analysis with antibodies to GFP or to p27 Kip1 . A portion (10%) of the input lysates was also subjected directly to immunoblot analysis with the same antibodies.
fraction of p27 Kip1 is exported from the nucleus by a mechanism independent of CRM1 and Ser 10 phosphorylation. These results indicate that phosphorylation of Ser 10 of p27 Kip1 is required for the binding of CRM1 and subsequent translocation of p27 Kip1 to the cytoplasm. However, the phosphorylation is not sufficient for the nuclear export, and another factor(s) may be necessary in addition to the phosphorylation of Ser 10 .
Nuclear Export Controlled by Ser 10 Phosphorylation Is Important for p27 Kip1 Down-regulation at the G 0 -G 1 Transition-We have recently shown that the ubiquitin-mediated proteolysis of p27 Kip1 at the G 0 -G 1 transition occurs normally in Skp2 Ϫ/Ϫ cells (23), whereas the degradation of this protein during S and G 2 phases is markedly impaired in these cells. Given also that Skp2 is not expressed in the early phase (G 0 -G 1 ) of p27 Kip1 degradation, this process appears to be independent of Skp2. In contrast, our previous data indicate that Skp2 is indispensable for the late phase (S-G 2 ) of p27 Kip1 degradation (23). The Skp2-and Thr 187 phosphorylation-dependent degradation of p27 Kip1 occurs in the nucleus, whereas the proteolysis independent of Skp2 and Thr 187 phosphorylation appears to occur in the cytoplasm. In vitro ubiquitination assays revealed that the polyubiquitination activity in the cytoplasm appears to be active throughout the cell cycle and that the substitution of Ser 10 with Asp, Glu, or Ala does not affect the p27 Kip1 polyubiquitination. 2 These data suggest that the phosphorylation of Ser 10 is important for the regulation of p27 Kip1 translocation but not for degradation itself; p27 Kip1 may be polyubiquitinated by the constitutively active ubiquitination machinery in the cytoplasm regardless of the phosphorylation status of Ser 10 once the protein has been translocated to the cytoplasm. Identification of the putative proline-directed kinase responsible for the phosphorylation of p27 Kip1 on Ser 10 as well as of the upstream signaling pathways that link to this kinase should provide further insight into the mechanism of the early phase of degradation of this CKI, which is required for the G 0 -G 1 transition.
During preparation of this manuscript, Rodier et al. (31) also reported that phosphorylation on Ser 10 is necessary for the nuclear export of p27 Kip1 . Although our present results are mostly consistent with those of Rodier et al., these latter researchers demonstrated that endogenous p27 Kip1 phosphorylated on Ser 10 is translocated from nucleus into cytoplasm by using the antibody that is specific for p27 Kip1 phosphorylated on Ser 10 . We added the finding that p27 Kip1 physically associates with the carrier protein CRM1 and that the formation of this complex is controlled by the phosphorylation of p27 Kip1 on Ser 10 . Both reports suggest that the nuclear export of p27 Kip1 is regulated by the phosphorylation on Ser 10 and plays a critical role to decrease the abundance of p27 Kip1 protein below a certain threshold to allow the activation of cyclin-CDK complexes.
FIG. 4. Ser 10 phosphorylation-dependent nuclear export of p27 Kip1 . A, expression of Tet-On FLAG-p27 Kip1 derivatives was induced by Dox (1 g/ml) during synchronization of NIH 3T3 cells at G 0 phase by serum deprivation for 96 h (upper panels). The cells were then restimulated to enter the cell cycle by exposure to 20% serum in Doxfree medium (lower panels) for 8 h. The subcellular localization of recombinant p27 Kip1 was determined by immunofluorescence staining with antibodies to the FLAG epitope, and the nuclei of the same cells were revealed by staining with Hoechst 33258. B, quantitative analysis of the subcellular localization of p27 Kip1 derivatives in experiments similar to that described in A. At least 300 cells were scored for each sample. Data represent the percentage of cells showing predominant localization of p27 Kip1 in the nucleus and are means Ϯ S.E. of values from three independent experiments. WT, wild type.