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J. Biol. Chem., Vol. 277, Issue 32, 28706-28713, August 9, 2002

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Akt-dependent Phosphorylation of p27^Kip1 Promotes Binding to 14-3-3 and Cytoplasmic Localization^*

Naoya Fujita, Saori Sato, Kazuhiro Katayama, and Takashi Tsuruo^§¶

From the  Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo 113-0032 and the ^§ Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo 170-8455, Japan

Received for publication, April 16, 2002, and in revised form, May 10, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In many human cancers, the cyclin-dependent kinase inhibitor p27^Kip1 is expressed at low or undetectable levels. The decreased p27^Kip1 expression allows cyclin-dependent kinase activity to cause cells to enter into S phase and correlates with poor patient survival. Inhibition of serine/threonine kinase Akt signaling by some pharmacological agents or by PTEN induces G₁ arrest, in part by up-regulating p27^Kip1. However, the role of Akt-dependent phosphorylation in p27^Kip1 regulation is not clear. Here, we show that Akt bound directly to and phosphorylated p27^Kip1. Screening p27^Kip1 phosphorylation sites identified the COOH-terminal Thr¹⁹⁸ residue as a novel site. Further analysis revealed that 14-3-3 proteins bound to p27^Kip1 through Thr¹⁹⁸ only when it was phosphorylated by Akt. Although Akt also phosphorylated p27^Kip1 at Ser¹⁰ and Thr¹⁸⁷, these two sites were not involved in the binding to 14-3-3 proteins. p27^Kip1 phosphorylated at Thr¹⁹⁸ exists only in the cytoplasm. Therefore, Akt promotes cell-cycle progression through the mechanisms of phosphorylation-dependent 14-3-3 binding to p27^Kip1 and cytoplasmic localization.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The characterization of the survival signal transduction pathways stimulated by growth factors and cytokines has revealed that the serine/threonine kinase Akt (also known as protein kinase B or RAC-PK) is involved in the pathway (1, 2). After stimulation with growth factors, Akt is recruited to the plasma membrane and binds to the phosphatidylinositide 3-OH kinase (PI3K)¹-generated phospholipid second messenger molecule, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P₃), through its pleckstrin homology domain (1, 2). Then, Akt is phosphorylated at two key regulatory sites, Thr³⁰⁸ and Ser⁴⁷³. Phosphorylation at both residues is necessary for full activation of Akt and the subsequent regulation of many biological responses, including glucose uptake, protein synthesis, and apoptosis inhibition (3). Akt phosphorylation at Thr³⁰⁸ is catalyzed by the ubiquitously expressed 3-phosphoinositide-dependent protein kinase-1 (PDK1) (reviewed in Ref. 4). The kinase responsible for phosphorylation of Akt at Ser⁴⁷³ is called PDK2. Recently, several reports suggested that Akt itself, integrin-linked kinase, or PDK1 complexed with the fragment of PRK2 (protein kinase C-related kinase-2) is associated with the Akt phosphorylation at Ser⁴⁷³ (5-7).

A number of molecules play an important role in regulating cell-cycle progression. Cell-cycle progression depends on the activity of kinase complexes composed of cyclins and cyclin-dependent kinases (CDKs). The CDK activity is suppressed in part by association with CDK inhibitors, including the INK4 family (p16^INK4a, p15^INK4b, p18^INK4c, and p19^INK4d) and the Cip/Kip family (p21^Waf1/Cip1, p27^Kip1, and p57^Kip2) (reviewed in Ref. 8). p27^Kip1, a Cip/Kip member, was identified as a CDK inhibitor that causes G₁ arrest by inhibiting the activities of G₁ cyclins/CDKs. The activity of p27^Kip1 is controlled by its concentration, its distribution among different cellular complexes, and its cellular localization (9, 10). In many human cancers, reduced p27^Kip1 expression is frequently observed (10). The reduced expression of p27^Kip1 is reported to correlate with tumor progression and poor patient survival (11, 12). Thus, p27^Kip1 may participate in tumor suppression by inhibiting abnormal cell-cycle progression.

The concentration of p27^Kip1 is transcriptionally and post-translationally regulated. Akt is known to down-regulate p27^Kip1 transcription by phosphorylation-dependent inhibition of the Forkhead family of transcription factors (13). However, the p27^Kip1 concentration is reported to be mainly regulated by post-translational mechanisms (14, 15). Phosphorylation of p27^Kip1 at Thr¹⁸⁷ by the cyclin E-CDK2 complex triggers p27^Kip1 degradation (16-19). Although p27^Kip1 needs to be transported into the nucleus to exert CDK inhibitory action, phosphorylation at Ser¹⁰ by unknown kinases was recently reported to increase nuclear export of p27^Kip1 through binding to CRM1 (20). Thus, some kinases regulate degradation and cytoplasmic localization p27^Kip1 through phosphorylation-dependent mechanisms.

The tumor suppressor gene PTEN (phosphatase and tensin homologue deleted in chromosome 10) is mutated in a wide range of human cancers. PTEN encodes a lipid phosphatase that removes the D-3-phosphate from PtdIns(3,4,5)P₃ (21). Thus, the loss or mutation of PTEN increases the amount of PtdIns(3,4,5)P₃, which in turn activates pleckstrin homology domain-containing Akt. PTEN induces growth arrest in part by up-regulating p27^Kip1 (22). Furthermore, inhibition of PI3K activity by the PI3K inhibitor LY294002 results in G₁ arrest with p27^Kip1 up-regulation (23). Thus, Akt might be involved in the down-regulation of p27^Kip1 expression. Although Akt transcriptionally regulates p27^Kip1 expression by phosphorylating and inhibiting Forkhead transcription factors (13), the post-translational regulation of p27^Kip1 expression remains unclear.

We sought to determine the Akt-mediated post-transcriptional regulation of p27^Kip1. We found that Akt directly phosphorylated p27^Kip1 in vivo and in vitro. Mutation and immunoblot analyses revealed that Akt phosphorylated p27^Kip1 at the previously identified Ser¹⁰ and Thr¹⁸⁷ residues. Furthermore, we identified the COOH-terminal Thr¹⁹⁸ residue as a novel Akt-dependent phosphorylation site. Screening of the p27^Kip1-binding protein identified that 14-3-3 proteins bound to p27^Kip1 only when p27^Kip1 was phosphorylated at Thr¹⁹⁸ by Akt. Because Thr¹⁹⁸-phosphorylated p27^Kip1 was localized only in the cytoplasm, Akt might promote 14-3-3 binding to p27^Kip1 by phosphorylation at Thr¹⁹⁸, allowing its cytoplasmic localization and degradation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Reagents and Cell Culture Conditions-- The recombinant human inactive Akt1, active Akt1, and active CDK2/cyclin A proteins were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). LY294002 was purchased from Sigma. Our previously identified PDK1 inhibitor UCN-01 was kindly provided by Kyowa Hakko Kogyo (Tokyo, Japan) (24). The synthetic PGLRRRQT peptide (Kiptide) corresponding to amino acids 191-198 of human p27^Kip1 sequence was obtained from Sawady Technology (Tokyo, Japan). The synthetic biotinylated peptides biotin-PKKPGLRRRQT-amide (RRRQT) and biotin-PKKPGLRRRQpT-amide (RRRQpT, where pT represents phosphorylated threonine) were also obtained from Sawady Technology. Human embryonic kidney 293T and African green monkey kidney COS-7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum.

Plasmid Construction-- The wild-type (WT), active (E40K), and dominant-negative (K179A/T308A/S473A, referred to below as AAA) human akt1 cDNAs in the pFLAG-CMV-2 vector (Sigma) or a pHM6 vector (Roche Molecular Biochemicals, Mannheim, Germany) were established in our laboratory (25, 26). The WT and NH₂-terminal myristoylated active mouse akt1 cDNAs in the pUSEamp vector were purchased from Upstate Biotechnology, Inc. The human WT skp2 cDNA in the pcDNA3.1GS vector was purchased from Invitrogen. Human full-length WT p27^kip1 cDNA (WT p27^Kip1) was generated by PCR with a human HeLa cDNA library (Invitrogen) as the template. The 1-26 (N26-p27^Kip1, amino acids 27-198) and 1-52 (N52-p27^Kip1, amino acids 53-198) deletion mutants of human p27^kip1 cDNAs were generated by PCR with WT p27^kip1 cDNA as the template. The PCR products were cloned into the pCRII vector (Invitrogen). The translation initiation codon ATG in WT p27^kip1 was converted to the isoleucine codon ATC by PCR-based mutagenesis using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Substitution of Ser¹³⁸, Thr¹⁵⁷, or Gln¹⁸⁶ with stop codons in WT p27^kip1 to generate the COOH-terminal deletion mutants (137STOP-p27^Kip1, 156STOP-p27^Kip1, and 185STOP-p27^Kip1, respectively) was accomplished by converting the appropriate codons to the stop codon TAG. Substitution of Ser¹⁰, Thr¹⁵⁷, Thr¹⁸⁷, or Thr¹⁹⁸ with Ala or Asp in WT p27^kip1 was accomplished using the PCR-based mutagenesis kit. The double- and triple-point mutants were established by further PCR mutagenesis. WT p27^kip1 and these p27^kip1 mutants were then subcloned into the pFLAG-CMV-2 vector or the pQBI50-fC vector (Wako Bioproducts, Richmond, VA). The WT 14-3-3, 14-3-3, 14-3-3, 14-3-3, and 14-3-3 cDNAs were generated by PCR with a human fetal brain cDNA library (Invitrogen) as the template. The PCR products were cloned into the pCRII vector. Substitutions of both Arg⁵⁶ and Arg⁶⁰ with Ala (R56A/R60A) in 14-3-3 were accomplished by converting the Arg codon AGG to the Ala codon GCG using the PCR-based mutagenesis kit. Before subcloning, they were used as templates in a double-stranded Sequenase reaction. All plasmid DNAs for transfection were purified using a QIAGEN plasmid maxi kit according to the manufacturer's protocol.

Transient Transfection, Immunoprecipitation, and Western Blot Analysis-- Cells were transfected with appropriate plasmids using Superfect transfection reagent (QIAGEN Inc.) or LipofectAMINE 2000 reagent (Invitrogen) according to the manufacturers' instructions.

Immunoprecipitation and Western blot analysis were performed as described previously (25, 26). In some experiments, nuclear and cytoplasmic fractions were separated using an NE-PER extraction kit (Pierce) according to the manufacturer's instructions. For Western blot analysis, we used the following: an antibody to phospho-Akt (Thr³⁰⁸) (Upstate Biotechnology, Inc.), antibodies to Akt or phospho-Ser/Thr Akt substrate (Cell Signaling Technology, Beverly, MA), an antibody to p27^Kip1 (Transduction Laboratories, Lexington, KY), an antibody to phospho-p27^Kip1 (Thr¹⁸⁷) (Zymed Laboratories Inc., South San Francisco, CA), an antibody to a V5 tag (Invitrogen), an antibody to a blue fluorescence protein tag (clone 11E5, Wako Bioproducts), an antibody to a hemagglutinin (HA) tag (clone 3F10, Roche Molecular Biochemicals), or an antibody to a FLAG tag (clone M2, Sigma). Subsequently, membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibody. After washing several times, the membranes were developed with an enhanced chemiluminescence system (ECL, Roche Molecular Biochemicals) according to the manufacturer's instructions.

Peptide Binding Experiments-- 293T cells were transfected with the pFLAG-CMV-2 empty vector or the pFLAG-CMV-2 vector encoding WT 14-3-3 or mutant R56A/R60A 14-3-3. After transfection for 24 h, transfectants were harvested and lysed with lysis buffer (20 mM Tris-HCl, pH 7.5, 0.2% Nonidet P-40, 10% glycerol, 1 mM EDTA, 1.5 mM magnesium chloride, 137 mM sodium chloride, 50 mM sodium fluoride, 1 mM sodium vanadate, 12 mM -glycerophosphate, 1 mM phenylmethylsulfonyl fluoride, and 1 mM aprotinin) for immunoprecipitation. The cell lysates were precleared by incubation with avidin-conjugated agarose beads (Sigma) for 2 h at 4 °C. After centrifugation, precleared lysates were mixed with 10 µM biotinylated peptides and incubated for an additional 2 h at 4 °C. To precipitate the peptides, the reactions were incubated with avidin-conjugated agarose beads for 2 h at 4 °C. The samples were centrifuged, and the beads washed five times with lysis buffer. Coprecipitated proteins were electrophoresed and immunoblotted.

In Vitro Phosphorylation of p27^Kip1 Protein or Peptide Substrate-- 293T cells were transfected with the pFLAG-CMV-2 empty vector or the pFLAG-CMV-2 vector encoding WT or mutant p27^Kip1. After transfection for 24 h, transfectants were harvested and lysed with lysis buffer for immunoprecipitation. After immunoprecipitation with anti-FLAG antibody M2-agarose, the proteins were incubated with recombinant inactive Akt (500 ng), active Akt (500 ng), or active CDK2 (10 units) in 40 µl of kinase reaction buffer (20 mM MOPS, 25 mM -glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM dithiothreitol, 112.5 µM ATP, and 17 mM magnesium chloride) at 30 °C in the presence or absence of 15 µCi of [-³²P]ATP. The levels of incorporated radioactivity were visualized and quantified with a BAS1000 bioimaging analyzer (Fuji Film, Tokyo, Japan). The reactions were also electrophoresed and immunoblotted using an anti-p27^Kip1 antibody. Phosphorylation of the synthetic PGLRRRQT peptide (Kiptide) was carried out according to a previously described method (25). In brief, recombinant inactive Akt (500 ng) or active Akt (500 ng) was incubated with 100 µM Kiptide in 40 µl of kinase reaction buffer containing 15 µCi of [-³²P]ATP for 30 min at 30 °C. Reactions were stopped by adding 20 µl of 40% trichloroacetic acid, spotted onto phosphocellulose P-81 paper, washed three times with 0.75% phosphoric acid, air-dried, and subjected to Cerenkov counting.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Akt Binds to and Phosphorylates p27^Kip1-- The loss of the tumor suppressor PTEN is often observed in tumor cells (27), and the loss increases the amount of PtdIns(3,4,5)P₃, which in turn activates Akt (21). PTEN induces growth arrest in part by up-regulating p27^Kip1 (22). Although Akt transcriptionally regulates p27^Kip1 expression by phosphorylating and inhibiting Forkhead transcription factors (13), the post-translational regulation of p27^Kip1 expression remains unclear.

Thus, we sought to determine whether p27^Kip1 is directly phosphorylated by Akt. When immunoprecipitated FLAG-tagged WT p27^Kip1 was incubated in vitro with recombinant active Akt, p27^Kip1 was phosphorylated in a time-dependent manner (Fig. 1A, lanes 5-8). This result indicates that p27^Kip1 is one of the substrates of Akt. We then checked the in vivo p27^Kip1 binding to Akt by immunoprecipitating FLAG-tagged p27^Kip1 following Western blot analysis with an anti-Akt antibody. Fig. 1B (upper panel, lane 4) shows that Akt was co-immunoprecipitated with p27^Kip1, suggesting that Akt interacts directly with and phosphorylates p27^Kip1 in vivo.

View larger version (59K): [in this window] [in a new window]   Fig. 1.   Phosphorylation of p27Kip1 by Akt in vitro and in vivo. A, Akt-dependent p27Kip1 phosphorylation in vitro. 293T cells were transfected with the pFLAG-CMV-2 vector alone (Mock; lanes 1-4) or encoding WT p27Kip1 (WT-p27; lanes 5-8). The cell lysates were incubated with anti-FLAG antibody-agarose following extensive washing. Then, the agarose was incubated with 500 ng of recombinant active Akt protein (lanes 1-8) in the presence of [-32P]ATP for the indicated times at 30 °C. The reactions were stopped, electrophoresed, and visualized by autoradiography. B, Akt binding to p27Kip1 in vivo. 293T cells were transfected with the pFLAG-CMV-2 vector alone (lanes 1 and 2) or encoding WT p27Kip1 (lanes 3 and 4) together with the pHM6 vector alone (lanes 1 and 3) or encoding WT Akt (lanes 2 and 4). The FLAG-tagged proteins were immunoprecipitated (IP) and subjected to immunoblot analysis using an anti-Akt antibody (upper panel) or an anti-FLAG antibody (second panel). The expression level of transfected Akt and FLAG-tagged p27Kip1 proteins was confirmed upon immunoblot analysis of the cell lysates using an anti-Akt antibody (third panel) or an anti-FLAG antibody (lower panel). C, analysis of Akt-dependent p27Kip1 phosphorylation in vivo. 293T cells were transfected with the pFLAG-CMV-2 vector alone (lane 1) or encoding p27Kip1 (lanes 2-8) together with the pUSEamp vector encoding WT Akt (lane 3) or myristoylated active Akt (Myr; lane 4) or with the pFLAG-CMV-2 vector encoding WT Akt (lane 6), active Akt (E40K) (lane 7), or dominant-negative Akt (AAA) (lane 8). The cell lysates were electrophoresed and immunoblotted using an anti-phospho-Ser/Thr Akt substrate antibody (upper panel), an anti-p27Kip1 antibody (second panel), an anti-phospho-Akt (Thr308) antibody (third panel), or an anti-Akt antibody (lower panel). The asterisk indicates the background band.

To confirm the p27^Kip1 phosphorylation in vivo, the pFLAG-p27^Kip1 plasmid was cotransfected with WT, active (myristoylated or E40K), or dominant-negative (AAA) akt cDNA into 293T cells. The phosphorylation of p27^Kip1 was estimated by immunoblot analysis using an anti-phospho-Ser/Thr Akt substrate antibody that can preferentially recognize the conserved Akt phosphorylation motif (RXRXX(S/T), where X is any amino acid) (28) only when Ser or Thr is phosphorylated by Akt. As shown in Fig. 1C (upper panel), the anti-phospho-Ser/Thr Akt substrate antibody recognized the phosphorylated form of p27^Kip1 only when 293T cells were cotransfected with WT or active (myristoylated or E40K) akt cDNA. The in vivo phosphorylation of p27^Kip1 was also observed in COS-7 cells when they were transfected with WT p27^kip1 and WT akt cDNAs (data not shown), suggesting that p27^Kip1 phosphorylation by Akt is not restricted to one particular cell line. By contrast, cotransfection with dominant-negative (AAA) akt cDNA did not induce p27^Kip1 phosphorylation (Fig. 1C, lane 8), indicating that Akt kinase activity is required for p27^Kip1 phosphorylation.

Incubation of the cells with the PI3K inhibitor LY294002 (50 µM) or the PDK1 inhibitor UCN-01 (1 µM) (24) decreased the level of Thr³⁰⁸-phosphorylated Akt within 2 h (Fig. 2A, third panel). LY294002 and UCN-01 decreased the phosphorylation of the endogenous Akt substrate glycogen synthase kinase-3 (data not shown), suggesting that Akt dephosphorylation is associated with its inactivation. Under this condition, we observed a decrease in the phospho-p27^Kip1 level (Fig. 2A, upper panel, lanes 3 and 4). These results strongly suggest that p27^Kip1 is one of the substrates of Akt in vivo. To confirm that endogenous p27^Kip1 was also phosphorylated in an Akt-dependent manner, we investigated the change in the endogenous phospho-p27^Kip1 level after treatment of the cells with LY294002 and UCN-01. We found that LY294002 and UCN-01 decreased the phospho-p27^Kip1 level in cells expressing only endogenous protein (Fig. 2B, upper panel). These results indicate that p27^Kip1 is indeed phosphorylated in an Akt-dependent manner in vivo.

View larger version (44K): [in this window] [in a new window]   Fig. 2.   Inhibition of p27Kip1 phosphorylation by suppressing the PI3K/PDK1/Akt pathway. A, inhibition of exogenous p27Kip1 phosphorylation by PI3K and PDK1 inhibitors. 293T cells were transfected with the pFLAG-CMV-2 vector encoding WT Akt (lanes 1-4) together with the pFLAG-CMV-2 vector alone (lane 1) or encoding p27Kip1 (lanes 2-4). After transfection for 24 h, cells were treated with vehicle (; lanes 1 and 2), 50 µM LY294002 (LY; lane 3), or 1 µM UCN-01 (UCN; lane 4) for 2 h prior to cell harvest. The cell lysates were electrophoresed and immunoblotted using an anti-phospho-Ser/Thr Akt substrate antibody (upper panel), an anti-p27Kip1 antibody (second panel), an anti-phospho-Akt (Thr308) antibody (third panel), or an anti-Akt antibody (lower panel). B, inhibition of endogenous p27Kip1 phosphorylation by PI3K and PDK1 inhibitors. 293T cells were treated with vehicle (lane 1), 50 µM LY294002 (lane 2), or 1 µM UCN-01 (lane 3) for 2 h prior to cell harvest. The cell lysates were electrophoresed and immunoblotted using an anti-phospho-Ser/Thr Akt substrate antibody (upper panel) or an anti-p27Kip1 antibody (lower panel).

Identification of the COOH-terminal Thr¹⁹⁸ Residue as a Novel Phosphorylation Site in p27^Kip1-- To identify the p27^Kip1 phosphorylation sites, we generated several p27^Kip1 point mutants in which Thr¹⁵⁷ was converted to Ala (T157A) or Asp (T157D) because p27^Kip1 contains the conserved Akt phosphorylation motif (RKRPAT¹⁵⁷) (Fig. 3B). Contrary to our expectation, the T157A and T157D p27^Kip1 mutants were similarly phosphorylated by Akt (Fig. 3A, lanes 5 and 7) compared with WT p27^Kip1 (lane 3). This result suggests either that Thr¹⁵⁷ in p27^Kip1 is not the Akt-mediated phosphorylation site or that the anti-phospho-Ser/Thr Akt substrate antibody cannot recognize phosphorylated Thr¹⁵⁷ in p27^Kip1. Thus, we prepared NH₂- and COOH-terminal deletion mutants (Fig. 3B) and transfected them into 293T cells together with WT or dominant-negative (AAA) akt cDNA. Although WT p27^Kip1 and the NH₂-terminal deletion mutants N26-p27^Kip1 and N52-p27^Kip1 were equally phosphorylated by Akt (Fig. 3C, upper panel, lanes 3 and 5), all the COOH-terminal deletion mutants (137STOP-p27^Kip1, 156STOP-p27^Kip1, and 185STOP-p27^Kip1) were not phosphorylated by Akt (lanes 7, 9, and 11). These results suggest that the residues around amino acids 186-198 of p27^Kip1 may contain the phosphorylation sites. Among these residues, p27^Kip1 contains two threonine residues (Thr¹⁸⁷ and Thr¹⁹⁸), but does not contain a Ser residue. Thus, we prepared several point mutants in which Thr¹⁸⁷ or Thr¹⁹⁸ of p27^Kip1 was converted to Ala (T187A and T198A, respectively) or Thr¹⁹⁸ was converted to Asp (T198D). Cotransfection of these mutants with WT akt cDNA revealed that the anti-phospho-Ser/Thr Akt substrate antibody could not detect the FLAG-tagged (in the pFLAG vector) or blue fluorescence protein-tagged (in the pQBI vector) T198A and T198D mutants (Fig. 3D, upper panel, lanes 3, 4, 6, and 7). By contrast, mutation of Thr¹⁸⁷ had no effects on the recognition capability of the anti-phospho-Ser/Thr Akt substrate antibody (Fig. 3D, upper panel, lanes 5 and 8). These results indicate that Thr¹⁹⁸ in p27^Kip1 is a novel phosphorylation site that is phosphorylated by Akt and that the anti-phospho-Ser/Thr Akt substrate antibody recognizes the site only when it is phosphorylated. Although the anti-phospho-Ser/Thr Akt substrate antibody preferentially recognized the peptides containing phospho-Thr/Ser preceded by Arg at positions 5 and 3, it had some cross-reactivity with those containing phospho-Thr/Ser preceded by Arg at positions 3 and 2. This could be why the antibody is able to recognize phospho-Thr¹⁹⁸ without Arg at position 5 (LRRRQpT¹⁹⁸).

View larger version (64K): [in this window] [in a new window]   Fig. 3.   Identification of the COOH-terminal Thr198 residue in p27Kip1 as an Akt-dependent phosphorylation site. A, Thr157 in p27Kip1 is not the Akt-dependent phosphorylation site. 293T cells were transfected with the pFLAG-CMV-2 vector alone (; lanes 1 and 2) or encoding WT p27Kip1 (lanes 3 and 4), T157A p27Kip1 (lanes 5 and 6), or T157D p27Kip1 (lanes 7 and 8) together with the pHM6 vector encoding WT Akt (lanes 1, 3, 5, and 7) or dominant-negative Akt (AAA) (lanes 2, 4, 6, and 8). The cell lysates were electrophoresed and immunoblotted using an anti-phospho-Ser/Thr Akt substrate antibody (upper panel) or an anti-p27Kip1 antibody (lower panel). The asterisk indicates the background band. B, the structural domains of WT p27Kip1, the NH2-terminal deletion p27Kip1 mutants (N26-p27Kip1 and N52-p27Kip1), and the COOH-terminal deletion p27Kip1 mutants (137STOP-p27Kip1, 156STOP-p27Kip1, and 185STOP-p27Kip1) used in the experiments are represented as black bars. C, the COOH-terminal domain in p27Kip1 contains Akt-dependent phosphorylation sites. 293T cells were transfected with the pFLAG-CMV-2 vector encoding WT p27Kip1 (lanes 1 and 2), N26-p27Kip1 (lanes 3 and 4), N52-p27Kip1 (lanes 5 and 6), 137STOP-p27Kip1 (lanes 7 and 8), 156STOP-p27Kip1 (lanes 9 and 10), or 185STOP-p27Kip1 (lanes 11 and 12) together with the pHM6 vector encoding WT Akt (lanes 1, 3, 5, 7, 9, and 11) or dominant-negative Akt (AAA) (lanes 2, 4, 6, 8, 10, and 12). The cell lysates were electrophoresed and immunoblotted using an anti-phospho-Ser/Thr Akt substrate antibody (upper panel), an anti-FLAG antibody (second panel), an anti-phospho-Akt (Thr308) antibody (third panel), or an anti-HA antibody (lower panel). The asterisk indicates the background band. D, Thr198 in p27Kip1 is identified as an Akt-dependent phosphorylation site. 293T cells were transfected with the pFLAG-CMV-2 vector encoding WT p27Kip1 (lanes 1 and 2), T198A p27Kip1 (lane 3), T198D p27Kip1 (lane 4), or T187A p27Kip1 (lane 5) or with the pQBI50-fC vector encoding T198A p27Kip1 (lane 6), T198D p27Kip1 (lane 7), or T187A p27Kip1 (lane 8) together with the pHM6 vector alone (; lane 1) or encoding WT Akt (+; lanes 2-8). The cell lysates were electrophoresed and immunoblotted using an anti-phospho-Ser/Thr Akt substrate antibody (upper panel), an anti-FLAG antibody (second panel), an anti-blue fluorescence protein (BFP) antibody (third panel), or an anti-Akt antibody (lower panel). Asterisks indicate the background bands. E, shown is the Akt-dependent phosphorylation of the p27Kip1 peptide containing Thr198. 500 ng of recombinant active or inactive Akt protein was incubated with buffer alone (No peptide) or with 100 µM PGLRRRQT peptide (Kiptide) in the presence of 15 µCi of [-32P]ATP for 30 min at 30 °C. The net radioactivities were determined by subtracting the radioactivity of the inactive Akt-treated samples from that of the active Akt-treated samples. The vertical bars represent S.D. of triplicate determinations.

To further confirm that Akt can phosphorylate Thr¹⁹⁸, a synthetic peptide around the hypothetical phosphorylation site (PGLRRRQT, Kiptide) was incubated with recombinant active or inactive Akt in vitro. As shown in Fig. 3E, Akt phosphorylated Kiptide. These results strongly indicate that Thr¹⁹⁸ in p27^Kip1 is a novel phosphorylation site.

Akt Phosphorylates p27^Kip1 at Ser¹⁰ and Thr¹⁸⁷ in Addition to Thr¹⁹⁸-- p27^Kip1 is known to be phosphorylated at Ser¹⁰ (20, 29, 30) and Thr¹⁸⁷ (16-19) by the cyclin E-CDK2 complex and by unknown kinases. We thus examined whether Akt can phosphorylate these sites using point mutants in which Thr¹⁹⁸, Thr¹⁸⁷, Thr¹⁵⁷, or Ser¹⁰ of p27^Kip1 was converted to Ala (T198A, T187A, T157A, and S10A, respectively). Immunoprecipitated WT p27^Kip1 and its point mutants were incubated in vitro with recombinant active or inactive Akt. Akt is known to be phosphorylated by itself at Ser⁴⁷³ (5). Consistent with the previous report, active (but not inactive) Akt was phosphorylated by itself (Fig. 4A, upper arrowhead); and under this condition, WT p27^Kip1 was phosphorylated by active Akt (but not by inactive Akt) (lower arrowhead). Conversion of Thr¹⁹⁸ or Ser¹⁰ to Ala (T198A and S10A, respectively) in p27^Kip1 drastically decreased the Akt-mediated phosphorylation (Fig. 4, A and B), but it was only slightly decreased by Thr¹⁸⁷ mutation (T187A). By contrast, the level of phospho-T157A p27^Kip1 was similar to that of WT p27^Kip1. Consistent with previous reports (16-19), we observed a drastic decrease in the phosphorylated level of the T187A mutant and a slight decrease in the phospho-S10A p27^Kip1 level when these mutants were incubated in vitro with recombinant active CDK2 (Fig. 4C, lanes 4 and 6, respectively). Thr¹⁹⁸ or Thr¹⁵⁷ mutation did not affect cyclin E-CDK2 complex-mediated phosphorylation (Fig. 4C, lanes 3 and 5). Thus, Thr¹⁹⁸ might be specifically phosphorylated by Akt. Moreover, Akt may be one of the unidentified Ser¹⁰ kinases.

View larger version (60K): [in this window] [in a new window]   Fig. 4.   Identification of Ser10 and Thr187 in p27Kip1 as additional Akt-dependent phosphorylation sites. A and B, Akt-dependent p27Kip1 phosphorylation in vitro. 293T cells were transfected with the pFLAG-CMV-2 vector alone (Mock; lanes 1 and 2) or encoding WT p27Kip1 (lanes 3 and 4), T198A p27Kip1 (lanes 5 and 6), T187A p27Kip1 (lanes 7 and 8), T157A p27Kip1 (lanes 9 and 10), or S10A p27Kip1 (lanes 11 and 12). The cell lysates were incubated with anti-FLAG antibody-agarose following extensive washing. Then, the agarose was incubated with 500 ng of active Akt (lanes 1, 3, 5, 7, 9, and 11) or inactive Akt (lanes 2, 4, 6, 8, 10, and 12) in vitro in kinase reaction buffer containing [-32P]ATP. After incubation for 90 min at 30 °C, the reactions were stopped and electrophoresed. The levels of incorporated radioactivity were visualized (A) and quantified with a BAS1000 bioimaging analyzer (B). The net radioactivities were determined by subtracting the radioactivity of the inactive Akt-treated samples from that of the active Akt-treated samples. C, CDK2-dependent p27Kip1 phosphorylation in vitro. Immunoprecipitated (IP) FLAG-tagged none (Mock), WT p27Kip1, and p27Kip1 point mutants (T198A, T187A, T157A, and S10A) were incubated with 10 units of active CDK2 protein as described under "Experimental Procedures." The levels of incorporated radioactivity were visualized. The amount of immunoprecipitated FLAG-tagged p27Kip1 protein was confirmed by immunoblot analysis using an anti-FLAG antibody (lower panels in A and C). WB, Western blot.

We also examined in vivo Akt-dependent Thr¹⁸⁷ phosphorylation using a phospho-Thr¹⁸⁷-specific p27^Kip1 antibody and Skp2 binding to p27^Kip1. Fig. 5 (A and B, upper panels) shows that Akt did induce p27^Kip1 phosphorylation at Thr¹⁸⁷ and Skp2 binding, indicating that Akt also phosphorylates p27^Kip1 at Thr¹⁸⁷ and possibly promotes proteasome-dependent degradation (31-33). Thus, Akt preferentially phosphorylates at both Ser¹⁰ and Thr¹⁹⁸ and slightly phosphorylates at Thr¹⁸⁷ in p27^Kip1 in vivo.

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Akt promotes p27Kip1 phosphorylation at Thr187 and Skp2 binding. A, shown is the phosphorylation of p27Kip1 at Thr187 by Akt. 293T cells were transfected with the pFLAG-CMV-2 vector alone (; lanes 1 and 2) or encoding WT p27Kip1 (+; lanes 3 and 4) together with the pUSEamp vector alone (; lanes 1 and 3) or encoding myristoylated active Akt (Myr-Akt; +; lanes 2 and 4). The cell lysates were electrophoresed and immunoblotted using an anti-phospho-p27Kip1 (Thr187) antibody (upper panel), an anti-FLAG antibody (middle panel), or an anti-Myc antibody (lower panel). B, 293T cells were cotransfected with the pcDNA3.1GS vector alone (; lanes 1 and 3) or encoding WT Skp2 (+; lanes 2 and 4) and the pUSEamp vector alone (; lanes 1 and 2) or encoding myristoylated active Akt (+; lanes 3 and 4) together with the pFLAG-CMV-2 vector encoding WT p27Kip1 (lanes 1-4). The FLAG-tagged p27Kip1 proteins were immunoprecipitated (IP), and co-immunoprecipitated proteins were analyzed by immunoblot analysis using an anti-V5 antibody (upper panel), an anti-FLAG antibody (middle panel), or an anti-Myc antibody (lower panel). The asterisk indicates the background band.

Akt Promotes p27^Kip1 Binding to 14-3-3 through Phosphorylation at Thr¹⁹⁸-- We then examined the role of Thr¹⁹⁸ phosphorylation. We first examined p27^Kip1 binding to 14-3-3 because many proteins translocate to the cytoplasm by binding to 14-3-3, and the identified phosphorylation site (LRRRQpT¹⁹⁸) has homology to 14-3-3-binding motifs (RXX(pS/pT) or RXXX(pS/pT), where X is any amino acid and pS/pT represents phosphorylated serine or threonine) (34). FLAG-tagged p27^Kip1 was immunoprecipitated from 293T cells that had been transfected with pFLAG-p27^kip1 and pHM6-14-3-3. As shown in Fig. 6A (upper panel, lane 3), 14-3-3 was co-immunoprecipitated with p27^Kip1 only when Akt was cotransfected into 293T cells, indicating that 14-3-3 binds to the phosphorylated form of p27^Kip1 in vivo. As the 14-3-3 mutant (R56A/R60A), which loses its ligand binding ability (35), failed to bind to p27^Kip1 (Fig. 6A, upper panel, lane 4), the binding to 14-3-3 became specific. We further examined the p27^Kip1 binding capability for other 14-3-3 isoforms. Although all bound to Raf-1 (data not shown), p27^Kip1 could not bind to 14-3-3 and 14-3-3 (Fig. 6B, upper panel, lanes 3 and 4). Moreover, p27^Kip1 binding to 14-3-3 and 14-3-3 was very weak (Fig. 6B, upper panel, lanes 5 and 6), suggesting that the not all 14-3-3 isoforms bind to phospho-p27^Kip1.

View larger version (43K): [in this window] [in a new window]   Fig. 6.   Akt-mediated phosphorylation induces p27Kip1 binding to 14-3-3 proteins. A, 14-3-3 binding to p27Kip1 in cells. 293T cells were cotransfected with the pHM6 vector encoding WT 14-3-3 (WT; lanes 1-3) or mutant R56A/R60A 14-3-3 (R56,60A; lane 4), which loses its ligand binding ability, and the pUSEamp vector alone (; lane 2) or encoding myristoylated active Akt (Myr-Akt; +; lanes 1, 3, and 4) together with the pFLAG-CMV-2 vector alone (; lane 1) or encoding WT p27Kip1 (+; lanes 2-4). The FLAG-tagged p27Kip1 proteins were immunoprecipitated (IP), and co-immunoprecipitated proteins were subjected to immunoblot analysis using an anti-HA antibody (upper panel) or an anti-FLAG antibody (second panel). The expression level of Myc-tagged Akt, p27Kip1 phosphorylated at Thr198, and HA-tagged 14-3-3 proteins was confirmed by immunoblot analysis of the cell lysates using an anti-Myc antibody (third panel), an anti-phospho-Ser/Thr Akt substrate antibody (fourth panel), or an anti-HA antibody (lower panel). The asterisk indicates the background band. B, isoform specificity for 14-3-3 binding to p27Kip1. 293T cells were transfected with the pHM6 vector alone (Mock; lane 1) or encoding 14-3-3 (lane 2), 14-3-3 (lane 3), 14-3-3 (lane 4), 14-3-3 (lane 5), or 14-3-3 (lane 6) together with the pUSEamp vector encoding myristoylated active Akt (lanes 1-6) and the pFLAG-CMV-2 vector encoding WT p27Kip1 (lanes 1-6). The FLAG-tagged p27Kip1 proteins were immunoprecipitated, and co-immunoprecipitated proteins were subjected to immunoblot analysis using an anti-HA antibody (upper panel). The expression level of transfected HA-tagged 14-3-3 proteins was confirmed by immunoblot analysis of the cell lysates using an anti-HA antibody (lower panel).

We then tried to identify the phosphorylation-dependent 14-3-3-binding site in p27^Kip1 by transfecting 14-3-3 together with WT p27^kip1 and its point mutants S10A, T187A, T198A, and S10A/T187A/T198A into 293T cells. 14-3-3 binding to p27^Kip1 was diminished only when Thr¹⁹⁸ in p27^Kip1 was converted to Ala (T198A and S10A/T187A/T198A (Triple)) (Fig. 7A, upper panel, lanes 11 and 12). To confirm the results, we carried out peptide binding experiments. We obtained a biotinylated threonine-phosphorylated peptide (RRRQpT) and a non-phosphorylated peptide (RRRQT). The threonine-phosphorylated peptide bound to WT 14-3-3 (Fig. 7B, lane 5), but not to the 14-3-3 mutant (R56A/R60A) (lane 6). However, the non-phosphorylated peptide did not bind to either WT or mutant 14-3-3 (Fig. 7B, lanes 2 and 3). Thus, 14-3-3 recognizes and binds to p27^Kip1 only when Thr¹⁹⁸ is phosphorylated by Akt.

View larger version (79K): [in this window] [in a new window]   Fig. 7.   Involvement of Akt-mediated p27Kip1 phosphorylation at Thr198 in p27Kip1 binding to 14-3-3 protein. A, phosphorylation-dependent p27Kip1 binding to 14-3-3. 293T cells were cotransfected with the pHM6 vector encoding WT 14-3-3 (+; lanes 1-12) and the pUSEamp vector alone (; lanes 1-6) or encoding myristoylated active Akt (Myr-Akt; +; lanes 7-12) together with the pFLAG-CMV-2 vector alone (Mock; lanes 1 and 7) or encoding WT p27Kip1 (lanes 2 and 8), S10A p27Kip1 (lanes 3 and 9), T187A p27Kip1 (lanes 4 and 10), T198A p27Kip1 (lanes 5 and 11), or triple-point mutant S10A/T187A/T198A p27Kip1 (Triple; lanes 6 and 12). The FLAG-tagged p27Kip1 proteins were immunoprecipitated (IP), and co-immunoprecipitated proteins were subjected to immunoblot analysis using an anti-HA antibody (upper panel) or an anti-FLAG antibody (second panel). The expression level of transfected Myc-tagged Akt, p27Kip1 phosphorylated at Thr198, and HA-tagged 14-3-3 proteins was confirmed by immunoblot analysis of the cell lysates using an anti-Myc antibody (third panel), an anti-phospho-Ser/Thr Akt substrate antibody (fourth panel), and an anti-HA antibody (lower panel). Asterisks indicate the background bands. B, a threonine-phosphorylated peptide of COOH-terminal p27Kip1 can interact with 14-3-3 protein. The cell lysates of 293T cells that had been transfected with the pFLAG-CMV-2 vector alone (Mock; lanes 1, 4, and 7) or encoding WT 14-3-3 (WT; lanes 2, 5, and 8) or mutant R56A/R60A 14-3-3 (R56,60A; lanes 3, 6, and 9), which loses its ligand binding ability, were incubated with the biotin-PKKPGLRRRQT-amide peptide (RRRQT) (lanes 1-3) or its threonine-phosphorylated peptide (RRRQpT) (lanes 4-6). The proteins bound to each peptide were precipitated with avidin-agarose (lanes 1-6), and the coprecipitated proteins were probed using an anti-FLAG antibody. The expression level of transfected FLAG-tagged 14-3-3 was confirmed by immunoblot analysis of the cell lysates using an anti-FLAG antibody (lanes 7-9).

Cytoplasmic Localization of p27^Kip1 Phosphorylated at Thr¹⁹⁸-- To examine the role of 14-3-3 binding to p27^Kip1, we investigated the subcellular localization of p27^Kip1 in vivo. After transfecting WT, single-point mutant (T198A or S10A), or double-point mutant (T198A/S10A) of p27^kip1, nuclear and cytoplasmic fractions were separated using the NE-PER extraction kit. Immunoblot analysis with an anti-phospho-Ser/Thr Akt substrate antibody clearly indicated that p27^Kip1 phosphorylated at Thr¹⁹⁸ was localized only in the cytoplasm (Fig. 8A, upper panel, lanes 1 and 5). These results indicate that Akt-mediated phosphorylation at Thr¹⁹⁸ promotes 14-3-3 binding and participates in cytoplasmic localization (Fig. 8B). Because Akt-mediated phosphorylation also promotes Skp2 binding to p27^Kip1 (Fig. 5B), Akt might induce the proteasome-dependent degradation of p27^Kip1 through promoting its cytoplasmic localization.

View larger version (52K): [in this window] [in a new window]   Fig. 8.   Cytoplasmic localization of p27Kip1 phosphorylated at Thr198. A, 293T cells were transfected with the pFLAG-CMV-2 vector encoding WT p27Kip1 (lanes 1 and 2), T198A p27Kip1 (lanes 3 and 4), S10A p27Kip1 (lanes 5 and 6), or double-point mutant T198A/S10A p27Kip1 (lanes 7 and 8). The cytoplasmic (C) and nuclear (N) fractions were separated, electrophoresed, and immunoblotted using an anti-phospho-Ser/Thr Akt substrate antibody (upper panel), an anti-FLAG antibody (middle panel), or an anti-Akt antibody (lower panel). B, shown is a model for Akt-induced cytoplasmic localization of p27Kip1 through phosphorylation at Thr198 and promotion of binding to 14-3-3 proteins.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

By activation of PI3K, PtdIns(3,4,5)P₃ and PtdIns(3,4)P₂ are synthesized at the plasma membrane, and the serine/threonine kinase Akt is recruited to the plasma membrane (1, 2). Interaction of Akt with these lipids induces a conformational change in Akt. Then, Akt is phosphorylated at two key regulatory sites, Thr³⁰⁸ in the activation loop of the catalytic domain and Ser⁴⁷³ in the COOH-terminal domain. Dual phosphorylation at both residues is necessary for full activation of Akt. Activated Akt prevents cells from undergoing apoptosis and contributes tumor formation and progression by phosphorylating Bad, procaspase-9, IB kinase, and Forkhead transcription factors (1, 2). In addition to suppressing apoptosis, Akt contributes to cell-cycle progression. For example, activated Akt translocates to the nucleus and phosphorylates MDM2 (murine double minute 2) and p21^Waf1/Cip1, resulting in p53 degradation and cytoplasmic localization of p21^Waf1/Cip1 (36, 37). Akt is often activated in tumor cells lacking PTEN expression. By contrast, p27^Kip1 expression is frequently down-regulated in PTEN-negative tumor cells. Because transfection of PTEN induces growth arrest in part by up-regulating p27^Kip1 expression (22), it is possible that Akt promotes cell-cycle progression by regulating p27^Kip1 function.

The function of p27^Kip1 is regulated by changes in its concentration and in its subcellular localization. The p27^Kip1 concentration is reported to be controlled mainly by proteasome-dependent degradation (16-19). Phosphorylation of p27^Kip1 at Thr¹⁸⁷ by the cyclin E-CDK2 complex triggers p27^Kip1 binding to Skp2, a member of the F-box family of proteins that associates with the SCF (Skp1/Cul1/F-box protein) ubiquitin-ligase complex (31-33). In addition to the ubiquitin-dependent pathway, p27^Kip1 is degraded by ubiquitin-independent proteolytic cleavage (38). We found here that Akt also phosphorylated p27^Kip1 at Thr¹⁸⁷ (Fig. 4) and promoted p27^Kip1 binding to Skp2 (Fig. 5). Because several mitogenic factors are known to decrease p27^Kip1 protein amounts upon transition from the G₁ to S phase of the cell cycle (39, 40), Akt might contribute to cell-cycle progression by promoting p27^Kip1 destabilization through directly phosphorylating Thr¹⁸⁷.

To exhibit CDK inhibitory action, p27^Kip1 needs to be transported into the nucleus. The nuclear import of p27^Kip1 is dependent on the nuclear localization signal localized near the COOH terminus (41) and the interaction with nuclear pore-associated protein-60 (42). By contrast, the association with Jab1 (Jun activation domain-binding protein-1) promotes cytoplasmic localization and degradation of p27^Kip1 (43). Recently, phosphorylation at Ser¹⁰ by unknown kinases was reported to increase nuclear export of p27^Kip1 (30) through binding to CRM1 (20). However, phosphorylation of Ser¹⁰ is not sufficient to promote cytoplasmic localization of p27^Kip1 because the S10D mutant is also localized in the nucleus in G₀/G₁ cells. Rodier et al. (30) suggested that another signal provided by serum growth factors appears to be necessary to direct p27 to the cytoplasm. Thus, some kinases regulate degradation and cytoplasmic localization of p27^Kip1 through phosphorylation-dependent mechanisms. Because Akt is also activated by serum stimulation, we hypothesized that Akt participates in the phosphorylation and cytoplasmic localization of p27^Kip1. We observed that Akt bound to and phosphorylated p27^Kip1 in vivo and in vitro (Figs. 1 and 2). Identification of p27^Kip1 phosphorylation sites revealed that Akt phosphorylated p27^Kip1 at Ser¹⁰ (Fig. 4). Therefore, Akt might participate in nuclear export of p27^Kip1 as well as p27^Kip1 degradation. Moreover, Akt might be one of the unidentified Ser¹⁰ kinases.

In addition to Ser¹⁰ and Thr¹⁸⁷, we identified the COOH-terminal Thr¹⁹⁸ residue as a novel phosphorylation site (Fig. 3). Because the identified phosphorylation site around Thr¹⁹⁸ has homology to 14-3-3-binding motifs (RXX(pS/pT) or RXXX(pS/pT)) (34), we investigated p27^Kip1 binding to 14-3-3. As expected, 14-3-3 could bind to p27^Kip1 through Thr¹⁹⁸ only when it was phosphorylated by Akt (Fig. 7A). This result is supported by the fact that the synthetic phospho-Thr¹⁹⁸ peptide (RRRQpT), but not the non-phospho-Thr¹⁹⁸ peptide (RRRQT), bound to 14-3-3 in vitro (Fig. 7B). Because 14-3-3, 14-3-3, and 14-3-3 (but not 14-3-3 and 14-3-3) could form a complex with p27^Kip1 (Fig. 6B), 14-3-3 proteins might have some isoform specificity for recognizing their partners. 14-3-3 binding is known to promote cytoplasmic localization of some cell-cycle regulators such as Cdc25, Wee1, and CDK2 (34). Immunoblot analysis of the cytosolic and nuclear fractions with an anti-phospho-Ser/Thr Akt substrate antibody clearly indicated that phospho-p27^Kip1 (Thr¹⁹⁸) was localized only in the cytoplasm (Fig. 8A). Therefore, Akt might accelerate p27^Kip1 cytoplasmic localization by phosphorylating Thr¹⁹⁸ in addition to Ser¹⁰.

In summary, we discovered that Akt-mediated p27^Kip1 phosphorylation directly induces p27^Kip1 binding to 14-3-3 and cytoplasmic localization through phosphorylating the newly identified Thr¹⁹⁸ residue. Because Akt also phosphorylates p27^Kip1 at Thr¹⁸⁷ and Ser¹⁰, which are involved in Skp2-mediated ubiquitinylation (31-33) and cytoplasmic localization (20, 30), respectively, Akt contributes cytoplasmic localization and degradation of p27^Kip1. Zhou et al. (37) have recently reported that Akt-dependent phosphorylation promotes cytoplasmic localization of p21^Waf1/Cip1 in Her-2/neu-overexpressing cells. Overexpression of Her-2/neu is also known to promote cytoplasmic localization and degradation of p27^Kip1 (44, 45). Therefore, Akt might be involved in abnormal cell proliferation of Her-2/neu-overexpressing cells by phosphorylation-dependent cytoplasmic localization and degradation of both p27^Kip1 and p21^Waf1/Cip1 cell-cycle inhibitors. The Akt-mediated pathway seems to be very important for tumor growth control and may be a promising target for tumor treatment.

	ACKNOWLEDGEMENT

We thank Dr. S. Akinaga (Kyowa Hakko Kogyo) for providing UCN-01 and for valuable discussions.

	FOOTNOTES

^* This work was supported in part by a special grant for advanced research on cancer from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to T. T.) and by the Foundation for Promotion of Cancer Research in Japan (to N. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Inst. of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan. Tel.: 81-3-5841-7861; Fax: 81-3-5841-8487; E-mail: ttsuruo@iam.u-tokyo.ac.jp.

Published, JBC Papers in Press, May 31, 2002, DOI 10.1074/jbc.M203668200

	ABBREVIATIONS

The abbreviations used are: PI3K, phosphatidylinositide 3-OH kinase; PtdIns(3, 4,5)P₃, phosphatidylinositol 3,4,5-trisphosphate; PDK, 3-phosphoinositide-dependent protein kinase; CDK, cyclin-dependent kinase; PTEN, phosphatase and tensin homologue deleted in chromosome 10; WT, wild-type; Skp, S phase kinase-associated protein; HA, hemagglutinin; MOPS, 4-morpholinepropanesulfonic acid.

	REFERENCES

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