PKB/Akt mediates cell-cycle progression by phosphorylation of p27^Kip1 at threonine 157 and modulation of its cellular localization

The sequential activation of cyclin-dependent kinases (CDKs) regulates the eukaryotic cell cycle. CDK inhibitors (CKIs) negatively regulate CDKs, and CDK-bound G1 cyclins can activate CDKs¹. The CKIs can be subdivided into two families: the Ink4 family, consisting of p16^INK4a, p15^INK4b, p18^INK4c, p19^INK4d, and the Cip/Kip family, including p21^Cip1, p27^Kip1 and p57^Kip2 (ref. 1). p27^Kip1, first identified in cells arrested by transforming growth factor- (TGF-), is a potent inhibitor of cyclin E/CDK2 and cyclin A/CDK2 (refs. 2,3 and references therein). Its expression is highest in quiescent cells, and decreases upon re-entry into the cell cycle⁴. Antiproliferative signals such as mitogen withdrawal, contact inhibition, TGF-, cyclin AMP and inhibitors of the erbB (HER) network lead to accumulation and stabilization of p27 (refs.1,2,5, 6, 7, 8). The intracellular levels of p27 are highly regulated by post-translational mechanisms^{3, 4}. These include ubiquitin−proteasome-mediated degradation and proteolysis to remove the cyclin-binding domain^{9, 10}. During the G1-to-S transition, p27 is phosphorylated at Thr¹⁸⁷ by CDK2 (refs. 11, 12), resulting in its dissociation from cyclin E/CDK2 complexes and binding of SCF^Skp2, a protein complex that targets p27 for ubiquitination and degradation^{13, 14}.

The phosphatidylinositol-3 kinase (PI3K)-Akt pathway is centrally involved in cell proliferation, survival and motility¹⁵. The serine/threonine kinase Akt inactivates several pro-apoptotic molecules, including Bad, caspase-9, forkhead transcription factors, IB kinase and p53 (through MDM2-mediated phosphorylation)^{16, 17, 18, 19, 20, 21}, resulting in enhanced cell survival. Akt also participates in cell-cycle progression. By phosphorylating forkhead transcription factors, it inhibits AFX-mediated transcription of p27 (ref. 19). Akt has been shown to induce E2F activity²² and transcription of c-Myc²³. Akt phosphorylates and inactivates GSK-3, relieving cyclin D1 from GSK-3-mediated nuclear exclusion and proteolysis²⁴. Phosphorylation of the CKI p21^Cip1 by Akt induces its retention in the cytoplasm, preventing CDK inhibition and growth arrest²⁵. In this study, we present evidence that p27 is phosphorylated at Thr¹⁵⁷ by Akt both in vitro and in vivo. This phosphorylation results in cytoplasmic retention of p27, abrogation of its CDK2 inhibitory activity and cell-cycle progression.

Blockade of the HER2/neu receptor has been shown to inhibit PI3K and Akt and to upregulate p27 levels^{6, 7, 8}. Because p27 contains a putative Akt consensus phosphorylation site (RXXRXXT¹⁵⁷D) in its nuclear localization sequence (NLS; amino acids 151−166), we examined whether Akt from HER2-overexpressing cells can phosphorylate p27 and whether this effect can be modulated by inhibition of HER2. Treatment of BT-474 cells with Herceptin or the PI3K inhibitor LY294002 inhibited Akt, as measured by the ability of Akt precipitates to phosphorylate GSK-3 in vitro as well as phospho-Ser⁴⁷³ (P-S473) Akt immunoblot (Fig. 1a). Akt from BT-474 cells was equally potent in phosphorylating wild-type p27 and T187A-p27, a mutant in which Thr¹⁸⁷ of the CDK2 phosphorylation site is replaced by alanine (Fig. 1b). Similar results were obtained with recombinant Akt1 (data not shown), indicating that active Akt from HER2-overexpressing tumor cells may phosphorylate p27 in vitro in a HER2-dependent manner and that this phosphorylation occurred at a residue that is not part of the CDK2 site. To confirm that Akt is responsible for the in vitro phosphorylation of p27, we infected MCF-7 cells with adenovirus encoding either -galactosidase (-gal) or myristoylated Akt (Myr-Akt). Myr-Akt consists of Akt1 ligated to a myristoylation sequence, resulting in an enzyme approximately tenfold more active than the wild-type enzyme²⁶. Akt1 precipitated from cells infected with Myr-Akt but not from control cells induced phosphorylation of both wild-type p27 and T187A-p27 in vitro (Fig. 1c).

Figure 1. Cellular Akt phosphorylates p27 in vitro. a, BT-474 cells were treated with 10 g/ml Herceptin or 40 M LY294002 for 20 h, washed and lysed in NP-40 lysis buffer. Akt was precipitated with immobilized-Akt 1G1 IgG2a and used for in vitro kinase assays against GSK-3 (top panel) or tested for P-S473 Akt and total Akt content by immunoblot (lower panels). b, Active Akt was precipitated from proliferating BT-474 cells with immobilized-Akt 1G1 IgG2a (as in a) or control IgG2 and used for in vitro kinase assays with GST-purified wild-type (WT) p27 or T187A-p27 as substrates. c, MCF-7 cells were infected with adenoviruses encoding -gal or Myr-Akt (multiplicity of infection (MOI) of 5). Akt was precipitated as in b above and assayed for kinase activity against His-tagged WT p27 or T187A-p27 (ref. 8). Full Figure and legend (25K)

Figure 2. Akt associates with and phosphorylates p27 on Thr157. a, GST was cleaved off from bacterially-produced GST-p27 (5 g) fusions. The resulting wild-type (WT) p27 and T157A-, S161A- and S162A-p27 mutants were used as substrates in an in vitro kinase assay using recombinant active Akt. Equal loading of p27 per reaction was confirmed by Coomassie brilliant blue staining. In vivo phosphorylation was assessed by labeling 293T cells transfected with FLAG-27 with [32P]orthophosphate. Labeled FLAG-p27 was precipitated with FLAG-M2 antibody and analyzed by SDS-PAGE and autoradiography. To normalize for transfection efficiency, FLAG-precipitates were subjected to p27 immunoblot (bottom panel). b, 293T cells were transfected with FLAG-tagged wild-type or mutant p27. 1 g of 293T cell lysates was incubated with 2 g of FLAG-M2 antibody. Co-precipitated Akt was detected by immunoblot analyses using antibodies specific for total Akt, P-S473 Akt and FLAG. Ectopic FLAG-p27 was detected in immunoblots of Akt precipitates. c, Endogenous total Akt and p27 were precipitated from BT-474 cell lysates. Both Akt and p27 were measured in the precipitates by immunoblot analysis. Full Figure and legend (25K)

Figure 3. Akt phosphorylates p27 at Thr157in vivo. 293T cells transfected with FLAG-tagged wild-type (WT) or T157A-p27 were metabolically labeled with [32P]orthophosphate. FLAG-p27 was precipitated from cell lysates and analyzed by two-dimensional phosphopeptide mapping. To activate Akt, cells were infected with adenovirus encoding Myr-Akt (MOI of 20). Arrowheads indicate phosphopeptide spots induced by Myr-Akt that are absent in the T157A-p27 mutant. TLC, thin layer chromatography; TLE, thin layer electrophoresis. Full Figure and legend (36K)

Figure 4. Phosphorylation of Thr157 in p27 is required for Akt-induced cytosolic localization. a, 293T cells were transfected with wild-type (WT) FLAG-p27 or T157A-FLAG-p27. Subcellular localization of FLAG-p27 was monitored by FLAG immunostaining. Where indicated, dn-Akt, CA-MEK1 or dn-MEK1 was co-transfected with FLAG-p27 (vector transfection efficiency on glass coverslips was approximately 30%). In other cases, cells were infected with Myr-Akt adenoviruses for 3 h after transfection with FLAG-p27. At 21 h later, fixed cells were stained with antibody against FLAG (FLAG-p27, green on merge), antibody against HA (HA dn-Akt, red on merge) or Hoechst dye for DNA (blue on merge). b, Total cell lysates (50 g) were prepared from 293T cells transfected with WT-p27 or T157A-p27 with or without CA-MEK1 or dn-MEK1. The samples were resolved by SDS-PAGE and subjected to immunoblotting with total MAPK or P-MAPK antibodies. c, 293T cells transfected with WT-p27 or T157A-p27 were infected with Myr-Akt adenoviruses or -gal for 3 h after transfection. At 21 h later the cells were fractionated into nuclear and cytosol fraction and subjected to immunoblot analysis with cyclin D1 antibody or c-Jun (as nuclear marker) antibodies. d, BT474 cells on coverslips were treated with 40 M LY294002 or 5 M UO126 for 24 h, fixed and immunostained for total p27, P-T157 p27 or Hoechst dye. e, Total cell lysates were prepared from BT474 cells treated as in−or with 20 g/ml Herceptin for 24 h and subjected to the indicated immunoblot procedures. Full Figure and legend (121K)

We next examined whether the predominant nuclear localization of T157A-p27 altered its CDK inhibitory activity. 293T cells were transfected with Flag-tagged wild-type p27 and T157A-p27 for 16 h, followed by transduction with -gal or Myr-Akt adenoviruses for 3 h. We examined CDK2 activity against histone H1 (HH1) in vitro 21 h later. CDK2 activity was reduced to a larger degree in cells transfected with T157A-27 as compared to wild-type p27 (Fig. 5a, lanes 2 versus 4). Myr-Akt partially rescued the CDK2 inhibitory effect of wild-type p27 but did not alter the inhibitory effect of T157A-p27 (lanes 3 versus 5). Similar results were obtained by examining cyclin E−associated kinase activity. The changes in kinase activity were not due to the changes in cyclin E or CDK2 protein levels, as determined by immunoblot analyses (Fig. 5a). These data suggested an Akt-resistant effect of T157A-p27 on cell-cycle progression. Therefore, 293T cells were transfected with FLAG-tagged wild-type p27 or T157A-p27 followed by infection with -gal or Myr-Akt adenoviruses. Cells were permeabilized and then double-labeled with propidium iodide and antibody against FLAG. The transfection efficiency of each of the 27 plasmids was >75%. FLAG-expressing cells were gated and analyzed by flow cytometry. Both wild-type p27 and T157A-p27 increased the proportion of cells in G1 phase and reduced the S phase fraction. Myr-Akt rescued 293T cells from the cell-cycle delay induced by wild-type p27 but not by T157A-p27 (Fig. 5b and Table 1), further supporting a role for Akt-mediated phosphorylation of Thr¹⁵⁷ of p27 in the derepression of CDK2.

Figure 5. Anti-proliferative effect of T157A-p27 is not rescued by active Akt. a, CDK2 or cyclin E was precipitated from 293T cells transfected with FLAG-tagged wild-type (WT) or T157A-p27. Where indicated, cells were transduced with Myr-Akt or -gal adenoviruses. CDK2- or cyclin E-associated kinase activities were determined by using HH1 as a substrate. Reaction products were resolved by 12.5% SDS-PAGE and analyzed by autoradiography. Protein levels were determined by immunoblotting with antibodies against CDK2 or cyclin E. For each immunoprecipitation, the top panel is a kinase assay and the bottom panel is an immunoblot. b, FLAG-tagged WT- or T157A-p27 were expressed in 293T cells and then transduced with either -gal of Myr-Akt adenoviruses for 3 h (MOI of 20). At 21 h later, FLAG-positive cells were analyzed for cell-cycle distribution by flow cytometry. A representative DNA histogram and mean cell cycle distribution values from 4 independent experiments are shown. c, Top, 293T cells were mock-transfected (lane 1), or transfected with Flag-tagged WT-p27 (Lane 2) or T157A-p27 (lane 3). CDK2 was precipitated from whole cell lysates and subjected to FLAG immunoblot. Bottom, cytosolic and nuclear fractions of 293T cells transfected with WT- or T157A-p27 were prepared. Where indicated, cells were transduced with Myr-Akt or -gal adeno-viruses or co-transfected with CA-MEK1 or dn-MEK1. 50 g of each fraction were subjected to immunoblot analyses for FLAG and c-Jun (nuclear marker). To determine the association of FLAG-p27 with CDK2, CDK2 was precipitated from 500 g of each cytosolic or nuclear fraction and subjected to FLAG and CDK2 immunoblot analyses. T157A-p27 localized predominantly in the nucleus and, in contrast to WT-p27, was resistant to Myr-Akt induced exclusion from the nucleus. Full Figure and legend (112K)

Figure 6. Expression of P-S473 Akt in invasive breast carcinomas correlates with cytosolic p27. a, Sections from 100 breast cancers were subjected to immunohistochemistry to detect P-S473 Akt and p27. Tumor I, invasive ductal carcinoma with nuclear staining for P-S473 Akt and presence of p27 in both nucleus and cytosol (N/C). Tumor II, P-S473 Akt−negative invasive ductal carcinoma with expression of p27 detectable exclusively in tumor-cell nuclei (N). b, Percentage of primary breast cancers (n = 96) showing expression of cytoplasmic p27 as a function of the percentage of P-Akt-positive tumors cells. 0%, n = 33; 1−10%, n = 17; 11−25%, n = 19; >25%, n = 27 (P < 0.0001). Full Figure and legend (44K)

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The human p27 gene in pET vector (from M. Pagano, New York University) was subcloned into the FLAG-tagged vector pCMV-Tag2B (Stratagene, La Jolla, California). Site-directed mutagenesis was done according to the manufacturer's protocol (Promega). Thr¹⁵⁷, Ser¹⁶¹ or Thr¹⁶² of p27 were replaced by alanine using the following primers: T157A, 5'-AAGCGACCTGCAGCCGACGATTCT-3'; S161A, 5'-ACCGACGATTCTGCTACTCAAAACAAAAGA-3'; T162A, 5'-GACGATTCTTCTGCTCAAAACAAAAGAGCC-3'. For expression, p27 fragments were subcloned into pGEX-2T (Amersham, Piscataway, New Jersey). GST-p27 fusions were produced in BL21 E. coli strain and purified by glutathione-Sepharose chromatography; the GST portion was cleaved off with thrombin (Amersham). The dn Akt plasmid⁴⁰ was from P. Tsichlis (Fox Chase Cancer Center). Myr-Akt and -galactosidase adenoviruses were from W. Ogawa (Kobe University)²⁶. Active MEK1 and dominant-negative MEK1 were described previously⁴¹.

Akt was precipitated with Akt 1G1 monoclonal IgG_2a (New England Biolabs). Immune complexes were incubated with 0.04 g GSK3 (Sigma) or 5 g of GST-p27 with 10 Ci [-³²P]ATP (Amersham) as described⁸. In other cases, recombinant Akt1 (0.4 g/reaction; Upstate Biotechnology, Waltham, Massachusetts) was tested for kinase activity against wild-type and mutant p27. CDK2 and cyclin E kinase reactions were done as previously described⁴².

293T cells were transfected with 2 g FLAG-p27 and labeled with 500 Ci/ml of [³²P]orthophosphate for 18 h in phosphate-free DMEM containing 10% dialyzed FBS. FLAG-p27 was precipitated with antibody against FLAG and prepared for phosphopeptide mapping as described⁴³. Dried samples were treated overnight at 37°C with 20 g of TPCK-trypsin (Sigma). Digested peptides were applied to cellulose thin-layer chromatography plates (Sigma) and separated by electrophoresis at 1,000 V for 45 min in a buffer containing 2.2% formic acid and 7.8% acetic acid, pH 1.9. Chromatography was done with a buffer containing 38% n-butanol, 25% pyridine and 7.5% acetic acid.

293T cells were transfected with 2 g of FLAG-p27 with or without dn-Akt (2 g), CA-MEK1 (2 g) or dn-MEK1 (2 g). Where indicated, cells transfected with FLAG-p27 alone were infected 18 h later with Myr-Akt adenoviruses for 3 h (MOI of 20). Cells were washed with PBS, fixed with 4% paraformaldehyde (10 min) and permeabilized with 0.1% Triton X-100 (15 min). Localization of p27 was determined using anti-FLAG (1:500). Expression of HA-dn-Akt was monitored with polyclonal anti-HA (1:250). Endogenous p27 in BT474 cells was stained with monoclonal anti-p27 (1:250) and anti-Thr¹⁵⁷ P-p27 (1:250). After washes, samples were treated with Texas Red−conjugated anti-rabbit IgG (1:250) or Oregon Green−conjugated anti-mouse IgG (1:250). For DNA staining, samples were incubated with Hoechst 33342 dye (1 g/ml, 10 min). Immunofluorescence was recorded with a Princeton Instruments (Monmouth Junction, New Jersey) cooled digital CCD camera on a Zeiss Axiophot upright microscope.

Nuclear and cytoplasmic fractions were prepared as described⁸. Whole cell lysates were prepared in NP-40 lysis buffer (0.5% Nonidet P-40, 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EGTA, 200 M Na₃VO₄, 1 mM PMSF and 2 g/ml leupeptin/aprotinin). Lysates were centrifuged at 4°C for 10 min at 10,000g in Eppendorf microfuge (Hamburg, Germany) to remove cellular debris. Immunoblot analyses and immunoprecipitations were done as described previously⁸.

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