Article Figures & Data
Figures
- FIG. 1.
K cyclin promotes the phosphorylation of p21Cip1 on serine 130 in vitro. (A) K cyclin/cdk6 was expressed in Sf9 cells by infection with recombinant baculoviruses and used in kinase assays with [γ-32P]ATP and GST-p21Cip1 from bacteria as a substrate. Sf9 cells infected with wild-type baculovirus were used for the negative control. Products of the phosphorylation reactions were resolved by SDS-PAGE and subjected to autoradiography. (B) GST-p21Cip1 phosphorylated by K cyclin/cdk6 was subject to two-dimensional tryptic phosphopeptide mapping with electrophoresis in the horizontal direction and chromatography in the vertical direction. The point of sample application is marked by “x.” (C) The phosphopeptide shown in panel B was eluted from the cellulose plate and subjected to acid hydrolysis. The resulting hydrolysate was resolved by electrophoresis and subjected to autoradiography. The positions of phosphoamino acid standards are shown. (D) Wild-type and indicated mutant forms of p21Cip1 [N indicates p21(1-103) and C indicates p21(102-164)] were synthesized in bacteria as GST fusion proteins and used as substrates in kinase reactions with K cyclin/cdk6, as shown in panel A.
- FIG. 2.
K cyclin can promote the phosphorylation of p21Cip1 on serine 130 in vivo. (A) NIH 3T3-K cells were cultured for 3 days in the absence (−) or presence (+) of IPTG to induce K cyclin expression. Cells were then lysed and subjected to immunoblotting for endogenous p21Cip1 and K cyclin. (B) NIH 3T3 cell lysates were heated to 95°C for 5 min, denatured protein was removed by centrifugation, and the resulting extract was incubated in the absence (−) or presence (+) of calf intestinal alkaline phosphatase prior to immunoblotting for p21Cip1. (C) U2-OS cells were transfected with plasmids directing the expression of HA-K cyclin, wild-type p21Cip1, and either wild-type cdk6 or kinase-dead cdk6. After 48 h, cells were lysed and subjected to immunoblotting for p21Cip1. (D) Poly(His)-tagged p21Cip1 (His-p21) was isolated from recombinant bacteria and used as a substrate in kinase reactions, as described in the legend to Fig. 1, with nonradioactive ATP. Products of the phosphorylation reactions were resolved by SDS-PAGE and immunoblotted using either an anti-carboxy-terminal p21Cip1 antibody (pan) or the anti-S130 phosphospecific serum (pS130). (E) NIH 3T3-K cells induced to express K cyclin were lysed and subjected to immunoprecipitation with either an anti-carboxy-terminal p21Cip1 antibody (pan) or the anti-S130 phosphospecific p21Cip1 serum (pS130). The products of the immunoprecipitates (IP) were immunoblotted with the anti-carboxy-terminal p21Cip1 antibody and compared to a sample of the starting material (Total).
- FIG. 3.
K cyclin associates with and promotes phosphorylation of p21Cip1 both in transfected cells and in PEL-derived cells. (A) Wild-type and indicated mutant forms of p21Cip1 were transfected into U2-OS cells, together with Myc-K cyclin or control expression vectors. After 48 h, cells were lysed and subjected to immunoprecipitation using anti-Myc antibody. Immunoprecipitates were assayed for kinase activity towards coprecipitated proteins and analyzed by SDS-PAGE and autoradiography (top). The K cyclin-associated p21Cip1 was analyzed by immunoblotting with anti-p21Cip1 antibody. Total lysates were immunoblotted with anti-p21Cip1 and anti-Myc antibodies. (B) Lysates of the PEL and JOK-1 cells were immunoprecipitated using either anti-K cyclin or anti-p21Cip1 antibodies and analyzed for associated proteins on SDS-PAGE by immunoblotting for K cyclin and p21Cip1. Total lysates of PEL cells (50 μg) were resolved by SDS-PAGE (12%) and subjected to immunoblotting for endogenous p21Cip1 and K cyclin.
- FIG. 4.
K cyclin/cdk6 phosphorylates p21Cip1 in PEL cells. (A) Lysates from the BC-3 cell line were separated by gel filtration chromatography on a Superdex 200 column. Fractions were resolved by SDS-PAGE (12%) and immunoblotted with antibodies to K cyclin and p21Cip1. The elution profile of the molecular mass standards is indicated in kilodaltons. (B) A representative fraction (eluting at 110 kDa) was immunoprecipitated with anti-K cyclin antibody and either assayed for kinase activity towards coprecipitated endogenous p21Cip1 by autoradiography or immunoblotted for the indicated proteins. “Block” indicates that the immunoprecipitating antibody was pretreated with the antigen (+) or treated with a nonspecific protein (−). (C) The same fraction shown in panel B was immunodepleted of p21Cip1 with three consecutive rounds of immunoprecipitation using anti-p21Cip1 antibody (depleted, +) or with rabbit immunoglobulin G for control (−). The two panels show immunoblots for the indicated protein after these cycles of immunodepletion. The p21Cip1- and control-depleted extracts were then immunoprecipitated with anti-K cyclin antibody and subjected to an in vitro kinase assay of coprecipitated endogenous p21Cip1. Kinase activity was determined by autoradiography after SDS-PAGE (12%) (right, top). Immunoprecipitated (IP) proteins were analyzed by immunoblotting for K cyclin and p21Cip1 (right, middle and bottom). (D) Lysates from BC-3 cells were immunodepleted for cdk2, cdk4, and cdk4 by three consecutive rounds of immunoprecipitation with anti-cdk2, anti-cdk4, or anti-cdk6 antibodies. In the control (−), lysate was immunoprecipitated with rabbit immunoglobulin G (IgG). Depleted lysates were subjected to immunoprecipitation by anti-K cyclin antibody and an in vitro kinase assay of GST-p21Cip1. Kinase activity was determined by autoradiography after SDS-PAGE (12%). cdk-depleted lysates (40 μg) were resolved by SDS-PAGE (12%) and immunoblotted with antibodies against cdk2, cdk4, and cdk6. (E) BC-3 cells were lysed and subjected to immunoprecipitation using anti-K cyclin antibody. Immunoprecipitates were assayed for kinase activity with wild-type or S130A mutant GST-p21Cip1 as a substrate. Products of the phosphorylation reactions were resolved by SDS-PAGE (12%) and subjected to autoradiography.
- FIG. 5.
S130 phosphorylation does not alter p21Cip1 turnover or subcellular localization but restricts binding to cyclin/cdk2 complexes. (A) Logarithmically growing cultures of NIH 3T3-K cells (cultured in either the absence or presence of IPTG for 24 h) were pulse labeled with [35S]methionine and cysteine and chased in medium containing unlabeled amino acids. At the indicated time, cell extracts were prepared and immunoprecipitated with p21Cip1 antiserum. The labeled proteins were fractionated by SDS-PAGE and subjected to autoradiography. (B) Quantitation of the immunoprecipitated p21Cip1 shown in panel A in the absence (squares) or presence (triangles) of K cyclin by phosphorimager analysis. (C) NIH 3T3-K cells (cultured in either the absence or presence of IPTG for 24 h) were fractionated into nuclear and cytoplasmic samples prior to immunoblotting for p21Cip1, E2F-1, and α-tubulin. (D) BC-3 cells were separated into cytosolic and nuclear extracts. The resulting fractions were resolved by SDS-PAGE (12%) and analyzed by immunoblotting for p21Cip1, Sp1, and β-tubulin. cdk2 or cyclin D1 (E) or p21Cip1 or S130-phosphorylated p21Cip1 (F) were immunoprecipitated from NIH 3T3-K cells (cultured in the presence of IPTG), and the immune complexes were immunoblotted for the indicated proteins.
- FIG. 6.
The K cyclin-dependent circumvention of a p21Cip1-imposed G1 arrest is dependent on S130 phosphorylation. (A) U2-OS cells were transfected with plasmids directing the expression of histone 2B-GFP (as a marker of transfected cells), wild-type or S130A mutant p21Cip1, and 2× Flag-K cyclin. BrdU was added after 24 h; after an additional 24 h, cells on coverslips were fixed, BrdU-positive cells were detected by indirect immunofluorescence to BrdU, and DNA was counterstained with DAPI. (B) Cells shown in panel A were lysed and subjected to immunoblotting for exogenous p21Cip1 and K cyclin. (C) U2-OS cells were transfected with Myc-tagged K cyclin and HA-tagged wild-type or S130A mutant p21Cip1 and treated with BrdU as in panel A. Indirect immunofluorescence was used to detect BrdU incorporation and p21Cip1 (via anti-HA antibodies). Nuclei were visualized by Hoechst staining. Representative cells are marked with arrows. (D) Myc-K cyclin was expressed in U2-OS cells alone or together with HA-tagged wild-type or S130A mutant p21Cip1. After 48 h, cells were lysed and subjected to immunoprecipitation with anti-Myc antibody. Immunoprecipitates were assayed for kinase activity towards pRb and histone H1 and analyzed by autoradiography following SDS-PAGE.
- FIG. 7.
K cyclin-dependent bypass of DNA damage-induced cell cycle arrest is accompanied by S130 phosphorylation of p21Cip1 and partial restoration of cyclin/cdk2 kinase activity. (A) NIH 3T3-K cells were cultured in the absence (−) or presence (+) of IPTG to induce K cyclin expression for ∼16 h. Cells were then treated with vehicle or hydrogen peroxide for 4.5 h before lysis and immunoblotting for the indicated proteins. (B) Cells shown in panel A were treated with BrdU for the final 30 min of hydrogen peroxide treatment, and indirect immunofluorescence was used to detect S-phase progression. Data represent the mean ± standard error of at least four separate experiments with at least 100 cells scored per experiment. (C) U2-OS-K cells were cultured in the absence (−) or presence (+) of IPTG to induce K cyclin expression for ∼20 h. Cells were then treated with vehicle or hydrogen peroxide for 20 h and lysed. Lysates were immunoprecipitated through cdk2 and in vitro kinase assays of pRb and histone H1. Kinase activity was determined by autoradiography after SDS-PAGE (12%). Immune complexes and total lysates were immunoblotted for the proteins indicated.
