Downregulation of p21^Cip1 by ERK activation. (A) p21^Cip1 levels are decreased in Ras transgenic mice. Hepatic tissue from normal and H-Ras^V12-transgenic mice was lysed and immunoblotted with anti-p21^Cip1 and anti-phospho-ERK antibodies and normalized with anti-β-actin antibody. (B) Ras overexpression reduces p21^Cip1 level. H-Ras-transformed and mock-transformed NIH 3T3 cells were lysed and immunoblotted with anti-p21^Cip1, anti-phospho-ERK, and anti-H-Ras antibodies and normalized with anti-β-actin antibody. (C) ERK inhibition blocks EGF stimulation-induced p21^Cip1 downregulation. HeLa cells were pretreated with U0126 (20 μM, 1 h) or LY294002 (5 μM, 1 h) and then incubated with or without EGF. Cell lysates were prepared 40 min after stimulation, analyzed by immunoblotting with anti-p21^Cip1 and anti-phospho-ERK antibodies, and normalized with anti-β-actin antibody. (D and E) ERK inhibition prolongs p21^Cip1 half-life. HeLa cells were pretreated with U0126 (10 μM) or dimethyl sulfoxide (vehicle) for 1 h and were incubated with cycloheximide (CHX) (10 μg/ml) in the absence or presence of EGF (50 ng/ml). At the indicated times, cell lysates were prepared, analyzed by immunoblotting with anti-p21^Cip1 antibody, and normalized with anti-β-actin antibody. A representative Western blot (D) was quantified by densitometry (E). All signals are normalized to the signal at time zero (100%).

p21^Cip1 interacts with ERK2. (A) HEK293T cells were cotransfected with HA-p21^Cip1 and FLAG-ERK2 or control vector. After 36 h, cells were lysed and ERK2 was immunoprecipitated (I/P) with anti-FLAG (α-FLAG) antibody. p21^Cip1 and ERK2 were detected by immunoblotting with anti-HA (α-HA) and anti-FLAG antibodies, respectively. (B) HeLa cells were pretreated with MG132 alone and along with U0126 (10 μM) for 2 h and were treated with EGF (50 ng/ml) or left untreated for 30 min. Cells were lysed, and p21^Cip1 was immunoprecipitated (I/P) with an anti-p21^Cip1 (α-p21^Cip1) antibody. ERK1/2, polyubiquitinated p21^Cip1, and native p21^Cip1 were detected by immunoblotting with anti-ERK1/2, antiubiquitin (α-ubiquitin), and anti-p21^Cip1 antibodies, respectively. Total cell extracts were analyzed by immunoblotting with anti-phospho-ERK1/2, anti-ERK1/2, and anti-p21^Cip1 antibodies. (C) Aligned amino acid sequences of the ERK-docking FXF motifs of human p21^Cip1 and other known ERK substrates: human mitogen-activated protein kinase phosphatase 1 (hMKP1), Elk-1, phosphodiesterase 4A (PDE4A), serum response factor accessory protein 1 (hSAP-1), and mouse kinase suppressor of Ras 1 (mKsr1). Conserved residues are highlighted. Numbers on the right indicate the positions of the final residues shown in each case. (D) Aligned amino acid sequences of the ERK-docking KIM motifs of human p21^Cip1 and other known ERK substrates: hMKP2, Elk-1, PDE4A, striatally enriched tyrosine phosphatase (STEP), and p90 ribosomal S6 kinase 1 (RSK1). (E) HEK293T cells were cotransfected with either HA-tagged wild-type (WT) p21^Cip1 (1 μg) or docking mutant (DM) p21^Cip1 (0.4 μg) and FLAG-ERK2 or control vector. Different amounts of p21^Cip1 constructs were used to adjust the levels of protein expression equally. After 36 h, cells were incubated with MG132 for 4 h prior to preparing cell extracts. p21^Cip1 was immunoprecipitated with an anti-HA antibody. p21^Cip1 and ERK2 were detected by immunoblotting with anti-HA and anti-FLAG antibodies, respectively.

ERK2 alters the cellular localization of p21^Cip1. (A and B) HeLa cells were cotransfected with HA-tagged wild-type p21^Cip1 (WT) or T57A S130A mutant p21^Cip1 together with FLAG-ERK2 or the control vector. At 42 h after transfection, cells were treated with MG132 for 2 h prior to fixation. The cellular localization of p21^Cip1 was detected using an anti-HA antibody. After washing extensively in PBS, samples were further incubated with an anti-FLAG antibody to detect ERK2. Nuclei were visualized by DAPI staining. Cells were observed by fluorescence microscopy. A representative microscopic field in the lower middle panel in A shows that nuclear and cytoplasmic localization of p21^Cip1 is indicated by two cells (arrows) coexpressing FLAG-ERK2 (red signals in ERK2 staining; right panel) and HA-p21^Cip1 and that nuclear localization of p21^Cip1 is indicated by two cells (starred) expressing HA-p21^Cip1 only (no signal in ERK2 staining; right panel). (C) HeLa cells were transfected with HA-tagged wild-type p21^Cip1. At 42 h after transfection, cells were treated with MG132 and U0126 (10 μM) or DMSO (vehicle) for 2 h prior to treatment with EGF (50 ng/ml) for 30 min. After fixation, the cellular localization of p21^Cip1 was detected using an anti-HA antibody. Nuclei were visualized by DAPI staining. Cells were observed by fluorescence microscopy. (D) Cells in A and B were fractionated, and cytoplasmic (C) and nuclear (N) proteins were resolved by SDS-PAGE and analyzed by immunoblotting with anti-HA (α-HA), anti-FLAG (α-FLAG), antitubulin, and anti-lamin A/C antibodies. Lamin A/C and tubulin are nuclear and cytoplasmic markers, respectively. (E) HeLa cells were treated with MG132 and U0126 (10 μM) or with DMSO (vehicle) for 2 h prior to treatment with EGF (50 ng/ml) for 30 min. Cytoplasmic (C) and nuclear (N) proteins were resolved by SDS-PAGE and analyzed by immunoblotting with anti-p21^Cip1, anti-pERK1/2, anti-ERK1/2, antitubulin, and anti-PARP-1 antibodies. PARP-1 and tubulin are nuclear and cytoplasmic markers, respectively.

ERK2 promotes cell cycle progression via degradation of p21^Cip1. (A) HEK293 cells were transfected with HA-tagged wild-type (WT) p21^Cip1 or T57A S130A mutant p21^Cip1 together with FLAG-ERK2 or a control vector. A vector encoding membrane-bound GFP was cotransfected, and cells were incubated for 32 h. During the last 16 h, cells were starved in serum-free medium and then returned to normal medium for an additional 8 h before harvesting. The cell cycle profile of GFP-positive cells was determined by fluorescence-activated cell sorting analysis. (B) HCT116 p21^−/− cells were transfected with HA-tagged wild-type (WT) p21^Cip1 or T57A S130A mutant p21^Cip1 together with FLAG-ERK2 or a control vector and a membrane-bound GFP vector. Cells were treated and analyzed as for panel A. The levels of HA-p21^Cip1 and FLAG-ERK2 were analyzed by immunoblotting (see Fig. S2 in the supplemental material). (C) Schematic representation of the proposed pathway, showing that ERK2 interacts with and phosphorylates p21^Cip1 at Thr57 and Ser130; phosphorylation of these residues enhances p21^Cip1 ubiquitination and subsequent degradation through the proteasome pathway, leading to cell proliferation.

ERK2 induces proteasome-dependent p21^Cip1 degradation. (A and B) ERK2 expression decreases the p21^Cip1 level, an effect that is blocked by the proteasome inhibitor, MG-132. HeLa cells were transfected with a plasmid encoding FLAG-ERK2 (A) or FLAG-ERK1 (B). After 36 h, cells were incubated with or without MG132 for 4 h prior to preparing cell extracts. Extracts were analyzed by immunoblotting (I/B) with anti-p21^Cip1, anti-phospho-ERK1/2 (pERK1/2), anti-FLAG (α-FLAG), anti-ERK1/2 (α-ERK1/2), and anti-β-actin antibodies. (C) DN MEK increases p21^Cip1 levels. HEK293T cells were cotransfected with HA-tagged p21^Cip1-encoding and increasing amounts of DN-MEK-encoding plasmids. After 40 h, cells were harvested and lysed. Extracts were analyzed by immunoblotting with anti-HA (α-HA), anti-phospho-ERK1/2, anti-phospho-MEK1/2 (pMEK), anti-MEK1/2, and anti-β-actin antibodies. (D) shRNA-mediated ERK2 knockdown increases p21^Cip1 levels. HeLa cells were transfected with an ERK2 shRNA (shERK2) expression plasmid or control vector. After 48 h, cells were harvested and lysed. Extracts were analyzed by immunoblotting with anti-p21^Cip1, anti-ERK2, and anti-β-actin antibodies. (E) ERK2 expression induces p21^Cip1 ubiquitination. HEK293 cells were cotransfected with HA-tagged p21^Cip1- and His₆-tagged ubiquitin-encoding plasmids together with a FLAG-ERK2 expression plasmid or control vector. After 36 h, cells were incubated with MG132 for 4 h prior to preparation of cell extracts. His₆-ubiquitin-conjugated HA-p21^Cip1 proteins were purified by nickel affinity chromatography and analyzed by immunoblotting with an anti-HA antibody. Total cell extracts were analyzed by immunoblotting with anti-phospho-ERK1/2, anti-HA, anti-FLAG, anti-ERK1/2, and antiubiquitin antibodies.

ERK2 phosphorylates Thr57 and Ser130 residues in p21^Cip1 in vitro. (A) HEK293T cells transfected with either HA-p21^Cip1 or HA-ERK2 were lysed and separately immunoprecipitated with anti-HA antibody. p21^Cip1 and ERK2 immunoprecipitates were incubated in kinase reaction buffer containing [γ-³²P]ATP. Phosphorylated proteins were resolved by SDS-PAGE and analyzed by autoradiography. The lower two panels show immunoblots (I/B) for the p21^Cip1 and ERK2 proteins used in the kinase reactions, detected with an anti-HA (α-HA) antibody. (B) Recombinant ERK2 and p21^Cip1 proteins were purified from E. coli and incubated in kinase reaction buffer containing [γ-³²P]ATP. Phosphorylated proteins were resolved by SDS-PAGE and analyzed by autoradiography. The lower panel shows an immunoblot with anti-p21^Cip1 antibody as a loading control. (C) The samples in panel B resolved by SDS-PAGE were transferred onto a polyvinylidene difluoride membrane. Radioactive protein was cut from the membrane, acid hydrolyzed, mixed with cold phosphoserine, phosphothreonine, and phosphotyrosine standards, and separated on cellulose-thin-layer chromatography plates (Merck) in two dimensions using an HTLE 7000 (CBS Scientific) apparatus. The positions of standard phosphoamino acids were determined by ninhydrin staining (left panel), and ³²P-amino acids were located by autoradiography (right panel). (D) Identification of the ERK-mediated phosphorylation sites in p21^Cip1. Recombinant p21^Cip1 protein was phosphorylated in vitro with ERK2. After the sample was resolved on SDS-PAGE, the p21^Cip1 protein band was excised and subjected to proteolysis with trypsin, and the resulting peptides were analyzed by LC-MS/MS. Two different phosphopeptides were detected. The presence of y- and b-type fragment ions in MS/MS spectra enabled identification of the tryptic peptides, SGEQAEGpSPGGPGDSQGR and ERWNFDFVTEpTPLEGDFAWER; phosphorylated Thr57 and Ser130 are indicated. (E) HEK293T cells transfected with either HA-tagged wild-type or T57A S130A p21^Cip1 were lysed and immunoprecipitated with anti-HA antibody. p21^Cip1 immunoprecipitates were incubated with recombinant active ERK2 (rec-ERK2) in kinase reaction buffer containing [γ-³²P]ATP. Phosphorylated proteins were resolved by SDS-PAGE and analyzed by autoradiography. The lower two panels show immunoblots for the p21^Cip1 and ERK2 proteins used in the kinase reactions, detected with anti-HA and anti-ERK2 antibodies, respectively.

ERK2 phosphorylation of p21^Cip1 at Thr57 and Ser130 induces p21^Cip1 ubiquitination and subsequent degradation. (A) HEK293 cells were cotransfected with HA-tagged wild-type (WT) p21^Cip1, T57A S130A mutant p21^Cip1, or docking mutant (DM) p21^Cip1 together with His₆-ubiquitin and FLAG-ERK2 or a control vector. After 36 h, cells were incubated with MG132 for 4 h prior to preparation of cell extracts. His₆-ubiquitin conjugates were purified by nickel affinity chromatography and analyzed by immunoblotting (I/B) with an anti-HA (α-HA) antibody. Total cell extracts were analyzed by immunoblotting with anti-HA, anti-FLAG (α-FLAG), and antiubiquitin antibodies. (B) HEK293 cells were cotransfected with either HA-tagged wild-type (WT) p21^Cip1 or T57A S130A mutant p21^Cip1 and FLAG-ERK2 or control vector. Cells were cotransfected with an enhanced-GFP vector for normalization. After 40 h, cells were harvested and lysed, and extracts were analyzed by immunoblotting with anti-HA, anti-phospho-ERK1/2, anti-FLAG, and anti-GFP antibodies. (C) HeLa cells were transfected with HA-tagged docking mutant p21^Cip1 together with FLAG-ERK2 or a control vector. After 48 h, cells were harvested, and cell extracts were analyzed by immunoblotting with anti-p21^Cip1 (α-p21^Cip1), anti-FLAG, and anti-β-actin antibodies.