The first-dimension separation was at 1

The first-dimension separation was at 1.5 kV for 20 min in TLE (pH 1.9) buffer containing 2.2% formic acid and 7.8% acetic acid; the second-dimension separation was at 1.3 kV for 13 min in TLE (pH 3.5) buffer containing 5% acetic acid and 0.5% pyridine. p21Cip1 translocation and degradation, thereby impairing ERK2-dependent cell cycle progression at the G1/S transition. These results indicate that ERK2 activation transduces mitogenic signals, at least in part, by downregulating the cell cycle inhibitory protein p21Cip1. The cyclin-dependent kinase (CDK) inhibitor p21Cip1 is usually important in the control of cell proliferation, differentiation, senescence, and apoptosis. p21Cip1 was initially identified as a component of a quaternary complex made up of CDK, cyclin, and proliferating cell nuclear antigen (PCNA) that regulates cell cycle progression MS023 and DNA replication. Overexpression of p21Cip1 results in cell cycle arrest (11), and p21Cip1 expression is induced at the transcriptional level by activation of p53 (10). Although the inhibitory role of p21Cip1 is usually well established, a positive role for p21Cip1 as an assembly factor for cyclin D1-CDK4/6 complexes has also been shown (8, 18). In addition to transcriptional regulation, p21Cip1 function can be regulated at the posttranslational level. AKT, protein kinase C zeta, MS023 CDK2, and glycogen synthase kinase 3 (GSK-3) phosphorylate p21Cip1 at Thr145, Ser146, Ser130, and Thr57/Ser114, respectively, resulting in inhibition, translocation, or destabilization of p21Cip1 (19, 27, 28, 31, 40, 41). Paradoxically, phosphorylation of Ser130 (by JNK1 or p38) or Ser146 (by AKT) has also been reported to enhance p21Cip1 stability (16, 20). p21Cip1 is usually a highly unstable protein (7, 21) that has been shown to accumulate following proteasome inhibition (3, 29). Multiple mechanisms appear to be involved in the proteasomal degradation of p21Cip. Some of these mechanisms are ubiquitination dependent, as well as others are ubiquitination impartial (33), including mechanisms mediated by an Skp2-made up of BMP7 SCF (Skp1, Cullin, and F-box MS023 protein) complex (2, 5) and by N-terminal ubiquitination (4) and a mechanism mediated by direct p21Cip1 interaction with the C8 subunit of the 20S proteasome (34). We previously exhibited that nucleocytoplasmic translocation of p21Cip1, mediated by two nuclear export sequences (NES), is required for p21Cip1 degradation (13). The Ras-dependent extracellular signal-regulated kinase 1/2 (ERK1/2) pathway plays a central role in controlling cell proliferation (22). Various mechanisms have been proposed to explain this action of the ERK1/2 pathway. For example, the ERK pathway has been shown to induce cyclin D1 MS023 transcription (1, 38) and to enhance the stability of the c-Myc protein (32), which play a central role in cell cycle progression and cell growth. A recent study has revealed that ERK associates with and phosphorylates GSK-3, resulting in inactivation of GSK-3 and upregulation of -catenin, which in turn stimulates c-Myc and cyclin D1 transcription (9). ERK also directly interacts with and phosphorylates FOXO3a, downregulating it by enhancing its degradation, thereby promoting cell proliferation (39). While these observations have provided tantalizing mechanisms, a complete picture of ERK1/2 regulation of cell proliferation has yet to emerge (22). Recently we observed that p21Cip1 protein levels were decreased in hepatocytes from H-RasV12-transgenic mice, which contain high levels of constitutively activated ERK. Here we focus on p21Cip1 downregulation as an alternative mechanism of Ras-ERK signaling-mediated cell proliferation. We demonstrate that ERK2 phosphorylates p21Cip1 on both Thr57 and Ser130 and show that this phosphorylation leads to cytoplasmic translocation, ubiquitination, and proteasome-dependent degradation of p21Cip1, thereby resulting in cell cycle progression. MATERIALS AND METHODS Reagents. DNase-free RNase A and protein A-agarose were purchased from Sigma. MG-132 was purchased from EMD Biosciences. Nickel affinity agarose from Qiagen and 4,6-diamidino-2-phenylindole (DAPI) from Roche were used. Cycloheximide, U0126, LY294002, and epidermal growth factor (EGF) were purchased from Calbiochem. Blasticidin, zeocin, and tetracycline were purchased from Invitrogen. The antibodies against green fluorescent protein (GFP) (FL), H-Ras (C20), lamin A/C (N18), p21Cip1 (C19 and F5), ubiquitin (P4D1), MEK1/2 (12B), and -actin (I19) were all obtained from Santa Cruz.