Rev. a timely control of mitosis onset. Unrestrained phosphorylation by JNK, as obtained by a cell cycle-stabilized form of JNK or as seen in some human tumors, results in aberrant cell cycle progression. Additionally, UV irradiation-induced G2/M checkpoint requires inactivation of Cdc25C by JNK phosphorylation. JNK phosphorylation of Cdc25C as well as Cdc25A establishes a novel link between stress signaling and unperturbed cell cycle and checkpoint pathways. (6). Moreover, activation of p38 was recently reported during the G2/M transition of the unperturbed cell cycle when it negatively targets Cdc25B (7). In addition, Rabbit Polyclonal to p38 MAPK JNKs (and in particular JNK2) have been found to be essential for the proliferation and survival of several cell lines obtained from gastrointestinal and prostate tumors, leukemia, melanoma, and glioblastoma (8,C16). Finally, initial studies in murine embryonic fibroblasts (MEFs) from JNK knock-out mice suggested that JNK1 is usually a SB-568849 negative and JNK2 a positive cell cycle regulator (17, 18), although more recent data generated using chemical genetic approaches suggest that both JNK1 and JNK2 are required for cell survival and proliferation (19, 20). Members of the Cdc25 family also appear to be exquisite targets of the G2/M checkpoint that delays entry into mitosis until DNA is usually faithfully and completely replicated. First, UV-induced G2/M checkpoint activation induces the phosphorylation and subsequent degradation of Cdc25A (21, 22). Moreover, Chk1 and Chk2 SB-568849 phosphorylates Cdc25B/C and negatively regulates their activity via cytoplasmic sequestration instigated by 14-3-3 proteins (23, 24). Finally, other kinases, such as p38s and MAPKAPK2, are also known to regulate Cdc25B/C localization and action by regulating, via phosphorylation, their conversation with 14-3-3s (25,C27). We have initially reported that stress-induced JNK activation (by anisomycin, sorbitol, or UV) causes phosphorylation of Cdc25C at serine 168 (Ser-168), inhibiting its phosphatase activity (28). However, the functional role of this event remained unclear. Here, we show that phosphorylation of Cdc25C at Ser-168 occurs during both unperturbed cell cycle progression and induction of the G2/M DNA damage checkpoint. Moreover, our data reveal that this phosphorylation is required for proper entry into mitosis and efficient checkpoint arrest after UV irradiation. EXPERIMENTAL PROCEDURES Cells and Transfection Human embryonic kidney 293T (HEK-293T) and HeLa cells were maintained in Dulbecco’s altered Eagle’s medium supplemented with 10% bovine serum without antibiotics. HFF-1 (diploid human normal skin fibroblasts) cells were purchased from the ATCC and cultured following the manufacturer’s recommendations. Freshly isolated primary MEFs, human malignant melanoma A375, and human glioblastoma/astrocytoma U87-MG cells were cultured in Dulbecco’s altered Eagle’s medium supplemented with 10% fetal bovine serum, 2 mm glutamine, 100 models/ml of penicillin, and 100 g/ml of streptomycin at 37 C. Transfections of HeLa and HEK-293T cells were performed with either Lipofectamine with the addition of Plus reagent (Invitrogen), Lipofectamine 2000 (Invitrogen), or FuGENE (Roche Diagnostics) following the manufacturer protocols. HFF-1 cells were nucleofected with the help of an Amaxa Nucleofector following the manufacturer protocols and indications. Efficiency of transfection was estimated by using a green fluorescent protein-encoding vector to be between 80 and 100% in all cases. All experiments performed with MEFs were done with cultures between passages 2 and 5. Cell viability assays were regularly performed in cell lines kept in culture by staining with crystal violet. All cell lines used in this study tested unfavorable for mycoplasma. Cell Cycle Synchronization For double-thymidine-block, either HeLa or HFF-1 cells were grown in the presence of thymidine (2 mm) for 18 h and then released into thymidine-free media for 6C8 h and finally grown again for 12 h with thymidine (2 mm) before their final release from G1/S-induced arrest. MEFs were synchronized by serum starvation (0.1% fetal bovine serum) for 48 h. For Fig. 1refers to exponentially growing cells, and fractions 1C4 correspond to cells in interphase, whereas fractions 5C10 correspond to cells in mitotic stages, as SB-568849 indicated by the FACS analysis (refers to the 54-kDa JNK2 isoform, and + refers to the 46-kDa JNK1 isoform present in HeLa cells. binding assay between JNKs and Cdc25C using bacterially purified recombinant proteins and reticulocyte extracts programmed to produce radiolabeled proteins as indicated. and show Cdk1 activity in the four different conditions (BL21-DE3-pLysS bacteria using standard techniques. All mutations described in this paper were generated by site-directed mutagenesis (QuikChange; Stratagene) and confirmed by sequencing. All chemicals, unless otherwise indicated, were purchased from Sigma. The cell-permeable peptide-based TAT-JIP1 (also known as JNK inhibitor VII) was purchased from Calbiochem (#420134) and used at 20 m. Antibodies The following antibodies were used in this study: cyclin B1 (Santa Cruz, GNS1 clone), Cdc2/Cdk1 (Santa Cruz: sc-54, sc-954, and POH-1 clone), pan-JNK (Santa Cruz,.