Cdc25C (cell division cycle 25C) phosphatase triggers entry into mitosis in the cell cycle by dephosphorylating cyclin B-Cdk1. Cdc25C exhibits basal phosphatase activity during interphase and then becomes activated at the G 2 /M transition after hyperphosphorylation on multiple sites and dissociation from 14-3-3. Although the role of Cdc25C in mitosis has been extensively studied, its function in interphase remains elusive. Here, we show that during interphase Cdc25C suppresses apoptosis signal-regulating kinase 1 (ASK1), a member of mitogen-activated protein (MAP) kinase kinase kinase family that mediates apoptosis. Cdc25C phosphatase dephosphorylates phospho-Thr-838 in the activation loop of ASK1 in vitro and in interphase cells. In addition, knockdown of Cdc25C increases the activity of ASK1 and ASK1 downstream targets in interphase cells, and overexpression of Cdc25C inhibits ASK1-mediated apoptosis, suggesting that Cdc25C binds to and negatively regulates ASK1. Furthermore, we showed that ASK1 kinase activity correlated with Cdc25C activation during mitotic arrest and enhanced ASK1 activity in the presence of activated Cdc25C resulted from the weak association between ASK1 and Cdc25C. In cells synchronized in mitosis following nocodazole treatment, phosphorylation of Thr-838 in the activation loop of ASK1 increased. Compared with hypophosphorylated Cdc25C, which exhibited basal phosphatase activity in interphase, hyperphosphorylated Cdc25C exhibited enhanced phosphatase activity during mitotic arrest, but had significantly reduced affinity to ASK1, suggesting that enhanced ASK1 activity in mitosis was due to reduced binding of hyperphosphorylated Cdc25C to ASK1. These findings suggest that Cdc25C negatively regulates proapoptotic ASK1 in a cell cycle-dependent manner and may play a role in G 2 /M checkpointmediated apoptosis.

Apoptosis signal-regulating kinase 1 (ASK1), a member of the mitogen-activated protein kinase kinase kinase family, plays a critical role in mediating apoptosis signals initiated by a variety of death stimuli such as hydrogen peroxide and tumor necrosis factor-a. Owing to its critical role in inducing apoptosis, the activity of ASK1 is tightly regulated by various mechanisms such as post-translational modifications and protein-protein interactions. Here we describe the identification of G 1 to S phase transition protein 1 (GSPT1), which is associated with protein translation, as a regulator of ASK1. GSPT1 interacts with ASK1 and enhances ASK1-induced apoptotic activity through the activation of caspase-3. In vitro kinase assay data show that GSPT1 enhances ASK1 autophosphorylation and its kinase activity. Cell cycledependent GSPT1 induction and small interfering RNA analyses show that ASK1 autophosphorylation is dependent on the expression level of endogenous GSPT1 in cells. GSPT1 inhibits the binding of ASK1 to the 14-3-3 protein, an ASK1 inhibitor, while GSPT1 has no effect on the interaction between ASK1 and TRAF2, a C-terminalbinding activator of ASK1. Thus, our results reveal a novel role of GSPT1 in the regulation of ASK1-mediated apoptosis.

Cystatin SN (CST1) is one of the several salivary cystatins that form tight equimolar complexes with cysteine proteases, such as the cathepsins. High expression of CST1 is correlated with advanced pTNM stage in gastric cancer. However, the functional role of CST1 in tumorigenesis has not been elucidated. In this study, we showed that CST1 was highly expressed in colon tumor tissues, compared with nontumor regions. Increased cell proliferation and invasiveness were observed in HCT116 cell lines stably transfected with CST1 cDNA (HCT116-CST1) but not in CST3-transfected cells. We also demonstrated that CST1-overexpressing cell lines exhibited increased tumor growth as well as metastasis in a xenograft nude mouse model. Interestingly, CST1 interacted with cystatin C (CST3), a potent cathepsin B (CTSB) inhibitor, with a higher affinity than the interaction between CST3 and CTSB in the extracellular space of HCT116 cells. CTSB-mediated cellular invasiveness and proteolytic activities were strongly inhibited by CST3, but in the presence of CST1 CTSB activities recovered significantly. Furthermore, domain mapping of CST1 showed that the disulfide-bonded conformation, or conserved folding, of CST1 is important for its secretion and for the neutralization of CST3 activity. These results suggest that CST1 upregulation might be involved in colorectal tumorigenesis and acts by neutralizing the inhibition of CTSB proteolytic activity by CST3.

-Jun N-terminal kinase (JNK) is activated by dual phosphorylation of both threonine and tyrosine residues in the phosphorylation loop of the protein in response to several stress factors. However, the precise molecular mechanisms for activation after phosphorylation remain elusive. Here we show that Pin1, a peptidyl-prolyl isomerase, has a key role in the JNK1 activation process by modulating a phospho-Thr-Pro motif in the phosphorylation loop. Pin1 overexpression in human breast cancer cell lines correlates with increased JNK activity. In addition, small interfering RNA (siRNA) analyses showed that knockdown of Pin1 in a human breast cancer cell line decreased JNK1 activity. Pin1 associates with JNK1, and then catalyzes prolyl isomerization of the phospho-Thr-Pro motif in JNK1 from trans-to cis-conformation. Furthermore, Pin1 enhances the association of JNK1 with its substrates. As a result, Pin1 À/À cells are defective in JNK activation and resistant to oxidative stress. These results provide novel insights that, following stress-induced phosphorylation of Thr in the Thr-Pro motif of JNK1, JNK1 associates with Pin1 and undergoes conformational changes to promote the binding of JNK1 to its substrates, resulting in cellular responses from extracellular signals. A variety of extracellular stimuli induce cellular activities such as survival, proliferation, differentiation, and apoptosis through the activation of a family of mitogen-activated protein kinases (MAPKs). Extracellular signal-regulated kinase (ERK), p38 MAPK, and c-Jun N-terminal kinase (JNK) are the members of MAPKs. 1,2 MAPKs are activated by phosphorylation of Thr and Tyr residues with the motif Thr-X-Tyr (X is Glu in ERK; Gly in p38; Pro in JNK) within a conserved phosphorylation loop. JNK, also known as stress-activated protein kinase, serves as a phosphorylation substrate for MAP kinase kinases (MAP2Ks) such as MKK4/7, which are activated in turn by phosphorylation via MAP2K kinases. [3][4][5] JNK is activated by phosphorylation of Thr and Tyr residues in the phosphorylation loop in response to several different stress factors including oxidative stress, inflammatory cytokines, protein synthesis inhibitors, growth factor withdrawal, chemotherapeutic drugs, and ultraviolet (UV) irradiation. 6 JNK phosphorylates and thereby regulates a variety of transcription factors, including c-Jun, ATF-2, Elk-1, p53, T-cell factor b1 (TCFb1), and c-Myc, as well as other factors such as BAD and Bcl-2. 7 However, how the duration of JNK activation is regulated has not been fully elucidated.The peptidyl-prolyl cis/trans-isomerase (PPIase) Pin1 is a small protein with an N-terminal WW domain and a C-terminal PPIase domain. 8 The WW domain binds phosphorylated Ser/ Thr-Pro (pSer/Thr-Pro) motifs 8-11 and the PPIase domain catalyzes cis/trans-isomerization of such proline-containing peptides. 8,12 Peptidyl-prolyl isomerization of phosphorylated moieties makes it possible to switch the conformation of a protein, thereby modulating protein activity, phosphorylation statu...