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Pancreatic cancer is characterized by constitutive activation of the mitogen-activated protein kinase (MAPK) pathway. Mutations of KRAS or BRAF and epigenetic abrogation of DUSP6 contribute synergistically to the constitutive activation of MAPK. Active MAPK induces the expression of a variety of genes that are thought to play roles in malignant phenotypes of pancreatic cancer. By blocking the functions of such induced genes, it is possible to attenuate the malignant phenotypes.The development of drugs targeting genes downstream of MAPK may provide a novel therapeutic option for pancreatic cancer. [ABSTRACT FROM AUTHOR]

N ε -carboxymethyllysine (CML) is an important driver of diabetic vascular complications and endothelial cell dysfunction. However, how CML dictates specific cellular responses and the roles of protein tyrosine phosphatases and ERK phosphorylation remain unclear. We examined whether endoplasmic reticulum (ER) localization of MAPK phosphatase-3 (MKP-3) is critical in regulating ERK inactivation and promoting NADPH oxidase-4 (Nox4) activation in CML-induced endothelial cell injury. We demonstrated that serum CML levels were significantly increased in type 2 diabetes patients and diabetic animals. CML induced ER stress and apoptosis, reduced ERK activation, and increased MKP-3 protein activity in HUVECs and SVECs. MKP-3 siRNA transfection, but not that of MKP-1 or MKP-2, abolished the effects of CML on HUVECs. Nox4-mediated activation of MKP-3 regulated the switch to ERK dephosphorylation. CML also increased the integration of MKP-3 with ERK, which was blocked by silencing MKP-3. Exposure of antioxidants abolished CML-increased MKP-3 activity and protein expression. Furthermore, immunohistochemical staining of both MKP-3 and CML was increased, but phospho-ERK staining was decreased in the aortic endothelium of streptozotocin-induced and high-fat diet-induced diabetic mice. Our results indicate that an MKP-3 pathway might regulate ERK dephosphorylation through Nox4 during CML-triggered endothelial cell dysfunction/injury, suggesting that therapeutic strategies targeting the Nox4/MKP-3 interaction or MKP-3 activation may have clinical implications for diabetic vascular complications. [ABSTRACT FROM AUTHOR]

Mitogen-activated protein kinase phosphatase-3 (MKP-3) negatively regulates ERK1/2 MAPK in a feedback loop. However, little is known about the molecular mechanism by which Ras signaling induces MKP-3 expression. In the present study, we demonstrate that exogenous expression of constitutively active H-Ras increases the level of MKP-3 mRNA. A transfection study using a series of MKP-3 promoter deletion constructs revealed that the c-Myb binding site is required for Ras-induced transcriptional activation of the MKP-3 gene promoter. Furthermore, we show that c-Myb directly binds to the MKP-3 promoter, as revealed by electrophoretic mobility shift assay and chromatin immunoprecipitation. Knock-down of c-Myb expression using siRNA abrogated Ras-induced MKP-3 promoter activity. These findings propose a novel mechanism through which Ras signaling activates c-Myb-dependent transcriptional activation of the MKP-3 gene. [ABSTRACT FROM AUTHOR]

DMKP-3 is a Drosophila dual-specificity phosphatase, which has high substrate specificity for Drosophila extracellular signal-regulated kinases (DERK). By in vitro reconstitution experiments, we found that DERK activates DMKP-3. Moreover, DMKP-3 was specifically activated by the addition of DERK but not by DJNK, Dp38, or Sevenmaker DERK D334N, a DMKP-3-binding mutant. The phosphatase activity of DMKP-3-R56A/R57A, a DERK-binding mutant, was not increased by DERK. Significantly, mammalian MKP-3 was also found to be activated by DERK. This cross-reactivity suggests a high level of conservation of the activation mechanism of ERK-specific phosphatases in Drosophila and mammals. When DMKP-3 was co-expressed with DERK in Drosophila Schneider cells, DMKP-3 protein levels increased, but this was not observed for the co-expressions of DJNK or Dp38. The stabilizations of the DERK binding mutants (DMKP-3-RR and DMKP-3-CA-RR) were not increased by DERK co-expression. Our results suggest that DERK specifically regulates DMKP-3 in terms of its enzyme activity and protein stability, and that direct protein-protein interaction is an essential aspect of this regulation. [ABSTRACT FROM AUTHOR]