Magic™ Anti-CAMK4 (Phospho T200) monoclonal antibody, clone 4F6.F9

Background: The signaling pathways most critical to prion disease pathogenesis are as yet incompletely characterized. We have developed a kinomics approach to identify signaling pathways that are dysregulated during prion pathogenesis. The approach is sensitive and specific enough to detect signaling pathways dysregulated in a simple in vitro model of prion pathogenesis. Here, we used this approach to identify signaling pathways dysregulated during prion pathogenesis in vivo. Methods: Mice intraperitoneally infected with scrapie (strain RML) were euthanized at 70, 90, 110, 130 days post-infection (dpi) or at terminal stages of disease (155-190 dpi). The levels of 139 protein kinases in brainstem-cerebellum homogenates were analyzed by multiplex Western blots, followed by hierarchical clustering and analyses of activation states. Results: Hierarchical and functional clustering identified CaMK4 beta and MST1 signaling pathways as potentially dysregulated. Targeted analyses revealed that CaMK4 beta and its downstream substrate CREB, which promotes neuronal survival, were activated at 70 and 90 dpi in cortical, subcortical and brainstem-cerebellum homogenates from scrapie-infected mice. The activation levels of CaMK4 beta/CREB signaling returned to those in mock-infected mice at 110 dpi, whereas MST1, which promotes neuronal death, became activated at 130 dpi. Conclusion: Pro-survival CaMK4 beta/CREB signaling is activated in mouse scrapie at earlier times and later inhibited, whereas pro-death MST1 signaling is activated at these later times.

Melanoma is an aggressive cancer with increasing incidence and a growing lifetime risk that arises from normal melanocytes or their precursors. A thorough understanding of the molecular mechanism of melanomagenesis and melanoma biology is essential for the diagnosis, prognostication, and therapy of melanoma. Cyclin-dependent protein kinase 5 (Cdk5) is one of the proteins highly expressed in B16-F10 melanoma cells that controls melanoma cell motility, invasiveness, and metastatic spread and might be a promising novel therapeutic target. The effect of Cdk5 on proliferation and migration, which are important for carcinogenesis, has not been reported. In the current study, we found that siRNA-mediated knockdown of Cdk5 in B16-F10 melanoma cells inhibited melanoma cell proliferation through downregulation of the CaMK4-p-CREB pathway, inhibited migration through downregulation of p-CREB, integrin beta 1, and integrin beta 5, and also inhibited tyrosinase activity and melanin production through p-CREB-MITF regulation. The results indicate that Cdk5 controls melanoma development, with an essential regulatory role for p-CREB.