Regulation and function of two membrane-associated protein kinases
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Protein kinases, many of which are regulated by autophosphorylation, modify their substrates by phosphorylation. Due to their roles in cellular signaling cascades controlling processes such as cellular proliferation, differentiation, metabolism, and apoptosis, protein kinases have caught the attention of scientists. In this study, we characterized the regulation and functions of two human membrane-bound protein kinases, TRPM7 and PERK. TRPM7 (transient receptor potential melastatin 7) is a non-selective cation channel fused to an atypical kinase domain at the C-terminus that is implicated in cellular magnesium homeostasis. While the channel properties of TRPM7 have been studied extensively, little is known about the mechanisms regulating its kinase activity. Furthermore, to date, no specific small molecule inhibitor of TRPM7 kinase has been identified. Therefore, we characterized the biochemical and functional properties of TRPM7 autoactivity and discovered the first group of small molecule compounds targeting the kinase activity. Through a broad range of biochemical assays, we demonstrated that early autophosphorylation on three serine residues is required for TRPM7 kinase activity. These phosphorylations promote a tetramer to dimer transition of the catalytic domain, as well as its cellular association with myosin. Aided by the discovery of effective kinase inhibitors a role for the kinase domain in actomyosin contractility and migration was examined. PERK (PKR-like endoplasmic reticulum kinase) is a serine/threonine kinase, resident in the ER membrane and activated by the unfolded protein response (UPR), which detects the accumulation of unfolded proteins in the ER. The UPR and PERK are up-regulated in a variety of tumor types and may be critical for cancer cell adaptation. Therefore, a goal of this study was to identify PERK inhibitors that will provide new potential therapeutic strategies for treating cancer. We discovered a number of lead compounds and validated their ability to inhibit PERK kinase activity in vitro. The most potent PERK inhibitor was further tested to evaluate the modulation of the PERK signaling pathway in pancreatic cancer cells.