TRPM7 kinase domain is part of Rac1-SSH2-cofilin complex regulating F-actin in neural system

Abstract Impairment in phosphorylation/dephosphorylation (inhibition/activation) of cofilin, an actin filaments (F-actin) severing protein, can result in numerous brain diseases. The α-kinase domain (M7CK) of transient receptor potential melastatin-like 7 (TRPM7) ion channel interacts with diverse signaling molecules regulating different cellular mechanisms. The kinase protects synaptic density, plasticity and learning and memory functions by phosphorylating cofilin, and hence protecting F-actin within spines. However, mechanisms underlying regulation of cofilin activity and/or F-actin by M7CK remain unknown. Using Western blot analysis in brain tissue, we found that knockout of TRPM7 in glutamatergic neurons of mice resulted in reduction in the phosphorylation levels of cofilin and its activator the protein phosphatase slingshot homolog 2 (SSH2). Co-immunoprecipitation experiments showed direct interaction between M7CK, cofilin and SSH2. Kinase enzymatic activity assay showed that both proteins are phosphorylation targets of M7CK. Upstream from this complex, we found that the GTPase Rac1 can also be co-immunoprecipitated. GTPase activity assay showed that M7CK is a phosphorylation target of Rac1. At the functional level, we found that knockdown (in vitro) or knockout (in vivo) of TRPM7 resulted in reduction in phalloidin puncta within neuronal dendritic areas indicating that F-actin is severed in the absence of M7CK. Expression of the kinase domain, but not the ion channel part, rescued phalloidin puncta in neuronal cell cultures following suppression of TRPM7. Our data suggest that the α-kinase domain of TRPM7 is activated by Rac1. Then the kinase protects F-actin by inhibiting cofilin; directly by phosphorylation and indirectly by preventing its dephosphorylation via SSH2-phosphatase.