Myosin phosphatase and RhoA-activated kinase modulate neurotransmitter release by regulating SNAP-25 of SNARE complexReport as inadecuate

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Reversible phosphorylation of neuronal proteins plays an important role in the regulation of neurotransmitter release. Myosin phosphatase holoenzyme MP consists of a protein phosphatase-1 PP1 catalytic subunit PP1c and a regulatory subunit, termed myosin phosphatase targeting subunit MYPT1. The primary function of MP is to regulate the phosphorylation level of contractile proteins; however, recent studies have shown that MP is localized to neurons, and is also involved in the mediation of neuronal processes. Our goal was to investigate the effect of RhoA-activated kinase ROK and MP on the phosphorylation of one potential neuronal substrate, the synaptosomal-associated protein of 25 kDa SNAP-25. SNAP-25 is a member of the SNARE soluble N-ethylmaleimide sensitive factor attachment protein receptor complex, along with synaptobrevin and syntaxin, and the primary role of SNAP25 is to mediate vesicle fusion. We showed that MYPT1 interacts with SNAP-25, as revealed by immunoprecipitation and surface plasmon resonance based binding studies. Mass spectrometry analysis and in vitro phosphorylation-dephosphorylation assays demonstrated that ROK phosphorylates, while MP dephosphorylates, SNAP-25 at Thr138. Silencing MYPT1 in B50 neuroblastoma cells increased phosphorylation of SNAP-25 at Thr138. Inhibition of PP1 with tautomycetin increased, whereas inhibition of ROK by H1152, decreased the phosphorylation of SNAP-25 at Thr138 in B50 cells, in cortical synaptosomes, and in brain slices. In response to the transduction of the MP inhibitor, kinase-enhanced PP1 inhibitor KEPI, into synaptosomes, an increase in phosphorylation of SNAP-25 and a decrease in the extent of neurotransmitter release were detected. The interaction between SNAP-25 and syntaxin increased with decreasing phosphorylation of SNAP-25 at Thr138, upon inhibition of ROK. Our data suggest that ROK-MP play a crucial role in vesicle trafficking, fusion, and neurotransmitter release by oppositely regulating the phosphorylation of SNAP-25 at Thr138.

Author: Dániel Horváth , István Tamás , Adrienn Sipos, Zsuzsanna Darula, Bálint Bécsi, Dénes Nagy, Judit Iván, Ferenc Erdődi, Be



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