Protein phosphatase‐1M and Rho‐kinase affect exocytosis from cortical synaptosomes and influence neurotransmission at a glutamatergic giant synapse of the rat auditory system

Abstract

Protein phosphatase‐1M (PP1M, myosin phosphatase) consists of a PP1 catalytic subunit (PP1c) and the myosin phosphatase target subunit‐1 (MYPT1). RhoA‐activated kinase (ROK) regulates PP1M via inhibitory phosphorylation of MYPT1. Using multidisciplinary approaches, we have studied the roles of PP1M and ROK in neurotransmission. Electron microscopy demonstrated the presence of MYPT1 and ROK in both pre‐ and post‐synaptic terminals. Tautomycetin (TMC), a PP1‐specific inhibitor, decreased the depolarization‐induced exocytosis from cortical synaptosomes. trans‐4‐[(1R)‐1‐aminoethyl]‐N‐4‐pyridinylcyclohexanecarboxamide dihydrochloride, a ROK‐specific inhibitor, had the opposite effect. Mass spectrometry analysis identified several MYPT1‐bound synaptosomal proteins, of which interactions of synapsin‐I, syntaxin‐1, calcineurin‐A subunit, and Ca2+/calmodulin‐dependent kinase II with MYPT1 were confirmed. In intact synaptosomes, TMC increased, whereas Y27632 decreased the phosphorylation levels of MYPT1Thr696, myosin‐II light chainSer19, synapsin‐ISer9, and syntaxin‐1Ser14, indicating that PP1M and ROK influence their phosphorylation status. Confocal microscopy indicated that MYPT1 and ROK are present in the rat ventral cochlear nucleus both pre‐ and post‐synaptically. Analysis of the neurotransmission in an auditory glutamatergic giant synapse demonstrated that PP1M and ROK affect neurotransmission via both pre‐ and post‐synaptic mechanisms. Our data suggest that both PP1M and ROK influence synaptic transmission, but further studies are needed to give a full account of their mechanism of action.

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

“…The effects of phosphatase inhibitors on neuronal exocytosis have been investigated. Tautomycetin, a PP1-specific inhibitor, decreases depolarization-induced exocytosis, while Y27632, a ROK-specific inhibitor, has the opposite effect [16]. Myosin phosphatase (MP) first identified in smooth muscles, is a Ser/Thr specific protein phosphatase 1 (PP1) consisting of a 38 kDa catalytic subunit (PP1c) of the β/δ isoform, a 130/133 kDa myosin phosphatase target subunit-1 (MYPT1) and a 20 kDa subunit of largely unknown function [17].…”

“…Myosin phosphatase (MP) first identified in smooth muscles, is a Ser/Thr specific protein phosphatase 1 (PP1) consisting of a 38 kDa catalytic subunit (PP1c) of the β/δ isoform, a 130/133 kDa myosin phosphatase target subunit-1 (MYPT1) and a 20 kDa subunit of largely unknown function [17]. MP (also termed PP1M) is involved in exocytotic processes; MYPT1 interacts and affects the dephosphorylation of several neuronal proteins [16]. Phosphorylation of Thr696 and/or Thr853 in MYPT1 by ROK results in the inhibition of MP [18, 19], and these regulatory events have also been identified in cortical synaptosomes [16].…”

“…MP (also termed PP1M) is involved in exocytotic processes; MYPT1 interacts and affects the dephosphorylation of several neuronal proteins [16]. Phosphorylation of Thr696 and/or Thr853 in MYPT1 by ROK results in the inhibition of MP [18, 19], and these regulatory events have also been identified in cortical synaptosomes [16]. MP is present in cortical synaptosomes in association with ROK [20], suggesting that MP is involved in synaptic signaling.…”

Interplay of myosin phosphatase and protein phosphatase-2A in the regulation of endothelial nitric-oxide synthase phosphorylation and nitric oxide production

“…In case of MP uncovering of MYPT1 binding protein partners led to the identification of several putative substrates24 and other interacting proteins important in the regulation of MP activity25. Thus, to prove eNOS as a possible substrate of MP we assessed initially if eNOS and MYPT1 interact in ECs or in tsA201 cells where these two proteins were co-expressed.…”

Identification of protein phosphatase interacting proteins from normal and UVA-irradiated HaCaT cell lysates by surface plasmon resonance based binding technique using biotin–microcystin-LR as phosphatase capturing molecule

“…Up to now isolation and analysis of phosphatase associated proteins from cell lysates has been carried out by conventional methods (immunoprecipitation, pull-down assays followed by SDS-PAGE, Western blot and/or mass spectrometry) which is quite time consuming and needs substantial amounts of samples and accessory proteins (i.e., antibodies, tagged recombinant proteins, etc.) [23,41]. A novel…”