Integrin-linked kinase phosphorylates the myosin phosphatase target subunit at the inhibitory site in platelet cytoskeleton

Abstract

The myosin phosphatase (MP) composed of the catalytic subunit of type 1 protein phosphatase and myosin phosphatase target subunit isoform 1 (MYPT1) was identified as the major serine/threonine phosphatase component in the platelet-cytoskeleton fraction. MYPT1 was phosphorylated by cytoskeletal kinase(s), but the identity of the kinase(s) and the effect of phosphorylation were not established. Incubation of platelet-cytoskeletal fraction with MgATP or MgATP[S] (magnesium adenosine 5'-[gamma-thio]triphosphate) caused a decrease in the 20 kDa light-chain of smooth-muscle myosin (MLC20) phosphatase and phosphorylase phosphatase activities. MYPT1 contains a phosphorylation site, Thr-695, involved in the inhibition of MP in a RhoA/Rho kinase-dependent manner. The cytoskeletal kinase(s) phosphorylated Thr-695 of glutathione S-transferase (GST)-MYPT1, as determined with an antibody specific for phosphorylated Thr-695. The level of Rho kinase was low in the cytoskeletal fraction and was detected primarily in the membrane and cytosolic fractions. The phosphorylation of Thr-695 by the cytoskeletal kinase(s) was not affected by Rho kinase inhibitor, Y-27632, suggesting that kinase(s) other than Rho kinase were involved. In-gel kinase assay identified a kinase at 54-59 kDa that phosphorylated the C-terminal fragment of MYPT1 (GST-MYPT1(667-1004)). Western blots detected both zipper-interacting protein kinase (ZIPK) and integrin-linked kinase (ILK) at 54-59 kDa in the cytoskeleton and membrane fractions. Cytoskeletal ZIPK and ILK were separated and partially purified by chromatography on SP-Sepharose and on MonoQ. ZIPK preferentially phosphorylated MLC20 and had low activity on MYPT1. ILK phosphorylated both MLC20 and MYPT1 and phosphorylation of MYPT1 occured on Thr-695. The above results raise the potential for regulation of MP activity in platelet cytoskeleton by ILK and suggest an alternative to the Rho-linked pathway.

Chronic Treatment with Interleukin-1β Attenuates Contractions by Decreasing the Activities of CPI-17 and MYPT-1 in Intestinal Smooth Muscle

“…It has been reported that MYPT-1 phosphorylation is regu- lated by endogenous kinases other than ROCKs, including ZIPK (54), ILK (55,56), and DMPK (57). Kitazawa et al (24) have reported that MYPT-1 phosphorylation in the resting state is not inhibited by the ROCKs inhibitor, Y-27632, in intact rabbit vas deferens, indicating the presence of ROCKindependent phosphorylation of MYPT-1.…”

The actin cytoskeleton in cancer cell motility

“…The major site of MYPT1 phosphorylation is Threonine 696 (numbering relates to the human form), which inhibits phosphatase function [57], possibly by blocking the active site or by disrupting interaction of the catalytic subunit with phosphorylated substrate [58]. Kinases that have been reported to phosphorylate Thr696 include: ROCK1 and ROCK2 [57], MRCKa and MRCKb [47,59], ILK [60,61], ZIPK [62] and the DMPK [63]. Phosphorylation of Threonine 853 by ROCK has also been reported to inhibit MLC dephosphorylation by decreasing MLC binding [57,64].…”

Roles of myosin phosphatase duringDrosophiladevelopment

“…Rho-kinase (ROCK) phosphorylates an inhibitory phosphorylation site on MYPT and inhibits the phosphatase activity in smooth muscle. This phosphorylation may occur through ZIPK (leucine zipper interacting protein kinase)-like kinase (MacDonald et al, 2001) or integrin-linked kinase (Kiss et al, 2002). Myotonic dystrophy protein kinase phosphorylates the same inhibitory phosphorylation site ), although it is not clear whether this phosphorylation event also goes through ZIPK.…”