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′-[γ-thio]triphosphate) caused a decrease in the 20kDa 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 54–59kDa that phosphorylated the C-terminal fragment of MYPT1 (GST-MYPT1667–1004). Western blots detected both zipper-interacting protein kinase (ZIPK) and integrin-linked kinase (ILK) at 54–59kDa 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.
- human platelet
- inhibitory phosphorylation
- protein phosphatase 1
- zipper-interacting protein kinase (ZIP kinase)
Abbreviations used: CL-A, calyculin A; GST, glutathione S-transferase; MP, myosin phosphatase; MYPT, myosin phosphatase target subunit; MYPT1-C, C-terminal fragment (residues 667–1004) of MYPT1; PP1, type 1 protein phosphatase; PP1c, catalytic subunit of PP1; PP2A, type 2A protein phosphatase; ILK, integrin-linked kinase; ZIPK, zipper-interacting protein kinase; MLC20, 20kDa light chain of non-muscle or smooth-muscle myosin; P-MLC20, phosphorylated MLC20; ATP[S], adenosine 5′-[γ-thio]triphosphate; SP-Sepharose, sulphopropyl-Sepharose.
- The Biochemical Society, London ©2002