Integrin-linked kinase phosphorylates the myosin phosphatase target subunit at the inhibitory site in platelet cytoskeleton.
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.
Kiss
Eniko
E
Department of Medical Chemistry, University of Debrecen, Medical and Health Science Center, H-4026 Debrecen, Bem tér 18/B, Hungary.
Murányi
Andrea
A
Csortos
Csilla
C
Gergely
Pál
P
Ito
Masaaki
M
Hartshorne
David J
DJ
Erdodi
Ferenc
F
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000328:Adult; D001665:Binding Sites; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology*; D003599:Cytoskeleton / Q000201:enzymology; D006801:Humans; D066298:In Vitro Techniques; D043263:Myosin-Light-Chain Phosphatase; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000097:blood* / Q000737:chemistry; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000097:blood*; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D000099200:Scaffold Protein ILK
D000255:Adenosine Triphosphate; D043263:Myosin-Light-Chain Phosphatase; D010749:Phosphoprotein Phosphatases; D017346:Protein Serine-Threonine Kinases; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D000099200:Scaffold Protein ILK