Differential phosphorylation of myosin light chain (Thr)18 and (Ser)19 and functional implications in platelets

Authors


Satya P. Kunapuli, Department of Physiology, and James L. Daniel, Department of Pharmacology, Temple University Medical School, 3420 N. Broad Street Philadelphia, PA 19140, USA.
Tel.: +1 215 707 4615; +215 707 4457; fax: +1 215 707 4003.
E-mail: spk@temple.edu; jdaniel@temple.edu

Abstract

Summary.  Background:  Myosin IIA is an essential platelet contractile protein that is regulated by phosphorylation of its regulatory light chain (MLC) on residues (Thr)18 and (Ser)19 via the myosin light chain kinase (MLCK).

Objective:  The present study was carried out to elucidate the mechanisms regulating MLC (Ser)19 and (Thr)18 phosphorylation and the functional consequence of each phosphorylation event in platelets.

Results:  Induction of 2MeSADP-induced shape change occurs within 5 s along with robust phosphorylation of MLC (Ser)19 with minimal phosphorylation of MLC (Thr)18. Selective activation of G12/13 produces both slow shape change and comparably slow MLC (Thr)18 and (Ser)19 phosphorylation. Stimulation with agonists that trigger ATP secretion caused rapid MLC (Ser)19 phosphorylation while MLC (Thr)18 phosphorylation was coincident with secretion. Platelets treated with p160ROCK inhibitor Y-27632 exhibited a partial inhibition in secretion and had a substantial inhibition in MLC (Thr)18 phosphorylation without effecting MLC (Ser)19 phosphorylation. These data suggest that phosphorylation of MLC (Ser)19 is downstream of Gq/Ca2+-dependent mechanisms and sufficient for shape change, whereas MLC (Thr)18 phosphorylation is substantially downstream of G12/13-regulated Rho kinase pathways and necessary, probably in concert with MLC (Ser)19 phosphorylation, for full contractile activity leading to dense granule secretion. Overall, we suggest that the amplitude of the platelet contractile response is differentially regulated by a least two different signaling pathways, which lead to different phosphorylation patterns of the myosin light chain, and this mechanism results in a graded response rather than a simple on/off switch.

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