Differential phosphorylation of myosin light chain (Thr)18 and (Ser)19 and functional implications in platelets.
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 5s along with robust phosphorylation of MLC (Ser)19 with minimal phosphorylation of MLC (Thr)18. Selective activation of G(12/13) 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 p160(ROCK) 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/Ca(2+) -dependent mechanisms and sufficient for shape change, whereas MLC (Thr)18 phosphorylation is substantially downstream of G(12/13) -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.
Getz
T M
TM
Department of Physiology, Temple University School of Medicine, Philadelphia, PA 19140, USA.
Dangelmaier
C A
CA
Jin
J
J
Daniel
J L
JL
Kunapuli
S P
SP
D000244:Adenosine Diphosphate / Q000737:chemistry; D001792:Blood Platelets / Q000378:metabolism*; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D048430:Cell Shape; D006801:Humans; D007700:Kinetics; D018994:Myosin Light Chains / Q000737:chemistry* / Q000235:genetics*; D010766:Phosphorylation; D010974:Platelet Aggregation; D012694:Serine / Q000737:chemistry*; D015398:Signal Transduction; D013912:Threonine / Q000737:chemistry*; D054460:rho-Associated Kinases / Q000378:metabolism
D018994:Myosin Light Chains; D013912:Threonine; D012694:Serine; D000244:Adenosine Diphosphate; D054460:rho-Associated Kinases; D002118:Calcium