In vivo phosphorylation of regulatory light chain of myosin II in sea urchin eggs and its role in controlling myosin localization and function during cytokinesis.
Phosphorylation of myosin regulatory light chain (RLC) at Ser19 (mono-phosphorylation) promotes filament assembly and enhances actin-activated ATPase activity of non-muscle myosin, while phosphorylation at both Ser19 and Thr18 (di-phosphorylation) further enhances the ATPase activity. However, it has not well been addressed which type of phosphorylation is important in regulating myosin during cytokinesis. Here, we investigated subcellular localization in sea urchin eggs of mono-phosphorylated and di-phosphorylated RLC by both quantitative biochemical and spatiotemporal cytological approaches. Mono-phosphorylated RLC was dominant in the equatorial cortex throughout the whole process of cytokinesis. Inhibition of myosin light chain kinase (MLCK) decreased mono-phosphorylated RLC both in the cortex and in the cleavage furrow, and blocked both formation and contraction of the contractile ring. Two different types of ROCK inhibitor gave inconsistent results: H1152 blocked both RLC mono-phosphorylation in the cleavage furrow and contraction of the contractile ring, while Y27632 affected neither the mono-phosphorylation nor cell division. These results suggest that there may be other targets of H1152 than ROCK, which is involved in the RLC phosphorylation in the cleavage furrow. Furthermore, it was revealed that localization of myosin heavy chain in the cleavage furrow, but not in the cortex, was perturbed by inhibition of RLC mono-phosphorylation. These results suggested that RLC mono-phosphorylation by more than two RLC kinases play a main role in regulation and localization of myosin in the dividing sea urchin eggs.
Uehara
Ryota
R
Department of Biology, School of Arts and Sciences, University of Tokyo, 3-8-1 Komaba, Tokyo 153-8902, Japan.
Hosoya
Hiroshi
H
Mabuchi
Issei
I
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000577:Amides / Q000494:pharmacology; D000814:Aniline Compounds; D000818:Animals; D001381:Azepines / Q000494:pharmacology; D024722:Cardiac Myosins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002455:Cell Division / Q000502:physiology; D002478:Cells, Cultured; D002970:Cleavage Stage, Ovum / Q000166:cytology* / Q000378:metabolism*; D003001:Cloning, Molecular; D048749:Cytokinesis / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018994:Myosin Light Chains / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D009281:Naphthalenes / Q000494:pharmacology; D010063:Ovum / Q000166:cytology* / Q000378:metabolism*; D010766:Phosphorylation; D011725:Pyridines / Q000494:pharmacology; D012617:Sea Urchins; D012694:Serine / Q000378:metabolism; D054460:rho-Associated Kinases / Q000378:metabolism
D000577:Amides; D000814:Aniline Compounds; D001381:Azepines; D004791:Enzyme Inhibitors; D018994:Myosin Light Chains; D009281:Naphthalenes; D011725:Pyridines; C107184:myosin light chain 2; C070571:ML 7; C108830:Y 27632; D012694:Serine; D054460:rho-Associated Kinases; D009219:Myosin-Light-Chain Kinase; D000251:Adenosine Triphosphatases; D024722:Cardiac Myosins; C004354:dicloran