A Novel Regulatory Mechanism of Myosin Light Chain Phosphorylation via Binding of 14-3-3 to Myosin Phosphatase

Yasuhiko Koga* ${dagger}$ and Mitsuo Ikebe*

^*Department of Physiology, University of Massachusetts Medical School, Worcester, MA 01655; Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Maebashi, Gunma 371-8511, Japan

Monitoring Editor: Erika Holzbaur

Myosin II phosphorylation-dependentcell motile events are regulated by myosin light chain (MLC)kinase and MLC phosphatase (MLCP). Recent studies have revealedmyosin phosphatase targeting subunit (MYPT1), a myosin bindingsubunit of MLCP, plays a critical role in MLCP regulation. Herewe report the new regulatory mechanism of MLCP via the interactionbetween 14–3-3 and MYPT1. The binding of 14–3-3 ${beta}$ to MYPT1 diminished the direct binding between MYPT1 and myosinII, and 14–3-3 ${beta}$ overexpression abolished MYPT1 localizationat stress fiber. Furthermore, 14–3-3 ${beta}$ inhibited MLCP holoenzymeactivity via the interaction with MYPT1. Consistently, 14–3-3 ${beta}$ overexpression increased myosin II phosphorylation in cells.We found that MYPT1 phosphorylation at Ser472 was critical forthe binding to 14–3-3. EGF-stimulation increased bothSer472 phosphorylation and the binding of MYPT1–14-3–3.Rho-kinase inhibitor inhibited the EGF-induced Ser472 phosphorylationand the binding of MYPT1–14-3–3. Rho-kinase specificsiRNA also decreased EGF-induced Ser472 phosphorylation correlatedwith the decrease in MLC phosphorylation. The present studyrevealed a new RhoA/Rho-kinase-dependent regulatory mechanismof myosin II phosphorylation by 14–3-3 that dissociatesMLCP from myosin II and attenuates MLCP activity.

Address correspondence to: Mitsuo Ikebe (Mitsuo.Ikebe{at}umassmed.edu)