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Vol. 19, Issue 3, 1062-1071, March 2008

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

Yasuhiko Koga^*,, and Mitsuo Ikebe^*

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

Submitted July 16, 2007; Revised November 26, 2007; Accepted December 12, 2007
Monitoring Editor: Erika Holzbaur

Myosin II phosphorylation–dependent cell motile events are regulated by myosin light-chain (MLC) kinase and MLC phosphatase (MLCP). Recent studies have revealed myosin phosphatase targeting subunit (MYPT1), a myosin-binding subunit of MLCP, plays a critical role in MLCP regulation. Here we report the new regulatory mechanism of MLCP via the interaction between 14-3-3 and MYPT1. The binding of 14-3-3β to MYPT1 diminished the direct binding between MYPT1 and myosin II, and 14-3-3β overexpression abolished MYPT1 localization at stress fiber. Furthermore, 14-3-3β inhibited MLCP holoenzyme activity via the interaction with MYPT1. Consistently, 14-3-3β overexpression increased myosin II phosphorylation in cells. We found that MYPT1 phosphorylation at Ser472 was critical for the binding to 14-3-3. Epidermal growth factor (EGF) stimulation increased both Ser472 phosphorylation and the binding of MYPT1-14-3-3. Rho-kinase inhibitor inhibited the EGF-induced Ser472 phosphorylation and the binding of MYPT1-14-3-3. Rho-kinase specific siRNA also decreased EGF-induced Ser472 phosphorylation correlated with the decrease in MLC phosphorylation. The present study revealed a new RhoA/Rho-kinase–dependent regulatory mechanism of myosin II phosphorylation by 14-3-3 that dissociates MLCP from myosin II and attenuates MLCP activity.

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

Abbreviations used: CBB, Coomassie blue staining; mcLR, microcystine LR; MLC, myosin light chain; MLCP, myosin light-chain phosphatase; MYPT1, myosin phosphatase targeting subunit 1; PP1, type 1 protein phosphatase; siRNA, small interfering RNA.