Abstract
BackgroundThe myosin phosphatase is a highly conserved regulator of actomyosin contractility. Zebrafish has emerged as an ideal model system to study the in vivo role of myosin phosphatase in controlling cell contractility, cell movement and epithelial biology. Most work in zebrafish has focused on the regulatory subunit of the myosin phosphatase called Mypt1. In this work, we examined the critical role of Protein Phosphatase 1, PP1, the catalytic subunit of the myosin phosphatase.Methodology/Principal FindingsWe observed that in zebrafish two paralogous genes encoding PP1β, called ppp1cba and ppp1cbb, are both broadly expressed during early development. Furthermore, we found that both gene products interact with Mypt1 and assemble an active myosin phosphatase complex. In addition, expression of this complex results in dephosphorylation of the myosin regulatory light chain and large scale rearrangements of the actin cytoskeleton. Morpholino knock-down of ppp1cba and ppp1cbb results in severe defects in morphogenetic cell movements during gastrulation through loss of myosin phosphatase function.Conclusions/SignificanceOur work demonstrates that zebrafish have two genes encoding PP1β, both of which can interact with Mypt1 and assemble an active myosin phosphatase. In addition, both genes are required for convergence and extension during gastrulation and correct dosage of the protein products is required.
Highlights
Reversible phosphorylation of the type II myosin regulatory light chain (MLC2) is a critical regulatory mechanism for controlling type II myosin and the actin cytoskeleton [1,2]
MLC2 is phosphorylated, primarily at serine 19 and threonine 18, by a number of protein kinases, including Myosin Light Chain Kinase (MLCK), Rho-Associated Protein Kinase (ROCK) and Zipper-Interacting Protein Kinase (ZIPK) [3,4]. The dephosphorylation of both sites on MLC2 is mediated by a highly conserved Myosin Phosphatase (MP) complex consisting of a targeting subunit Mypt1, the catalytic subunit Protein Phosphatase 1 β (PP1β) and an associated 20 kD protein (m20) [5,6]
All other amino acid differences are in the N- or C-terminal tails, the amino acids Threonine 197, Serine 232 and Asparagine 236, which are critical for the βisoform specific interaction with Mypt1 [36], are all conserved
Summary
Reversible phosphorylation of the type II myosin regulatory light chain (MLC2) is a critical regulatory mechanism for controlling type II myosin and the actin cytoskeleton [1,2]. MLC2 is phosphorylated, primarily at serine 19 and threonine 18, by a number of protein kinases, including Myosin Light Chain Kinase (MLCK), Rho-Associated Protein Kinase (ROCK) and Zipper-Interacting Protein Kinase (ZIPK) [3,4] The dephosphorylation of both sites on MLC2 is mediated by a highly conserved Myosin Phosphatase (MP) complex consisting of a targeting subunit Mypt, the catalytic subunit Protein Phosphatase 1 β (PP1β) and an associated 20 kD protein (m20) [5,6]. Conclusions/Significance: Our work demonstrates that zebrafish have two genes encoding PP1β, both of which can interact with Mypt and assemble an active myosin phosphatase Both genes are required for convergence and extension during gastrulation and correct dosage of the protein products is required
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