Par-4: a new activator of myosin phosphatase.
Myosin phosphatase (MP) is a key regulator of myosin light chain (LC20) phosphorylation, a process essential for motility, apoptosis, and smooth muscle contractility. Although MP inhibition is well studied, little is known about MP activation. We have recently demonstrated that prostate apoptosis response (Par)-4 modulates vascular smooth muscle contractility. Here, we test the hypothesis that Par-4 regulates MP activity directly. We show, by proximity ligation assays, surface plasmon resonance and coimmunoprecipitation, that Par-4 interacts with the targeting subunit of MP, MYPT1. Binding is mediated by the leucine zippers of MYPT1 and Par-4 and reduced by Par-4 phosphorylation. Overexpression of Par-4 leads to increased phosphatase activity of immunoprecipitated MP, whereas small interfering RNA knockdown of endogenous Par-4 significantly decreases MP activity and increases MYPT1 phosphorylation. LC20 phosphorylation assays demonstrate that overexpression of Par-4 reduces LC20 phosphorylation. In contrast, a phosphorylation site mutant, but not wild-type Par-4, interferes with zipper-interacting protein kinase (ZIPK)-mediated MP inhibition. We conclude from our results Par-4 operates through a "padlock" model in which binding of Par-4 to MYPT1 activates MP by blocking access to the inhibitory phosphorylation sites, and inhibitory phosphorylation of MYPT1 by ZIPK requires "unlocking" of Par-4 by phosphorylation and displacement of Par-4 from the MP complex.
Vetterkind
Susanne
S
Department of Health Sciences, Sargent College of Health and Rehabilitation Sciences, Boston University, Boston, MA 02215, USA.
Lee
Eunhee
E
Sundberg
Eric
E
Poythress
Ransom H
RH
Tao
Terence C
TC
Preuss
Ute
U
Morgan
Kathleen G
KG
D000818:Animals; D001011:Aorta / Q000378:metabolism; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism / Q000502:physiology*; D049109:Cell Proliferation; D004247:DNA / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004789:Enzyme Activation; D008856:Microscopy, Fluorescence / Q000379:methods; D009131:Muscle, Smooth, Vascular / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism*; D010766:Phosphorylation; D012313:RNA / Q000737:chemistry; D051381:Rats
D051017:Apoptosis Regulatory Proteins; C101296:prostate apoptosis response-4 protein; D012313:RNA; D004247:DNA; D043263:Myosin-Light-Chain Phosphatase