title: Myosin phosphatase modulates the cardiac cell fate by regulating the subcellular localization of Nkx2.5 in a Wnt/Rho-associated protein kinase-dependent pathway.
abstract: BACKGROUND
  Nkx2.5 is a transcription factor that regulates cardiomyogenesis in vivo and in embryonic stem cells. It is also a common target in congenital heart disease. Although Nkx2.5 has been implicated in the regulation of many cellular processes that ultimately contribute to cardiomyogenesis and morphogenesis of the mature heart, relatively little is known about how it is regulated at a functional level.
  
  
  OBJECTIVE
  We have undertaken a proteomic screen to identify novel binding partners of Nkx2.5 during cardiomyogenic differentiation in an effort to better understand the regulation of its transcriptional activity.
  
  
  RESULTS
  Purification of Nkx2.5 from differentiating cells identified the myosin phosphatase subunits protein phosphatase 1β and myosin phosphatase targeting subunit 1 (Mypt1) as novel binding partners. The interaction with protein phosphatase 1 β/Mypt1 resulted in exclusion of Nkx2.5 from the nucleus and, consequently, inhibition of its transcriptional activity. Exclusion of Nkx2.5 was inhibited by treatment with leptomycin B and was dependent on an Mypt1 nuclear export signal. Furthermore, in transient transfection experiments, Nkx2.5 colocalized outside the nucleus with phosphorylated Mypt1 in a manner dependent on Wnt signaling and Rho-associated protein kinase. Treatment of differentiating mouse embryonic stem cells with Wnt3a resulted in enhanced phosphorylation of endogenous Mypt1, increased nuclear exclusion of endogenous Nkx2.5, and a failure to undergo terminal cardiomyogenesis. Finally, knockdown of Mypt1 resulted in rescue of Wnt3a-mediated inhibition of cardiomyogenesis, indicating that Mypt1 is required for this process.
  
  
  CONCLUSIONS
  We have identified a novel interaction between Nkx2.5 and myosin phosphatase. Promoting this interaction represents a novel mechanism whereby Wnt3a regulates Nkx2.5 and inhibits cardiomyogenesis.
authors.affiliation: Department of Biochemistry, Microbiology, and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada.
authors.forename: Tammy
authors.initials: T
authors.lastname: Ryan
authors.forename: Michael
authors.initials: M
authors.lastname: Shelton
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Lambert
authors.forename: Barbora
authors.initials: B
authors.lastname: Malecova
authors.forename: Sophie
authors.initials: S
authors.lastname: Boisvenue
authors.forename: Marc
authors.initials: M
authors.lastname: Ruel
authors.forename: Daniel
authors.initials: D
authors.lastname: Figeys
authors.forename: Pier Lorenzo
authors.initials: PL
authors.lastname: Puri
authors.forename: Ilona S
authors.initials: IS
authors.lastname: Skerjanc
mesh_terms: D000818:Animals; D053595:Embryonic Stem Cells / Q000201:enzymology / Q000378:metabolism / Q000502:physiology; D006131:Growth Inhibitors / Q000502:physiology*; D057809:HEK293 Cells; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins / Q000378:metabolism*; D006801:Humans; D051379:Mice; D032383:Myocytes, Cardiac / Q000201:enzymology / Q000378:metabolism / Q000502:physiology*; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism / Q000502:physiology*; D054645:Protein Phosphatase 1 / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D013347:Subcellular Fractions / Q000201:enzymology / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D060449:Wnt Signaling Pathway / Q000502:physiology*; D060509:Wnt3A Protein / Q000502:physiology*; D054460:rho-Associated Kinases / Q000502:physiology*
chemical_list: D006131:Growth Inhibitors; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins; C095552:NKX2-5 protein, human; D014157:Transcription Factors; C555510:WNT3A protein, human; D060509:Wnt3A Protein; D054460:rho-Associated Kinases; C517188:PPP1CB protein, human; D054645:Protein Phosphatase 1; D043263:Myosin-Light-Chain Phosphatase; C490372:PPP1R12A protein, human