Myosin Phosphatase Modulates the Cardiac Cell Fate by Regulating the Subcellular Localization of Nkx2.5 in a Wnt/ROCK-Dependent Pathway
Rationale: 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.
Methods and Results: Purification of Nkx2.5 from differentiating cells identified the myosin phosphatase subunits PP1β and Mypt1 as novel binding partners. The interaction with PP1β/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 LeptomycinB and was dependent on a Mypt1 nuclear export signal. Furthermore, in transient transfection experiments, Nkx2.5 co-localized outside the nucleus with phosphorylated Mypt1 in a manner dependent on Wnt signalling 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.
- Cardiac differentiation
- Cardiac transcription factors
- Embryonic stem cells
- Gene regulation
- Signal transduction
- Stem cells
- Received July 4, 2012.
- Accepted November 20, 2012.
- Copyright © 2012, American Heart Association