Protein phosphatase activity is necessary for myofibrillogenesis.
During myofibrillogenesis, myosin light-chain kinase (MLCK) phosphorylates the regulatory light chain (RLC) of myosin II, enabling patterned assembly of myosin thick filaments. Aprotein phosphatase (PP) has been shown to mediate RLC dephosphorylation in adult smooth and striated muscle. A role for PP activity in regulating myofibrillogenesis during embryonic development, however, has not been investigated. Tautomycin (TM) was used to inhibit both PP1 and PP2A activities, whereas okadaic acid (OA) and fostriecin (FOS) were used to inhibit PP2A. TM affected both actin and myosin assembly at 5 nM; the IC50 value was 20 and 8.5 nM, respectively. In contrast, OA applied at 10 times above its reported Ki for PP2A caused no significant disruption. There was also no disruption when FOS was applied at a concentration 30 times above its reported Ki for PP2A. Thus, our results suggest a primary role for PP1 isoforms during myofibrillogenesis. Although rho kinase (RK) regulates PP activity in embryonic smooth and cardiac muscle, application of the RK inhibitor Y27632 did not affect actin or myosin assembly in skeletal myocytes. Collectively, our pharmacological results suggest that PP1 is involved in dynamic regulation of RLC phosphorylation. To specifically test involvement of the myosin-targeted isoform (PP1M), we used a morpholino antisense approach to knock down the myosin targeting (M) subunit of PP1. Embryos injected with morpholino targeted to the 110-kDa M targeting subunit had fewer somites, and myosin organization was significantly perturbed. The combined pharmacological and molecular results suggest a dynamic equilibrium between MLCK and PP1M activities is required for proper myofibrillogenesis.
Terry
Monica
M
Division of Molecular Biology and Biochemistry, School of Biological Sciences, 5007 Rockhill Road, University of Missouri, Kansas City, MO 64110, USA.
Walker
Danielle D
DD
Ferrari
Michael B
MB
D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D004789:Enzyme Activation; D018485:Muscle Fibers, Skeletal / Q000166:cytology* / Q000502:physiology*; D018482:Muscle, Skeletal / Q000166:cytology / Q000196:embryology* / Q000502:physiology*; D009210:Myofibrils / Q000000981:diagnostic imaging* / Q000201:enzymology*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D014463:Ultrasonography; D014982:Xenopus laevis
D010749:Phosphoprotein Phosphatases