Inhibition of Myogenesis by Depletion of the Glycogen-associated Regulatory Subunit of Protein Phosphatase-1 in Rat Skeletal Muscle Cells

Louis Ragolia,Qian Zuo,Najma Begum

doi:10.1074/jbc.m001955200

Abstract

In this study, we examined the role of the glycogen-associated regulatory subunit of protein phosphatase-1 (PP-1(G)) in L6 rat skeletal muscle cell myogenesis. The level of PP-1(G) was depleted by transfection with an inducible antisense-oriented PP-1(G) gene. Western blot analysis of the PP-1(G)-depleted cell line revealed a >90% depletion of PP-1(G) protein and a 45% reduction in cellular PP-1 activity and abolished the ability of L6 myoblasts to differentiate into multinucleated myotubes. PP-1(G)-depleted cells also exhibited a marked reduction in the expression of the differentiation marker myogenin as well as creatine kinase. After 7 days in culture, PP-1(G)-depleted cells sustained myoblast levels of inhibitor of differentiation-2, whereas control L6 cells had a severely lower inhibitor of differentiation-2 level and progressed into myotubes. Myoblasts were unable to exit the cell cycle, as measured by the impaired induction of p27 cyclin-dependent kinase inhibitor, a >2-fold increase in DNA synthesis, and elevated levels of phosphorylated retinoblastoma protein (pRb). Replacement of the PP-1(G) gene restored PP-1(G) protein expression, PP-1 enzymatic activity, and the ability to differentiate into myotubes. We conclude that PP-1(G) plays a definite role in L6 myogenesis via its regulation of PP-1 catalytic activity.

Highlights

Progression of myoblasts into myotubes involves the regulation of several skeletal muscle-specific genes encoding a group of proteins known collectively as the basic helix-loop-helix family [1, 2]
We examined the effects of PP-1C is found associated with glycogen particles (PP-1G) depletion on the process of myogenesis using PP-1G antisense mRNA expression
PP-1G protein was depleted from the insulin-sensitive rat skeletal muscle cell line L6 by transfection with an IPTG

Summary

The abbreviations used are

Id2, inhibitor of differentiation-2; MEM, minimal essential medium; Cdk, cyclin-dependent kinase; FBS, fetal bovine serum; IPTG, isopropyl-␤-D-thiogalactopyranoside; PI3kinase, phosphatidylinositol 3-kinase; PP-1, protein phosphatase-1; in proliferating cells, appears to preferentially bind E2a and thereby prevent myogenic activity [5]. PP-1C is found associated with glycogen particles (PP-1G), sarcoplasmic reticulum (PP-1SR), myofibrils (PP-1M), the cytosolic inhibitor-2 protein (PP-1I), and the nucleus (PP-1N) through a set of nuclear inhibitor proteins [18] These studies indicate that the intracellular distribution of PP-1 activity, via its regulatory subunits, may be an important aspect of its regulation. PP-1 has long been implicated in the regulation of cell growth and myogenesis in mammalian cells, the form of PP-1 that participates in these processes and the exact molecular mechanism remains unclear Recent studies from this laboratory have shown that the PP-1G subunit is expressed in L6 cells upon fusion and is absent in proliferating L6 myoblasts [19]. These results, together with the above studies indicating a potential role for PP-1 in cell cycle arrest and myogenesis, prompted us to further explore the role of PP-1G, a known regulator of PP-1 in myogenesis

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION