Two-dimensional gel analysis of nuclear proteins during muscle differentiation in vitro: II. Changes in protein phosphorylation

Abstract

Isolated nuclei from chick skeletal muscle cell cultures at different stages of differentiation were labelled in vitro with γ-[ 32P]ATP. Nuclear proteins were separated into saline-soluble and non-histone (NHP) fractions and analysed by two-dimensional gel electrophoresis and autoradiography. Relatively few proteins (10–12) were phosphorylated to any extent and most of these corresponded to minor, or even undetectable, components on stained gels. 32P incorporation into most of these proteins increased between 24 and 40 h and decreased sharply between 40 and 66 h, the changes being more marked in some phosphoproteins than in others. Pulse-chase data suggest that these overall changes in phosphorylation reflect changes in protein kinase, rather than protein phosphatase activities. One highly phosphorylated acidic protein (mol. wt 10 000 D; pI 4.2) which was undetectable on stained gels, showed a continuous increase in 32P incorporation between 24 and 66 h in both saline-soluble and NHP fractions. This protein may be the muscle counterpart of a phosphorylated nuclear protein partially characterized in other cell types. Both nuclear protein phosphorylation and low molecular weight non-histone proteins have been particularly implicated in activation of gene transcription in other systems, although we have no direct evidence, as yet, to connect them with control of myoblast growth and differentiation.