Characterisation of an Endogenous Protein Kinase Activity in Ribonucleoprotein Structures Containing Heterogenous Nuclear RNA in HeLa Cell Nuclei

Abstract

Ribonucleoprotein particles containing heterogeneous nuclear RNA (hnRNA) were isolated from sonically disrupted HeLa cells nuclei by sedimentation through a triple sucrose cushion. We found that these particles were able to catalyse the incorporation of radioactive phosphate from [γ32P]ATP into phosphoserine and phosphothreonine residues of endogenous proteins. Optimal conditions for enzymatic activity were defined after investigation of the influence of several parameters of the reaction. The requirement for a divalent cation was most efficiently met by 10 mM Mg2+, to a lower extent by Mn2+. An apparent Km value for ATP of 0.02 mM was determined and the enzyme was found to be sensitive to p‐hydroxymercuribenzoate. Neither cyclic AMP nor cyclic GMP stimulated the reaction over a wide range of concentrations and the same protein substrates were phosphorylated in their presence. After excluding possible contamination by other subcellular fractions, considerable evidence for the association of kinase and phosphate acceptor proteins with ribonucleoprotein structures containing hnRNA was gathered along several lines by showing that the enzymatic activity closely followed tritiated RNA pulse‐labeled in the presence of low doses of actinomycin D in the following circumstances: (a) sedimentation in sucrose gradients, (b) isopycnic banding in metrizamide density gradients in the presence or absence of Mg2+ ions and (c) adsorption to oligo(dT)‐cellulose columns under conditions where poly(A)‐containing material is adsorbed. Analysis of phosphorylated substrates by electrophoresis on polyacrylamide gels containing sodium dodecylsulphate revealed that, although they spanned a wide range of molecular weights, the most intensely labeled species were localized in bands corresponding to molecular weights of 37000 and 28000. The above pattern of polypeptides phosphorylated in vitro is compared with that of phosphoproteins labeled in vivo after exposure of cells to [32P]phosphate. Copyright © 1977, Wiley Blackwell. All rights reserved