Phosphorylation of the C proteins in heterogeneous ribonucleoprotein (hnRNP) particles in HeLa cells: Characterization of in vivo phosphorylation, comparison with in vitro phosphorylation using casein kinase II, and preliminary studies on the effects of phosphorylation on particle structure

Newly formed pre-messenger RNA associates with protein to form heterogeneous ribonucleoprotein (hnRNP) particles. In HeLa cells, hnRNP particles contain six core proteins. Two proteins, termed C1 and C2, are phosphorylated in vitro by casein kinase 11 (CKII). C1 protein became 32P-labeled after HeLa cells were incubated with [32P]-orthophosphate in vivo (ibid). Because phosphorylation is a ubiquitous regulatory mechanism, C protein phosphorylation was studied in greater detail. C protein phosphorylation in hnRNP particles was investigated in HeLa cells incubated with [32P]-orthophosphate in vivo. Immunoblotting in pH 3.5-10 isoelectric focusing (IEF) gels indicated that C proteins focus only at pH 5.0. In pH 4.5-5.5 IEF gels, individually purified C, and 2 proteins resolve into the same four closely spaced, 32P-labeled bands. A fifth, unlabeled, more basic species was detached when hnRNP particles were purified without NaF. All 32P-labeled species contained identical amounts of 32P per unit protein suggesting that charge heterogeneity is not due to differential phosphorylation. Attempts to detect bound carbohydrate were unsuccessful. 32P-labeled phosphate was readily removed by potato acid phosphatase. E. coli alkaline phosphatase and snake venom phosphodiesterase were ineffective. 32P-label was found exclusively in phosphoserine. One-dimensional peptide mapping with chymotrypsin and S. aureus protease detected two phosphorylated peptides. C protein phosphorylation was also investigated in vitro. Incubation of hnRNP particles with rabbit liver CKII and 32P-ATP followed by IEF in pH 4.5-5.5 gels indicated that all four C protein species were 32P-labeled. 32P-label was found exclusively in phosphoserine