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Title: 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 C{sub 1} and C{sub 2}, are phosphorylated in vitro by casein kinase 11 (CKII). C{sub 1} protein became {sup 32}P-labeled after HeLa cells were incubated with ({sup 32}P)-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 ({sup 32}P)-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, {sup 32}P-labeled bands. A fifth, unlabeled, more basic species was detached when hnRNP particles were purified without NaF. All {sup 32}P-labeled species contained identical amounts of {sup 32}P per unit protein suggesting that charge heterogeneity is not due to differential phosphorylation. Attempts to detect bound carbohydrate were unsuccessful. {sup 32}P-labeled phosphate was readily removed by potato acid phosphatase. E. coli alkaline phosphatase and snake venom phosphodiesterase were ineffective. {sup 32}P-label was found exclusively in phosphoserine. One-dimensional peptidemore » 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 {sup 32}P-ATP followed by IEF in pH 4.5-5.5 gels indicated that all four C protein species were {sup 32}P-labeled. {sup 32}P-label was found exclusively in phosphoserine.« less
Authors:
Publication Date:
OSTI Identifier:
5604418
Resource Type:
Miscellaneous
Resource Relation:
Other Information: Thesis (Ph. D.)
Publisher:
Nashville, TN (US); Vanderbilt Univ.
Research Org:
Vanderbilt Univ., Nashville, TN (United States)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; NUCLEOPROTEINS; PHOSPHORYLATION; PHOSPHOTRANSFERASES; ENZYME ACTIVITY; ACID PHOSPHATASE; COMPARATIVE EVALUATIONS; HELA CELLS; IN VITRO; IN VIVO; PHOSPHATES; PHOSPHORUS 32; RNA; TRACER TECHNIQUES; BETA DECAY RADIOISOTOPES; BETA-MINUS DECAY RADIOISOTOPES; CHEMICAL REACTIONS; DAYS LIVING RADIOISOTOPES; ENZYMES; ESTERASES; HYDROLASES; ISOTOPE APPLICATIONS; ISOTOPES; LIGHT NUCLEI; NUCLEI; NUCLEIC ACIDS; ODD-ODD NUCLEI; ORGANIC COMPOUNDS; OXYGEN COMPOUNDS; PHOSPHATASES; PHOSPHORUS COMPOUNDS; PHOSPHORUS ISOTOPES; PHOSPHORUS-GROUP TRANSFERASES; PROTEINS; RADIOISOTOPES; TRANSFERASES; 550501* - Metabolism- Tracer Techniques