Open AccessLocalization of Phosphoproteins and of Protein Kinases in Chromatin from Hepatoma Tissue‐Cultured Cells
Author(s) -
KITZIS Alain,
TICHONICKY Lydie,
DEFER Nicole,
KRUH Jacques
Publication year - 1980
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1980.tb06098.x
Subject(s) - chromatin , micrococcal nuclease , nucleosome , biochemistry , gel electrophoresis , kinase , histone , microbiology and biotechnology , polyacrylamide gel electrophoresis , phosvitin , histone h1 , biology , in vitro , chemistry , protein kinase a , dna , enzyme
An important role in the control of gene expression has been attributed to phosphoproteins present among chromatin, non‐histone proteins. In a previous work we have shown that at least part of these phosphoproteins are associated with nucleosomes. In this work we wanted to establish whether this association occurs with all nucleosomes or with the nucleosomes present in fragments preferentially released by a mild micrococcal nuclease digestion, which originated essentially from active parts of chromatin. Phosphoproteins were labelled in vivo by incubating hepatoma tissue‐cultured cells with [ 32 P]phosphate and chromatin was submitted to a limited micrococcal nuclease digestion. The released fragments were fractionated by preparative gel electrophoresis. [ 32 P]Phosphoproteins were essentially found in the smallest released fragments; monomers and dimers of nucleosomes. The same result was obtained when the phosphoproteins were labelled in vitro by incubating each fragment obtained by the preparative electrophoresis in the presence of [γ‐ 32 P]ATP. It indicates that part of the protein kinase activity was strongly bound to the particles. The bound phosphoproteins were analysed by sodium dodecylsulfate/polyacrylamide gel electrophoresis. Two main polypeptides were characterized: phosphopeptide a, M r 41000, present in all small fragments; phosphopeptide b, M r 31000, present in all small fragments, except in the fastest moving nucleosomes. Phosvitin kinase was found associated with the small released fragments, its specific activity was by far the highest in the fraction which includes the dieters of nucleosomes. It is concluded that phosphoproteins and protein kinases are associated with the nucleosomes of the active parts of chromatin, which suggests a role of these proteins in the control of gene expression.