A novel protein was extracted with 5% perchloric acid from rat liver and kidney. It is absent from other rat organs. Its apparent molecular mass is 23 kDa as determined by HPLC gel filtration. A single band, corresponding to 10 kDa, was observed after SDSPAGE, suggesting that the protein consists of two subunits with similar molecular masses.This protein can neither be phosphorylated by ATP, nor acetylated. The sequence of the cDNA encoding this protein was determined. Southern-blot analysis showed that the corresponding gene spanned at least 10 kb and contained at least five introns. Zoo-blot analysis at medium stringency strongly suggests that the gene has been conserved during evolution. The amino-acid sequence of this protein with a highly conserved region is similar to that of a heat-shock protein.High-mobility group (HMG) proteins are major chromatin components in all higher organisms. During the study of rat chromatin we have isolated from liver a novel protein which is co-extracted with the HMG protein by 5% perchloric acid.This protein does not meet the criteria that characterize the HMG protein group : monomeric structure, bipolar distribution of charged amino acids [ 1, 21. Since this protein is expressed at a high level in liver and kidney, but present in very low amounts in, or even absent from, other organs, it constitutes a good model for the study of tissue-specific regulation of protein synthesis.In this study we describe the purification of the protein whose apparent molecular mass is 23 kDa and which consists of two 10-kDa subunits, and the cloning and sequencing of the corresponding cDNA.This cDNA consists of 859 bp. The first half of the deduced polypeptide sequence presents 27% similarity with a region of the 83-90-kDa heat-shock protein (hsp) which is highly conserved from Drosophila to man [3 -51.
A new protein kinase has been characterized among the proteins tightly bound to rat liver DNA and released by DNase I and RNase A treatment. This enzyme was separated by gel filtration from this released material. Its apparent molecular mass was found to be 34 kDa and it is made of a single unit.The main characteristic of this protein kinase is that it is arginine-specific. Isolation of phosphoarginine required the use of proteolytic enzymes at alkaline pH since the phosphate bond is highly acid-labile. This protein kinase is able to autophosphorylate and to phosphorylate a single chromosomal protein of 11 kDa also tightly bound to DNA. It uses ATP and dATP as phosphate donors and is CAMP-independent. Its optimal activity requires Mn2 + ions. Vanadate, spermine and heparin have no effect on its activity.Chromosomal protein phosphorylation is a major posttranslational modification which could be implicated in the control of gene expression. It is, then, of major interest to characterize the nuclear protein kinases which are involved in this process.The best-documented enzymes are the CAMP-independent protein kinases NI and NII which use chromosomal nonhistone proteins as substrates [l -51. They phosphorylate the substrates on serine and threonine residues. Several histone kinases have been described but their subcellular localization is in general not known.We have recently described in rat liver a CAMP-independent protein kinase tightly bound to DNA, which is released by DNase I digestion and which is able to phosphorylate histones H2B and H3 and a set of chromosomal non-histone proteins on serine residues [6].In our attempt to purify this enzyme we found a second CAMP-independent protein kinase which is able to autophosphorylate and to phosphorylate a single 1 1-kDa chromosomal protein. The highest activity of this enzyme was observed in the presence of Mn2+. The main characteristic of this enzyme is that the bound phosphate is alkali-stable and highly acid-labile. The phosphorylated residue has been identified as arginine.Phosphorylated basic residues have been described in various cell types in rat liver fractions [7,9] in carcinoma cells [lo, 111 in bovine liver mitochondria [12] and in smooth muscle Phosphoarginine has been found in brain myelin [14] and in VP12, a capsidic protein of granulosis virus [15, 161. This viral protein is also a substrate of the enzyme described in this paper.
Rat liver nuclear protein kinases NI and NII have been purified to homogeneity by an improved method. This method includes a casein-phosvitin-Sepharose column step, which separates the enzymes from the other chromosomal non-histone proteins, and a gel filtration at high ionic strength in the presence of a high concentration of protease inhibitors to separate the two enzymes from each other. NI has an apparent molecular mass of approximately 50 kDa and is composed of a single subunit. NII has an apparent molecular mass of 133 kDa and is composed of two subunits of identical molecular mass. The V and the Km of the two enzymes were determined for several substrates. Both enzymes phosphorylate chromosomal non-histone proteins with partly different specificities as shown by two-dimensional electrophoreses. When incubated in the absence of protease inhibitors, the enzymes were degraded into discrete polypeptides. Autophosphorylation of a polypeptide derived from NII was observed after incubation of the enzyme with ATP. This phosphorylation stimulated the enzyme activity. Several chromosomal proteins coeluted with NII from the casein-phosvitin-Sepharose column. They remained associated with the enzyme in sucrose gradients, during gel filtration performed at physiological ionic strength, and are dissociated at high ionic strength. These proteins were highly phosphorylated when the protein-NII complex was incubated with ATP.
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