Characterization of an arginine‐specific protein kinase tightly bound to rat liver DNA

Abstract

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.

Focus on Phosphoarginine and Phospholysine

“…Upon separation by gel filtration, the enzyme was found to have a nominal molecular weight of 34 kDa and was thought to exist as a single subunit. The protein arginine kinase was able to autophosphorylate as well as phosphorylate an 11 kDa chromosomal protein (most likely histone H4) using ATP and deoxyATP as phosphoryl donors, with a requirement for Mn 2+ as a cofactor [76]. Phosphoarginine was identified by thin-layer electrophoresis, but the absence of phosphoamino acid standards needed to confirm this analysis weakens the credibility of the findings of this particular experiment.…”

Phosphoarginine stimulation of Na(+)‐Ca2+ exchange in squid axons‐‐a new pathway for metabolic regulation?

“…In eukaryotic cells, several proteins undergo covalent modification through phosphorylation in the side chain of basic N-amino acids such as arginine (Scot, et al 1976), histidine (Fujitaki et al 1981) and lysine (Chen et al 1977). Phosphorylated basic residues have been found in several cell types including rat liver (Levy-Favatier et al 1987), carcinoma cells, smooth muscle and bovine liver mitochondria (Boyer et al 1962;Urushizaki & Seifer, 1985). Specifically, phosphoarginine residues have been found in brain myelin (Scott et al 1976) and in VP12, a protein of granulosis virus (Wilson & Consigli, 1985).…”

Protein histidine phosphatase activity in rat liver and spinach leaves

“…However, almost all other studies in eukaryotes have focused on the phosphorylation of serine, threonine and tyrosine residues since partial acid hydrolysis, as used for conventional phosphoamino acid analysis, destroys the phosphoramidate bonds found in phosphohistidine, phospholysine or phosphoarginine [7]. Phosphoramidate bonds and protein kinases that phosphorylate arginine, histidine or lysine have been reported in eukaryotes [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] and reviewed recently [26]. Saccharomyces cerevisiae contains a kinase that transfers phosphate from ATP to histidine-75 in the protein, histone H4, in vitro [27].…”