Harry R. Matthews

Protein kinases and phosphatases that act on histidine, lysine, or arginine residues in eukaryotic proteins: A possible regulator of the mitogen-activated protein kinase cascade

Phosphohistidine goes undetected in conventional studies of protein phosphorylation, although it may account for 6% of total protein phosphorylation in eukaryotes. Procedures for studying protein N- kinases are described. Genes whose products are putative protein histidine kinases occur in a yeast and a plant. In rat liver plasma membranes, activation of the small G-protein, Ras, causes protein histidine phosphorylation. Cellular phosphatases dephosphorylate phosphohistidine. One eukaryotic protein histidine kinase has been purified, and specific proteins phosphorylated on histidine have been observed. There is a protein arginine kinase in mouse and protein lysine kinases in rat. Protein phosphohistidine may regulate the mitogen-activated protein kinase cascade.

Nuclear protein kinases

SummaryNuclear protein kinases include enzymes that transfer the γ-phosphate of ATP to serine, threonine, lysine or histidine in proteins. Nuclear kinases with a preference for basic proteins are known as histone kinases; those preferring acidic protein substrates are casein kinases. Histone kinases include both cyclic AMP-independent protein kinases and cyclic AMP-dependent protein kinases. The best-characterized cyclic AMP-independent nuclear protein kinase is associated with cell proliferation and is activated (or transported to the nucleus) in G2 phase of the cell cycle. It phosphorylates specific serine and threonine residues in the non globular domains of histone H1 and appears to promote chromosome condensation. The cyclic AMP-dependent protein kinase has unknown nuclear function(s), although it may be translocated from cytoplasm to nucleus in response to specific hormonal stimuli which are also associated with changes in transcriptional activity. There is a massive peak of nuclear cyclic AM P-dependent protein kinase activity in G2 phase of the cell cycle. Nuclear casein kinases are apparently very heterogeneous. Two of these enzymes have been purified to homogeneity. They phosphorylate non-histone chromosomal proteins, including RNA polymerase and ornithine decarboxylase. Phosphorylated ornithine decarboxylase is inactive enzymatically but, in Physarum, it binds to the rDNA minichromosome and stimulates rRNA transcription. Kinases forming phosphoramidate bonds occur in a variety of rat tissues and form phosphohistide in historic H4 and phospholysine in histone H1.

Histidine phosphorylation of annexin I in airway epithelia.

Although [Cl−] i regulates many cellular functions including cell secretion, the mechanisms governing these actions are not known. We have previously shown that the apical membrane of airway epithelium contains a 37-kDa phosphoprotein (p37) whose phosphorylation is regulated by chloride concentration. Using metal affinity (chelating Fe3+-Sepharose) and anion exchange (POROS HQ 20) chromatography, we have purified p37 from ovine tracheal epithelia to electrophoretic homogeneity. Sequence analysis and immunoprecipitation using monoclonal and specific polyclonal antibodies identified p37 as annexin I, a member of a family of Ca2+-dependent phospholipid-binding proteins. Phosphate on [32P]annexin I, phosphorylated using both [γ-32P]ATP and [γ-32P]GTP, was labile under acidic but not alkaline conditions. Phosphoamino acid analysis showed the presence of phosphohistidine. The site of phosphorylation was localized to a carboxyl-terminal fragment of annexin I. Our data suggest that cAMP and AMP (but not cGMP) may regulate annexin I histidine phosphorylation. We propose a role for annexin I in an intracellular signaling system involving histidine phosphorylation.

Association constants for the interaction of double-stranded and single-stranded DNA with spermine, spermidine, putrescine, diaminopropane, N1- and N8-acetylspermidine, and magnesium: determination from analysis of the broadening of thermal denaturation curves

The effect of Mg2+, putrescine, diaminopropane, N1-acetylspermidine, N8-acetylspermidine, spermidine, and spermine on the thermal denaturation of calf thymus DNA was investigated. As in a previous study with magnesium [W.F. Dove and N. Davidson, (1962) J. Mol. Biol. 5, 467-478], these ligands were found to raise the thermal denaturation temperature of the DNA and to broaden the thermal denaturation curve dramatically at the point where 10 to 20% of the DNA charge had been neutralized. At higher levels of charge neutralization the curves became sharper again. This behavior was due to differential binding of the ligands to single- and double-stranded DNA. The broadening was used to determine the ratio of the association constants of each ligand to the two forms of DNA using either an independent sites model of binding or an excluded sites model. The results show that the primary mode of binding of the ligands to DNA is electrostatic but that important secondary, nonelectrostatic, effects are also present.

A filter-based protein kinase assay selective for alkali-stable protein phosphorylation and suitable for acid-labile protein phosphorylation

Alkali-stable phosphorylation of proteins, particularly phosphotyrosine and phosphohistidine, is an important phenomenon in cells. In the case of phosphohistidine and some other phosphoamino acids, the phosphorylation is acid-labile and in these cases studies have been severely limited by the absence of a rapid assay suitable for acid-labile phosphorylation. The assay presented here involves a conventional kinase assay reaction followed by mild alkaline hydrolysis and adsorption of the product to washed Nytran paper at high pH. After further washing, at pH 9, the radioactivity on the papers is determined by liquid scintillation counting. Hence, acid-labile phosphorylation is preserved. The assay is selective for alkali-stable phosphorylation but not fully specific, mainly due to the need to balance the severity of the partial alkaline hydrolysis with the stability of the protein-peptide bonds. The assay has been used for the purification and characterization of a protein histidine kinase from Saccharomyces cerevisiae.

Rapid separation of phosphoamino acids including the phosphohistidines by isocratic high-performance liquid chromatography of the orthophthalaldehyde derivatives

Amino acids were derivatized with orthophthalaldehyde and separated by high-performance liquid chromatography on a polymer-based reverse-phase column (Hamilton PRP-1) at pH 7.2 using isocratic elution with 14.3 mM sodium phosphate, 1.1% tetrahydrofuran, 6.6% acetonitrile. Phosphorylated amino acids were eluted with baseline resolution in the following order: 1-phosphohistidine, phosphoserine, 3-phosphohistidine, phosphotyrosine, phosphothreonine, and phosphoarginine. Each of the phosphoamino acids was separated from its parent amino acid but aspartate and glutamate eluted in the same region as the phosphoamino acids. The sensitivity is in the picomole range and the separation time, injection to injection, is 15 min. The linearity for phosphothreonine extends at least from 30 pmol to 30 nmol. Quantitation by radioactivity is good for each of the phosphoamino acids except in the case of [1-32P]phosphohistidine, which coelutes with inorganic phosphate.

Application of sodium dodecyl sulfate-gel electrophoresis to low molecular weight polypeptides

Experiments with nine polypeptides with molecular weights between 2000 and 10,760 confirm the value of sodium dodecy sulfate (SDS)-gel electrophoresis for separating polypeptides in this molecular weight range. In one case, electrophoretic blotting and microsequencing were successfully carried out. However, molecular weight determination in the low molecular weight range (less than 10,000) is much less reliable than that in the conventional molecular weight range (greater than 10,000) for SDS gels. Information provided by suppliers of horse heart myoglobin fragment kits is potentially misleading.

Protein histidine phosphatase activity in rat liver and spinach leaves

Whole cell extracts from rat liver or spinach leaves contain divalent ion‐independent protein histidine phosphatase activity due to phosphatases of the PP1/PP2A family. In the rat liver extract, almost all the activity was found in the PP1, PP2A1 and PP22 peaks. In the spinach leaf extract, four phosphorylase phosphatase activity peaks were resolved — three containing PP1 and one containing PP2A — and all showed histidine phosphatase activity. Thus, protein histidine phosphatase activity is expressed in the cytosolic forms of protein phosphatases of the PP1/PP2A family in mammalian and plant cells.

Removal of phosphate from phosphohistidine in proteins

Kinetic constants of KM = 0.8 microM, 3 microM and 1.6 microM, and kcat = 9 s-1, 7 s-1 or 9 s-1 were determined for histidine dephosphorylation by protein phosphatases 1, 2A and 2C respectively. IC50 values were determined for the inhibition of protein phosphatase 1 by inhibitor 1 (IC50 = 1 nM), inhibitor-2 (IC50 = 3 nM) and okadaic acid (IC50 = 30 nM) and for the inhibition of protein phosphatase 2A by okadaic acid (IC50 = 0.02 nM) and microcystin-LR (IC50 = 1 nM). Inhibitor-1 (Ki = 0.7 nM) and okadaic acid (Ki = 32 nM) are noncompetitive with protein phosphatase 1. Some of the IC50 values were low enough to violate the assumptions of the usual inhibition equations and a more general approach to the analysis of the data was used. On the basis of these kinetic parameters and the presence of phosphohistidine, the major cellular protein serine/threonine phosphatases are likely to act as protein histidine phosphatases in the cell.

Studies of histidine phosphorylation by a nuclear protein histidine kinase show that histidine-75 in histone H4 is masked in nucleosome core particles and in chromatin

Histone H4 is a good substrate in vitro for the protein histidine kinase activity found both in Physarum polycephalum nuclear extracts and in Saccharomyces cerevisiae cell extracts. However, histone H4 in nucleosome core particles is not a substrate for these kinases. Isolated chromatin was also not a substrate for the protein histidine kinase. The results significantly limit possible interpretations of histidine phosphorylation on histone H4 in vivo and provide a new, sharper focus for future work. In addition, a polynucleotide kinase activity was identified in the Physarum extracts.