In vitro activation of a 60-70 kDa histone H4 protein kinase from neutrophils by limited proteolysis.
Neutrophils stimulated with the chemotactic peptide fMet-Leu-Phe (fMLP) are known to exhibit a rapid and transient activation of a histone H4 kinase that may function in a stimulatory pathway downstream of phosphatidylinositol 3-kinase. The activity of this histone kinase in unstimulated neutrophils and cells treated with 1.0 microM fMLP for 10 sec was 8.8 +/- 5 and 43 +/- 2 pmol P/min per 10(7) cells, respectively. In this paper, we report that unstimulated neutrophils contain a latent H4 kinase in the 100,000 x g soluble fraction that can be markedly activated by treatment with trypsin. The values for the untreated and trypsin treated enzyme were 5.5 +/- 1.0 and 63.6 +/- 18 pmol P/min per 10(7) cell-equivalents, respectively. This kinase was insensitive to a selective antagonist of protein kinase C (i.e., 50 microM 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7)) but completely blocked by 100 nM staurosporine. Only a single peak of activity was observed for this enzyme when the 100,000 x g supernatant fraction was fractionated on either an exclusion (KW-803) or an anion exchange column (DEAE), or during isoelectric focusing. The molecular weight of the latent kinase was 64 +/- 6 kDa and the isoelectric point was 7.6 +/- 0.1. During all fractionation procedures, the H4 kinase co-chromatographed with a trypsin-activated kinase that catalyzed the phosphorylation of a peptide which corresponds to residues 297-331 of the 47 kDa subunit of the NADPH-oxidase complex (p47-phox). The properties of the trypsin-activated H4 kinase from unstimulated neutrophils are very similar to those reported for this enzyme from fMLP-stimulated cells.
Liu
R
R
Boston Biomedical Research Institute, MA 02114, USA.
Leavis
P
P
Badwey
J A
JA
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000470:Alkaloids / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002458:Cell Fractionation; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006168:Guinea Pigs; D006657:Histones / Q000378:metabolism; D007526:Isoelectric Point; D015636:Magnesium Chloride / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D006656:Protamine Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D019311:Staurosporine; D014357:Trypsin / Q000378:metabolism
D000470:Alkaloids; D004791:Enzyme Inhibitors; D006657:Histones; D010750:Phosphoproteins; D015636:Magnesium Chloride; D000255:Adenosine Triphosphate; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D006656:Protamine Kinase; D014357:Trypsin; D019311:Staurosporine