Ca(2+)-calmodulin-dependent phosphorylation of arginine in histone 3 by a nuclear kinase from mouse leukemia cells.
A Ca(2+)-calmodulin dependent histone 3 kinase was partially purified from a low salt (150 mM NaCl) nuclear extract of mouse leukemia cells by calmodulin-Sepharose affinity chromatography. In vitro, the kinase activity transferred gamma-phosphate from ATP to histone 3 to form an acid-labile and alkaline-stable linkage. Under the assay conditions 1.8 mol of phosphate are incorporated per mol of histone 3. Upon modification of arginine residues with phenylglyoxal prior to phosphorylation, a considerable decrease in the amount of phosphate transferred to histone 3 was observed. Amino acid analysis revealed that H3 was phosphorylated on arginine residues. To identify the phosphorylated peptide(s), histone 3 was cleaved with cyanogen bromide prior to phosphorylation. The phosphorylated mixture was then separated by gel filtration high-performance liquid chromatography under denaturing conditions. Fragments I (N-terminal 10.3-kDa peptide) and III (C-terminal 1.7-kDa peptide) were both phosphorylated. Amino acid sequencing further revealed that the molar yields of 3 of the 4 arginines present in the phosphorylated cyanogen bromide fragment III were reduced by a factor of about 10 compared with the corresponding arginines from the unphosphorylated fragment. In the case of fragment I, 25 cycles of Edman degradation revealed that the recovery of only arginine 2 was reduced by a factor of 20. The putative phosphorylation sites are arginines 2, 128, 129, and 131. The sequence information offered an indirect evidence that these arginines were the sites of phosphorylation. The kinase described in this report represents a first member of a potentially important new class of kinases which are Ca(2+)-calmodulin dependent and which phosphorylate arginine.
Wakim
B T
BT
Macromolecular Laboratory, Stritch School of Medicine, Loyola University of Chicago, Maywood, Illinois 60153.
Aswad
G D
GD
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D002467:Cell Nucleus / Q000201:enzymology*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D006657:Histones / Q000302:isolation & purification / Q000378:metabolism*; D007939:Leukemia L1210 / Q000201:enzymology*; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D010748:Phosphopeptides / Q000302:isolation & purification; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D006656:Protamine Kinase / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D006657:Histones; D010446:Peptide Fragments; D010748:Phosphopeptides; D010750:Phosphoproteins; D001120:Arginine; D006656:Protamine Kinase; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C095895:calcium-calmodulin-activated histone 3 arginine kinase; D003488:Cyanogen Bromide