Phosphorylase kinase, a metal ion-dependent dual specificity kinase.
Phosphorylase kinase is shown to be a dual specificity kinase. The specificity of phosphorylation is determined by divalent cation. Mg2+ causes seryl phosphorylation of phosphorylase b, but Mn2+ activates tyrosine phosphorylation of angiotensin II. In contrast to seryl phosphorylation, the tyrosine kinase activity of holoenzyme is not regulated by Ca2+. Preincubation of the holoenzyme with Ca2+, Mg2+ and ATP that causes autophosphorylation activates tyrosine kinase activity. The tyrosyl kinase activity is a property of the gamma subunit. Addition of varying amounts of Mn2+ to a truncated form of the gamma subunit of phosphorylase kinase containing MgATP inhibits serine kinase but activates tyrosine kinase activity. This result along with an oxidative reaction caused by Cu2+ and site-directed mutagenesis of the putative catalytic base inhibiting both serine and tyrosine kinase activity suggest that one active site is involved in both activities. Kinetic studies with Mn2+ and ATP show that Km for nucleotide is not changed with a seryl or tyrosyl substrate. The Vm values are different, and the value for tyrosyl phosphorylation is similar to other tyrosyl kinases. We propose two conformations for the active site; one favors seryl phosphorylation, and the second tyrosyl phosphorylation is caused by the binding of divalent cation at a second metal ion binding site.
Yuan
C J
CJ
Department of Biochemistry and Biophysics, Iowa State University, Ames 50011.
Huang
C Y
CY
Graves
D J
DJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D000804:Angiotensin II; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D008345:Manganese / Q000494:pharmacology*; D009132:Muscles / Q000201:enzymology*; D010764:Phosphorylase Kinase / Q000302:isolation & purification / Q000378:metabolism*; D010763:Phosphorylase b / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D011994:Recombinant Proteins; D000804:Angiotensin II; D008345:Manganese; D000255:Adenosine Triphosphate; D010763:Phosphorylase b; D010764:Phosphorylase Kinase; D011505:Protein-Tyrosine Kinases; D008274:Magnesium; D002118:Calcium