Peptide specificity determinants at P-7 and P-6 enhance the catalytic efficiency of Ca2+/calmodulin-dependent protein kinase I in the absence of activation loop phosphorylation.
Phosphorylation of Ca2+/calmodulin-dependent protein kinase I (CaM KI) at Thr-177 by recombinant rat Ca2+/calmodulin-dependent kinase kinase B (CaM KKB) modulates the kinetics of synapsin-(4-13) peptide phosphorylation by reducing the Km 44-fold and decreasing the KCaM 4-fold. There is also a slight decrease in Km for ATP and increase in enzyme Vmax. A synthetic peptide substrate from the yeast transcription factor, ADR1-(222-234)G233 is a 15-fold better substrate for the Thr-177 dephospho-form of CaM KI than synapsin-(4-13). The Thr-177 dephospho-enzyme has a Km and Vmax for ADR1-(222-234)G233 similar to the values with synapsin-(4-13) using the Thr-177 phosphorylated enzyme. Likewise, with ADR1-(222-234)G233 as substrate, phosphorylation of Thr-177 or substitution of T177A had very little effect on the kinetic values. Using chimeric peptides between synapsin-(4-13) and ADR1-(222-234)G233 we found that N-terminal basic residues at P-7 and P-6 positions were sufficient to allow efficient phosphorylation by the Thr-177 dephospho-form of CaM KI. Phosphorylation of Thr-177 expands the substrate specificity of CaM KI and is not merely an "on-off" switch for kinase activity.
Hook
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Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Kemp
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BE
Means
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AR
D000595:Amino Acid Sequence; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002384:Catalysis; D004789:Enzyme Activation; D018922:HL-60 Cells; D006801:Humans; D007700:Kinetics; D010455:Peptides / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D010455:Peptides; D017871:Calcium-Calmodulin-Dependent Protein Kinases