Abstract
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.
Highlights
Protein phosphorylation plays a regulatory role in signal transduction for many physiological processes in eukaryotic cells
Mechanism of Activation of calmodulin-dependent protein kinase I (CaM KI) by KKB—CaM KI has been shown to be activated by phosphorylation on Thr-177, a residue residing in the activation loop [28], by two distinct Ca2ϩ/CaM-dependent protein kinases
We asked whether recombinant calmodulin-dependent kinase kinase B (CaM KKB) can phosphorylate CaM KI as does its tissuepurified counterpart [28, 30]
Summary
Aletta et al [31] has shown that elevated intracellular Ca2ϩ concentrations, resulting from either ionomycin or KCl treatment of PC12 cells, lead to phosphorylation of CaM KI on Thr-177. CaM KIV induction of AP-1 [34] and cAMP response element-binding protein-mediated transcription [35] is inhibited by a T200A mutation in human CaM KIV These data provide compelling evidence that phosphorylation of CaM KI and IV is a physiological response to signals that increase intracellular Ca2ϩ with these kinases serving as intermediates in signal transduction cascades. Recent data from Chin et al [36] revealed that Thr-177dephosphorylated CaM KI has one of the highest specific activities reported for any protein kinase, and peptide substrates could show greater than 400-fold differences in specificities These surprising observations raised the possibility that activation loop phosphorylation of CaM KI may not be essential for kinase activity but could modulate substrate specificity. CaM KI and IV, represent the first kinases whose peptide substrate specificity has been shown to be regulated by activation loop phosphorylation and raises the possibility that such control may be extended to members of other protein kinase families
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