CaMKII Phosphorylates Phospholamban via an Ordered Sequential Kinetic Mechanism

Abstract

Many of the cellular responses to Ca++ signal are modulated by a family of multifunctional Ca++/calmodulin dependent protein kinases (CaMKs): CaMKI, CaMKII and CaMKIV. CaMKII has an autoregulatory domain that restricts enzyme activity in the absence of Ca++/CaM. Ca++/CaM binding alone produce maximal activity of CaMKII, whereas CaMKI and CaMKIV have an activation loop that requires phosphorylation by an upstream CaMK kinase for maximal activity. In addition to its role in memory and other neuronal functions, CaMKII appears to mediate many of the pathologic changes that occur during cardiac hypertrophy and heart failure. In order to further understand the role of CaMKII, we investigated the kinetic mechanism of CaMKII (IIα, IIβ, IIδ, IIγ) in comparison with those of CaMKI and CaMKIV by analyzing their steady state kinetics using phospholamban as a phosphoacceptor. The results indicated that (a) CaMK family's reaction mechanisms were of the sequential type in which all substrates must bind to enzyme before any product is released; (b) While CaMKIIs catalyzed Ordered Bi Bi reaction where phospholamban was the first substrate to bind and ADP was the last product to be released, CaMKI and CaMKIV exhibited random sequential mechanism where either phospholamban and ATP can bind to the free enzyme and (c) Constant α (ratio of apparent dissociation constants for binding peptide in the presence and absence of the second ligand) of all isozymes for ATP and peptide was higher than 1 indicating that the binding of phospholamban to CaMK decreased its affinities toward ATP. Our results suggested a unique kinetic mechanism for CaMKIIs in which peptide substrate binding may be an initiating factor for the catalysis and may play a role in the regulation of the enzyme activity in cells.