Calmodulin (CaM) and C-kinase (protein kinase C, C-K) are universally important calcium-binding proteins which are intimately involved in the regulation of cell proliferation, among other events. These proteins, which have analogous calcium-binding domains, undergo Ca2+-induced structural rearrangement to expose binding sites capable of interacting with many of the same regulatory ligands. Recent evidence has suggested that polyamines, a class of ubiquitous aliphatic cations, may be endogenous regulators of CaM and C-K. Polyamines have previously been strongly implicated in the regulation of protein and DNA synthesis, and accumulate in high intracellular concentrations during the crucial transition from G1 to S phase of the cell cycle. If polyamines modulate the activity of C-K and CaM-dependent enzymes during this interval, this could partially account for their significant influence on cell growth processes. We will examine the role of polyamines in terms of their interaction with CaM and C-K, and determine the nature of their influence on phosphorylation reactions regulated by these important calcium-binding proteins. We will characterize polyamine binding to CaM and C-K. Using fluorescence-based binding assays and equilibrium dialysis techniques, we will determine the calcium dependence, affinity, and stoichiometry of polyamine binding, and evaluate allosteric interaction among polyamine sites and hydrophobic regulatory ligand binding sites. We will also study the effects of polyamines on the activity of C-K and several CaM-dependent enzymes in a cell-free system. We will examine the influence of intracellular polyamine levels on patterns of phosphorylation by C-K and CaM-dependent kinases duyring an interval of transient polyamine accumulation in late G1 phase. We will also determine the effects of drug-induced polyamine depletion on phosphorylation. These investigations should provide insight into the mechanism of action of polyamines, and of these key calcium-dependent regulatory proteins, in modulating cell proliferation and differentiation.
