TYROSINE-PHOSPHORYLATED CALMODULIN HAS REDUCED BIOLOGICAL-ACTIVITY

Calmodulin is phosphorylated by the purified insulin receptor on tyros ine residues with a maximum stoichiometry of 1 mol phosphate/mol of ca lmodulin. Isolated tryptic phosphopeptides were sequenced by manual Ed man degradation and demonstrated that calmodulin is equally phosphoryl ated on tyrosine 99 and tyrosine 138. Phosphorylated calmodulin has a decreased affinity (K-0.5 = 4.2 nM) for the 63-kDa isozyme of cyclic n ucleotide phosphodiesterase compared to nonphosphorylated calmodulin ( K-0.5 = 2.1 nM). The K-0.5 for Ca2+ is marginally increased from 2.8 t o 3.2 mu M in the presence of phosphotyrosyl calmodulin. The effect of the calmodulin antagonist, mastoparan, was investigated to determine whether mastoparan would differentially inhibit calmodulin- or phospho calmodulin-dependent enzyme activity. The IC50 of mastoparan is fourfo ld lower for phosphotyrosyl calmodulin compared to nonphosphorylated c almodulin. Phosphorylation of calmodulin may provide a mechanism for t he differential regulation of calmodulin-dependent enzymes. These obse rvations further support a potentially important regulatory function o f calmodulin phosphorylation in signal transduction. (C) 1994 Academic Press, Inc.