The role of polybasic compounds in determining the tyrosyl phosphorylation of calmodulin by the human insulin receptor.

A highly purified human insulin receptor preparation was shown to effect receptor autophosphorylation and the phosphorylation of poly(GluTyr) but not that of calmodulin. Addition of poly-L-lysine allowed for the stoichiometric tyrosyl phosphorylation of calmodulin in a dose-dependent fashion (EC50 approximately 83 nM) with the single target residue identified at tyr99. Higher concentrations of poly-L-lysine elicited the dose-dependent inhibition of calmodulin phosphorylation (IC50 approximately 0.7 microM) by a process which did not apparently involve either stimulation of calmodulin phosphatase activity or diminished receptor kinase activity. Polybasic substances such as poly-L-arginine, histone H1 and protamine sulphate all promoted calmodulin phosphorylation by the insulin receptor in a similar biphasic dose-dependent fashion. Poly-lysine's actions proved to lack stereo-specificity in that both the D- and L-forms were equally as effective. Reduction in the chain length of poly-L-lysine species attenuated their ability to promote calmodulin phosphorylation with L-lysine proving to be ineffective. Optimal promotion of calmodulin phosphorylation was achieved at an apparently constant ratio of calmodulin to poly-L-lysine of approximately 1:4 over a 100-fold range of calmodulin concentrations. Poly-L-lysine promoted the precipitation and subsequent resolubilization of calmodulin in a fashion whose biphasic dose-dependence paralleled that seen for its action in promoting calmodulin's phosphorylation. NaCl attenuated, in apparently identical dose-dependent fashions, poly-L-lysine's ability to both elicit the precipitation of calmodulin and to promote its phosphorylation. The presence of added Ca2+ led to a small potentiation of poly-L-lysine-dependent calmodulin phosphorylation at low concentrations, with inhibition occurring at higher concentrations where Ca2+ was shown to block calmodulin precipitation by poly-L-lysine. It is suggested that calmodulin can be phosphorylated by the insulin receptor only when it is cross-linked in a multivalent fashion to a suitable polybasic substance so that it forms large multimeric aggregates. Such a requirement for the formation of an aggregate between calmodulin and a suitable polybasic species may place specific constraints on the ability of calmodulin to serve as a substrate for receptor tyrosyl kinases within the cell.