Analysis of Differential Effects of Pb2+on Protein Kinase C Isozymes

Protein kinase C has been implicated as a cellular target for Pb2+toxicity. We have previously proposed that Pb2+modulates PKC activity by interacting with multiple sites within the enzyme. In order to further characterize the Pb–PKC interactions we compared the effects of Pb2+on the CA-dependent and -independent protein kinase C isozymes using recombinant human PKC-α, PKC-ϵ, and PKC-ζ as well as the catalytic fragment of bovine brain protein kinase C, the PKC-M. The results demonstrate that, whereas at pM concentrations Pb2+activates PKC-α half maximally (KAct≈ 2 pM), it has no effect on PKC-ϵ, PKC-ζ, or PKC-M activities. The activation of PKC-α by Pb2+is additive with Ca2+in a manner indicating interaction with half of the calcium activation sites. In the micromolar range of concentrations, Pb2+inhibits all PKCs with estimatedK0.5of 1.0, 2.3, 28, and 93 μM for PKC-M, PKC-α, PKC-ϵ, and PKC-ζ, respectively. Examination of Pb2+effects on PKC-M kinetics indicates a mixed type inhibition with respect to ATP and noncompetitive inhibition with respect to histone. Taken together with the results of our previous study (Tomsig and Suszkiw,J. Neurochem.64, 2667–2673, 1995) and the evidence for the existence of two Ca2+coordination sites Ca1 and Ca2 within the C2 domain (Shaoet al., Science [Washington, D.C.]273, 248–251, 1996), the results of the current study provide further support for a multisite Pb–PKC interaction scheme wherein lead (1) partially activates the enzyme through pM-affinity interactions with the Ca1 site and inhibits the divalent cation-dependent activity through nM-affinity interactions with Ca2 site in the C2 domain and (2) inhibits the constitutive kinase activity through μM-affinity interactions with the catalytic domain. The concentration dependence of the differential effects of Pb2+on the calcium-dependent and -independent PKCs underscores the importance of the C2 motif as a high affinity molecular target for Pb2+.