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Analysis of differential effects of Pb2+ on protein kinase C isozymes
1College of Medicine, University of Cincinnati, Cincinnati, Ohio, 45267-0576, USA.
Abstract:
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-alpha, PKC-epsilon, and PKC-zeta 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-alpha half maximally (KAct approximately 2 pM), it has no effect on PKC-epsilon, PKC-zeta, or PKC-M activities. The activation of PKC-alpha 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 estimated K0.5 of 1.0, 2.3, 28, and 93 microM for PKC-M, PKC-alpha, PKC-epsilon, and PKC-zeta, 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 (Shao et 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 microM-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+.
Insights
Lead (Pb2+) selectively activates and inhibits Protein Kinase C (PKC) at different concentrations. This study reveals Pb2+ interacts with multiple sites on PKC, affecting its activity and highlighting the C2 motif as a key target.
Area of Science:
- Biochemistry
- Molecular Biology
- Toxicology
Background:
- Protein Kinase C (PKC) is a key enzyme in cellular signaling.
- Lead (Pb2+) is known to cause cellular toxicity, with PKC implicated as a target.
- Previous work suggested Pb2+ interacts with multiple sites on PKC.
Purpose of the Study:
- To further characterize the interactions between lead (Pb2+) and various Protein Kinase C (PKC) isozymes.
- To compare the effects of Pb2+ on calcium-dependent and calcium-independent PKC isoforms.
- To elucidate the specific binding sites and mechanisms of Pb2+-PKC interaction.
Main Methods:
- Utilized recombinant human PKC-alpha, PKC-epsilon, and PKC-zeta.
- Employed the catalytic fragment of bovine brain protein kinase C (PKC-M).
- Assessed the effects of varying Pb2+ concentrations on enzyme activity and kinetics.
Main Results:
- Pb2+ activated PKC-alpha at picomolar (pM) concentrations, with additive effects to calcium (Ca2+).
- Pb2+ inhibited all tested PKC isoforms (PKC-M, PKC-alpha, PKC-epsilon, PKC-zeta) at micromolar (µM) concentrations.
- Kinetic analysis indicated mixed-type inhibition for ATP and noncompetitive inhibition for histone.
Conclusions:
- Pb2+ interacts with multiple sites on PKC, leading to concentration-dependent activation and inhibition.
- Pb2+ activates PKC-alpha via pM-affinity interactions with the Ca1 site and inhibits via nM-affinity interactions with the Ca2 site in the C2 domain.
- Pb2+ inhibits constitutive kinase activity through µM-affinity interactions with the catalytic domain, underscoring the C2 motif's role as a high-affinity target.