Analysis of differential effects of Pb2+ on protein kinase C isozymes
| Autor: | J. L. Tomsig, Xintian Tian, Xiaoyan Sun, Janusz B. Suszkiw |
|---|---|
| Rok vydání: | 1999 |
| Předmět: |
Molecular Sequence Data
Biology Toxicology Divalent Non-competitive inhibition Ca2+/calmodulin-dependent protein kinase Catalytic Domain Animals Humans Amino Acid Sequence Kinase activity Protein kinase C Protein Kinase C C2 domain Pharmacology chemistry.chemical_classification Binding Sites Dose-Response Relationship Drug Rats Isoenzymes Enzyme chemistry Biochemistry Lead Calcium Cattle Signal transduction |
| Zdroj: | Toxicology and applied pharmacology. 156(1) |
| ISSN: | 0041-008X |
| Popis: | 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+. |
| Databáze: | OpenAIRE |
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