Sun X, Tian X, Tomsig J L, Suszkiw J B
College of Medicine, University of Cincinnati, Cincinnati, Ohio, 45267-0576, USA.
Toxicol Appl Pharmacol. 1999 Apr 1;156(1):40-5. doi: 10.1006/taap.1999.8622.
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+.
蛋白激酶C被认为是铅离子（Pb2+）毒性作用的细胞靶点。我们之前曾提出，Pb2+通过与该酶内的多个位点相互作用来调节蛋白激酶C（PKC）的活性。为了进一步明确铅与PKC之间的相互作用，我们使用重组人PKC-α、PKC-ε和PKC-ζ，以及牛脑蛋白激酶C（PKC-M）的催化片段，比较了Pb2+对依赖钙（CA）和不依赖钙的PKC同工酶的影响。结果表明，在皮摩尔浓度下，Pb2+能使PKC-α达到最大激活的一半（激活常数KAct约为2皮摩尔），但对PKC-ε、PKC-ζ或PKC-M的活性没有影响。Pb2+对PKC-α的激活作用与Ca2+具有加和性，这表明它与一半的钙激活位点相互作用。在微摩尔浓度范围内，Pb2+抑制所有的PKC，对PKC-M、PKC-α、PKC-ε和PKC-ζ的半数抑制浓度（K0.5）估计分别为1.0、2.3、28和93微摩尔。对Pb2+对PKC-M动力学影响的研究表明，它对ATP表现为混合型抑制，对组蛋白表现为非竞争性抑制。结合我们之前研究的结果（Tomsig和Suszkiw，《神经化学杂志》64卷，2667 - 2673页，1995年）以及C2结构域内存在两个钙配位位点Ca1和Ca2的证据（Shao等人，《科学》[华盛顿特区]273卷，248 - 251页，1996年），本研究结果进一步支持了一种多位点铅 - PKC相互作用模式，即铅（1）通过与Ca1位点的皮摩尔亲和力相互作用部分激活该酶，并通过与C2结构域中Ca2位点的纳摩尔亲和力相互作用抑制二价阳离子依赖性活性；（2）通过与催化结构域的微摩尔亲和力相互作用抑制组成型激酶活性。Pb2+对依赖钙和不依赖钙的PKC产生不同影响的浓度依赖性，突出了C2基序作为铅的高亲和力分子靶点的重要性。
