Knapp L T, Kanterewicz B I, Hayes E L, Klann E
Department of Neuroscience, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
Biochem Biophys Res Commun. 2001 Aug 31;286(4):764-70. doi: 10.1006/bbrc.2001.5448.
Protein kinase C (PKC) is an important intracellular signaling molecule whose activity is essential for a number of aspects of neuronal function including synaptic plasticity. We investigated the regulation of PKC activity by reactive nitrogen species in order to examine whether such species regulate PKC in neurons. Neither autonomous nor cofactor-dependent PKC activity was altered when either hippocampal homogenates or rat brain purified PKC were incubated briefly with three different nitric oxide donor compounds. However, brief incubation of either hippocampal homogenates or purified PKC with peroxynitrite (ONOO(-)) inhibited cofactor-dependent PKC activity in a manner that correlated with the nitration of tyrosine residues on PKC, suggesting that this modification was responsible for the inhibition of PKC. Consistent with this idea, reducing agents had no effect on the inhibition of PKC activity caused by ONOO(-). Because there are numerous PKC isoforms that differ in the composition of the regulatory domain, we studied the effect of ONOO(-) on various PKC isoforms. ONOO(-) inhibited the cofactor-dependent activity of the alpha, betaII, epsilon, and zeta isoforms, indicating that inhibition of enzymatic activity by ONOO(-) was not PKC isoform-specific. We also were able to isolate nitrated PKCalpha and PKCbetaII from ONOO(-)-treated hippocampal homogenates via immunoprecipitation. Collectively, our findings support the hypothesis that ONOO(-) inhibits PKC activity via tyrosine nitration in neurons.
蛋白激酶C（PKC）是一种重要的细胞内信号分子，其活性对于包括突触可塑性在内的神经元功能的多个方面至关重要。我们研究了活性氮物质对PKC活性的调节作用，以检验此类物质是否在神经元中调节PKC。当海马匀浆或大鼠脑纯化PKC与三种不同的一氧化氮供体化合物短暂孵育时，自主型或辅因子依赖性PKC活性均未改变。然而，海马匀浆或纯化PKC与过氧亚硝酸盐（ONOO⁻）短暂孵育会抑制辅因子依赖性PKC活性，其抑制方式与PKC上酪氨酸残基的硝化作用相关，这表明这种修饰是PKC受抑制的原因。与这一观点一致，还原剂对ONOO⁻引起的PKC活性抑制没有影响。由于存在多种在调节结构域组成上不同的PKC同工型，我们研究了ONOO⁻对各种PKC同工型的影响。ONOO⁻抑制了α、βII、ε和ζ同工型的辅因子依赖性活性，表明ONOO⁻对酶活性的抑制并非PKC同工型特异性的。我们还能够通过免疫沉淀从ONOO⁻处理的海马匀浆中分离出硝化的PKCα和PKCβII。总体而言，我们的研究结果支持以下假设：ONOO⁻通过神经元中的酪氨酸硝化作用抑制PKC活性。
