Florez J C, Nelson R B, Routtenberg A
Cresap Neuroscience Laboratory, Northwestern University, Evanston, Illinois 60208.
Exp Neurol. 1991 Jun;112(3):264-72. doi: 10.1016/0014-4886(91)90126-w.
We introduce a new procedure to study kinase substrates in postmortem human brain. By adding purified exogenous protein kinase C (PKC) and the phospholipid phosphatidylserine to brain homogenates in vitro we are able to analyze PKC substrates. A human 53-kDa phosphoprotein is described that appears to be homologous to rat and monkey protein F1 (GAP-43). This identity is based on molecular weight, isoelectric point, phosphorylation by exogenous protein kinase C, enhancement of its phosphorylation by three activators (phospholipids, calcium and phorbol esters), phosphopeptide maps, and cross-reactivity with an antibody raised against rat protein F1. Protein F1 is a PKC substrate associated with synaptic plasticity and nerve growth. Its phosphorylation in rat brain has been correlated with long-term potentiation, an electrophysiological model of memory. In the present study of normal brain, human protein F1 shows an occipitotemporal in vitro phosphorylation gradient. This is consistent with previous observations in nonhuman primates. This gradient is less pronounced in Alzheimer's disease (AD). Changes in the in vitro phosphorylation pattern of three other non-PKC substrates in Alzheimer's disease, including one with characteristics similar to microtubule-associated protein tau, are also reported. These results suggest that protein phosphorylation can be studied in postmortem human brain and that PKC-mediated phosphorylation of protein F1, already linked to synaptic plasticity and memory, may be altered in AD.
我们介绍一种研究人死后大脑中激酶底物的新方法。通过在体外向脑匀浆中添加纯化的外源蛋白激酶C（PKC）和磷脂酰丝氨酸，我们能够分析PKC底物。本文描述了一种人53 kDa磷蛋白，它似乎与大鼠和猴的蛋白F1（GAP - 43）同源。这种同源性基于分子量、等电点、外源蛋白激酶C的磷酸化作用、三种激活剂（磷脂、钙和佛波酯）对其磷酸化作用的增强、磷酸肽图谱以及与针对大鼠蛋白F1产生的抗体的交叉反应性。蛋白F1是一种与突触可塑性和神经生长相关的PKC底物。其在大鼠脑中的磷酸化作用已与长时程增强（一种记忆的电生理模型）相关联。在对正常大脑的当前研究中，人蛋白F1在枕颞区显示出体外磷酸化梯度。这与先前在非人类灵长类动物中的观察结果一致。这种梯度在阿尔茨海默病（AD）中不太明显。本文还报道了阿尔茨海默病中其他三种非PKC底物的体外磷酸化模式变化，其中一种具有与微管相关蛋白tau相似的特征。这些结果表明，可以在人死后大脑中研究蛋白质磷酸化，并且已经与突触可塑性和记忆相关联的PKC介导的蛋白F1磷酸化在AD中可能发生改变。
