Ortiz J, Harris H W, Guitart X, Terwilliger R Z, Haycock J W, Nestler E J
Department of Psychiatry, Yale University School of Medicine, New Haven, Connecticut.
J Neurosci. 1995 Feb;15(2):1285-97. doi: 10.1523/JNEUROSCI.15-02-01285.1995.
Quantitative blot immunolabeling techniques were used to determine the concentrations of ERK1 (M(r) 44 kDa) and ERK2 (M(r) 42 kDa), the two major extracellular signal-regulated protein kinases, in different regions of rat brain. The aggregate ERK concentrations (ERK1 and ERK2) were relatively high in each of the brain regions studied, ranging from approximately 0.35 ng/microgram protein in cerebellum to approximately 1.2 ng/microgram protein in nucleus accumbens. However, differences in the regional distributions of ERK1 and ERK2 resulted in ratios of their relative abundance that differed by close to 10-fold among the regions studied. The ratios of ERK1 protein to ERK2 protein varied along a rostral-caudal gradient from a low of 0.16 in frontal cortex to a high of 1.5 in pons/medulla. In hypotonic homogenates from regions at either extreme of the gradient, ERK1 and ERK2 were both found to be predominantly (> 80%) soluble. In subcellular fractions prepared from sucrose homogenates of frontal cortex and pons/medulla, both ERK1 and ERK2 were enriched in the synaptosomal and cytosolic fractions, whereas ERK2 was also enriched in the microsomal fraction. By contrast, in subfractions containing purified nuclei, levels of ERK1 and ERK2 were about one-third of those seen in homogenates and, in subfractions enriched in mitochondria, both ERK1 and ERK2 were barely detectable. The catalytic activity of the ERKs paralleled their protein levels in all of the brain regions except the hippocampus, in which the activity and phosphotyrosine content were disproportionately high. As a possible explanation for this apparent disparity, the regional distribution of ERK kinase (MEK), which phosphorylates and activates the ERKs, was also investigated. The levels of immunoreactivity of the M(r) 45 kDa ERK kinase band differed by about threefold among the brain regions, with the highest levels being present in nucleus accumbens, hippocampus, substantia nigra, and caudate/putamen. Therefore, a higher concentration of ERK kinase immunoreactivity did not appear to account for the disproportionate levels of ERK activity and phosphotyrosine content in the hippocampus. Potential regulation of ERK and ERK kinase levels was also investigated in rats subjected to chronic morphine treatment. ERK1 and ERK2 levels were increased selectively in locus coeruleus and caudate/putamen after chronic morphine treatment, whereas ERK kinase immunoreactivity remained unchanged in all of the brain regions analyzed. In summary, the regional differences in ERK and ERK kinase expression and the region-specific regulation of ERK expression suggest that ERK-related signaling may play an important role in CNS function and its adaptive responses.
采用定量印迹免疫标记技术测定大鼠脑不同区域中两种主要的细胞外信号调节蛋白激酶ERK1（分子量44 kDa）和ERK2（分子量42 kDa）的浓度。在所研究的每个脑区中，ERK的总浓度（ERK1和ERK2）相对较高，范围从小脑的约0.35 ng/μg蛋白到伏隔核的约1.2 ng/μg蛋白。然而，ERK1和ERK2区域分布的差异导致其相对丰度的比值在各研究区域中相差近10倍。ERK1蛋白与ERK2蛋白的比值沿头-尾梯度变化，从额叶皮质的低比值0.16到脑桥/延髓的高比值1.5。在梯度两端区域的低渗匀浆中，ERK1和ERK2均主要（>80%）为可溶性。在由额叶皮质和脑桥/延髓的蔗糖匀浆制备的亚细胞组分中，ERK1和ERK2均在突触体和胞质组分中富集，而ERK2也在微粒体组分中富集。相比之下，在含有纯化细胞核的亚组分中，ERK1和ERK2的水平约为匀浆中的三分之一，而在富含线粒体的亚组分中，ERK1和ERK2几乎检测不到。除海马体外，ERK的催化活性与所有脑区中的蛋白水平平行，在海马体中其活性和磷酸酪氨酸含量异常高。作为对这种明显差异的一种可能解释，还研究了磷酸化并激活ERK的ERK激酶（MEK）的区域分布。分子量45 kDa的ERK激酶条带的免疫反应性水平在各脑区中相差约三倍，最高水平出现在伏隔核、海马体、黑质和尾状核/壳核中。因此，较高浓度的ERK激酶免疫反应性似乎不能解释海马体中ERK活性和磷酸酪氨酸含量的不成比例水平。还研究了慢性吗啡处理大鼠中ERK和ERK激酶水平的潜在调节。慢性吗啡处理后，蓝斑和尾状核/壳核中的ERK1和ERK2水平选择性增加，而在所分析的所有脑区中，ERK激酶免疫反应性保持不变。总之，ERK和ERK激酶表达的区域差异以及ERK表达的区域特异性调节表明，ERK相关信号传导可能在中枢神经系统功能及其适应性反应中起重要作用。
