Chen S J, Klann E, Gower M C, Powell C M, Sessoms J S, Sweatt J D
Division of Neuroscience, Baylor College of Medicine, Houston, Texas 77030.
Biochemistry. 1993 Feb 2;32(4):1032-9. doi: 10.1021/bi00055a006.
The neuronal protein neurogranin, also known as RC3, is a selective substrate for protein kinase C (PKC). We synthesized a peptide corresponding to the phosphorylation domain of neurogranin (amino acids 28-43) and characterized its properties as a PKC substrate. Neurogranin(28-43) was phosphorylated by purified PKC with a Km of 150 nM. No significant phosphorylation of the peptide by either cAMP-dependent protein kinase or by calcium/calmodulin-dependent protein kinase II could be detected. Thus, neurogranin(28-43) is a potent and selective substrate for PKC. We tested several peptide analogues of neurogranin(28-43) for their substrate potency and specificity as kinase substrates, in order to help elucidate the structural determinants involved in the phosphorylation of substrates by PKC. Substituting Arg36 with Ile caused a significant reduction in the affinity for PKC. Replacing Lys30 with Arg enhanced the catalytic efficiency (Vmax/Km) for PKC but diminished the selectivity of the substrate for PKC. These results support the generally held model that basic amino acids on both sides of the phosphorylated Ser are important structural determinants in PKC substrates. However, the data also suggest that the presence of particular basic amino acids (Arg vs Lys) can contribute to the degree of selectivity of a substrate for PKC. Replacement with Ala of Phe35, the amino acid adjacent to the Ser34 phosphorylation site, resulted in a peptide with greatly diminished potency as a PKC substrate. This finding indicates a critical role of Phe35 in modulating binding and phosphorylation of neurogranin-derived peptides by PKC.(ABSTRACT TRUNCATED AT 250 WORDS)
神经元蛋白神经颗粒素，也称为RC3，是蛋白激酶C（PKC）的选择性底物。我们合成了一段与神经颗粒素磷酸化结构域相对应的肽（氨基酸28 - 43），并对其作为PKC底物的特性进行了表征。神经颗粒素（28 - 43）被纯化的PKC磷酸化，Km为150 nM。未检测到该肽被环磷酸腺苷依赖性蛋白激酶或钙/钙调蛋白依赖性蛋白激酶II显著磷酸化。因此，神经颗粒素（28 - 43）是PKC的有效且选择性底物。我们测试了神经颗粒素（28 - 43）的几种肽类似物作为激酶底物的底物活性和特异性，以帮助阐明PKC使底物磷酸化所涉及的结构决定因素。用异亮氨酸取代精氨酸36会导致对PKC的亲和力显著降低。用精氨酸取代赖氨酸30提高了对PKC的催化效率（Vmax/Km），但降低了底物对PKC的选择性。这些结果支持了普遍持有的模型，即磷酸化丝氨酸两侧的碱性氨基酸是PKC底物中的重要结构决定因素。然而，数据也表明特定碱性氨基酸（精氨酸与赖氨酸）的存在会影响底物对PKC的选择性程度。将丝氨酸34磷酸化位点相邻的苯丙氨酸35替换为丙氨酸，会产生一种作为PKC底物活性大大降低的肽。这一发现表明苯丙氨酸35在调节PKC对神经颗粒素衍生肽的结合和磷酸化中起关键作用。（摘要截断于250字）
