Huang K P, Huang F L, Chen H C
Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
Arch Biochem Biophys. 1993 Sep;305(2):570-80. doi: 10.1006/abbi.1993.1463.
A 7.5-kDa heat- and acid-stable rat brain protein kinase C (PKC) substrate was purified to near homogeneity by a two-step procedure using DEAE-cellulose and hydroxylapatite column chromatography. This 78-amino-acid protein has a sequence identical to that deduced from rat brain RC3 cDNA identified with a cortex-minus-cerebellum subtracted cDNA probe (J. B. Watson et al., J. Neurosci. Res. 26, 397-408, 1990) and exhibits extensive sequence identity to bovine brain neurogranin (J. Baudier et al., J. Biol. Chem. 266, 229-237, 1991). On sodium dodecyl sulfate-polyacrylamide gel electrophoresis this protein, RC3, migrated as a M(r) 15-18K species in the presence of reducing agent and as heterogeneous species of M(r) 13-28K in the absence of reducing agent. Phosphorylation of RC3 by PKC alpha, beta, or gamma was stimulated by Ca2+, phospholipid, and diacylglycerol. A single site, Ser36, which is adjacent to the predicted calmodulin (CaM)-binding domain, was phosphorylated by these enzymes. Phosphorylation of RC3 by PKC or PKM, a protease-degraded PKC, was inhibited by CaM. The effect of CaM apparently targets at RC3, as phosphorylation of protamine sulfate by PKM was not inhibited by CaM. In the absence of Ca2+, RC3 formed a stoichiometric complex with CaM as evidenced by an increase in the M(r) determined by gel filtration chromatography. In the presence of Ca2+, the affinity of RC3 toward CaM is greatly reduced and Ca2+/CaM becomes less inhibitory of the PKM-catalyzed phosphorylation of RC3. Phosphorylation of RC3 by PKM prevented the interaction of this protein with CaM even in the absence of Ca2+. A 20-amino-acid synthetic peptide (AS-20F-W) containing the PKC phosphorylation site and CaM-binding domain of RC3 (Ala29-Ser48) with a substitution of Phe37 with tryptophan was used to monitor the interaction of this peptide with CaM by spectrofluorometry. In the absence of Ca2+, CaM caused negligible change in tryptophan fluorescence of the peptide; however, an enhancement and blue-shift of the emission fluorescence was observed in the presence of Ca2+. It seems that this synthetic peptide, as well as RC3 holoprotein, interacts with CaM through electrostatic interaction in the absence of Ca2+ but through hydrophobic interaction in the presence of Ca2+. In rat brain homogenate, RC3 formed a stable complex with CaM in the presence of Ca2+, as demonstrated by immunoblot analysis following gel filtration chromatography.(ABSTRACT TRUNCATED AT 400 WORDS)
一种7.5 kDa的热稳定且耐酸的大鼠脑蛋白激酶C（PKC）底物，通过使用DEAE - 纤维素和羟基磷灰石柱色谱的两步法纯化至接近均一。这种由78个氨基酸组成的蛋白质，其序列与用皮质减去小脑消减cDNA探针鉴定的大鼠脑RC3 cDNA推导的序列相同（J. B. 沃森等人，《神经科学研究杂志》26，397 - 408，1990），并且与牛脑神经颗粒蛋白具有广泛的序列同一性（J. 鲍迪尔等人，《生物化学杂志》266，229 - 237，1991）。在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳中，这种蛋白质RC3在还原剂存在下以M(r) 15 - 18K的条带迁移，在无还原剂时以M(r) 13 - 28K的异质条带迁移。PKCα、β或γ对RC3的磷酸化受Ca2 +、磷脂和二酰甘油的刺激。这些酶磷酸化了单个位点Ser36，该位点与预测的钙调蛋白（CaM）结合结构域相邻。CaM抑制PKC或PKM（一种蛋白酶降解的PKC）对RC3的磷酸化。CaM的作用显然靶向RC3，因为PKM对硫酸鱼精蛋白的磷酸化不受CaM抑制。在无Ca2 +时，RC3与CaM形成化学计量复合物，凝胶过滤色谱法测定的M(r)增加证明了这一点。在有Ca2 +时，RC3对CaM的亲和力大大降低，并且Ca2 + / CaM对PKM催化的RC3磷酸化的抑制作用减弱。即使在无Ca2 +时，PKM对RC3的磷酸化也阻止了该蛋白质与CaM的相互作用。一种含有RC3（Ala29 - Ser48）的PKC磷酸化位点和CaM结合结构域且用色氨酸取代Phe37的20个氨基酸的合成肽（AS - 20F - W），通过荧光光谱法用于监测该肽与CaM的相互作用。在无Ca2 +时，CaM对该肽色氨酸荧光的影响可忽略不计；然而，在有Ca2 +时观察到发射荧光增强且蓝移。似乎这种合成肽以及RC3全蛋白在无Ca2 +时通过静电相互作用与CaM相互作用，但在有Ca2 +时通过疏水相互作用。在大鼠脑匀浆中，如凝胶过滤色谱后的免疫印迹分析所示，在有Ca2 +时RC3与CaM形成稳定复合物。（摘要截断于400字）
