Okada M, Nakagawa H
Division of Protein Metabolism, Osaka University, Japan.
J Biol Chem. 1989 Dec 15;264(35):20886-93.
We recently identified a novel protein tyrosine kinase that specifically phosphorylates truncated pp60c-src (Mr = 53,000) at a tyrosine residue(s) distinct from its autophosphorylation site. In this study, we examined whether this enzyme phosphorylates intact pp60c-src (Mr = 60,000) and determined its phosphorylation site. Non-neuronal and neuronal forms of intact pp60c-src were separately purified from the membrane fraction of neonatal rat brain by sequential column chromatographies. The novel kinase phosphorylated tyrosine residues of both forms of intact pp60c-src. The phosphorylation occurred in parallel with autophosphorylation of pp60c-src, and in both forms the final stoichiometry estimated was quite similar to that of autophosphorylation (about 5%). The enzyme also phosphorylated pp60c-src in which the kinase activity had been destroyed by an ATP analogue, p-fluorosulfonylbenzoyl 5'-adenosine. The phosphorylation site of the non-neuronal form was analyzed by sequential peptide mapping with tosylphenylalanyl chloromethyl ketone-treated trypsin and alpha-chymotrypsin. Tryptic digestion of the phosphorylated pp60c-src yielded a unique phosphopeptide that cross-reacted with an antibody specific for the carboxyl-terminal sequence of chicken pp60c-src. Digestion of the phosphopeptide with chymotrypsin yielded a product that comigrated with a synthetic phosphopeptide corresponding to the carboxyl-terminal 15 residues of chicken pp60c-src. These results clearly indicate that the carboxyl-terminal sequence of rat pp60c-src is identical to that of chicken pp60c-src, and a tyrosine residue corresponding to chicken Tyr527 is the phosphorylation site. This phosphorylation resulted in a decrease in the enolase phosphorylating activity of pp60c-src. Kinetic experiments indicated that this decrease in activity was due to a decrease in the Vmax value of pp60c-src. These findings support our previous proposal that the novel tyrosine kinase acts as a specific regulator of pp60c-src in cells.
我们最近鉴定出一种新型蛋白酪氨酸激酶，它能在与自身磷酸化位点不同的酪氨酸残基处特异性地磷酸化截短的pp60c-src（分子量 = 53,000）。在本研究中，我们检测了这种酶是否能磷酸化完整的pp60c-src（分子量 = 60,000）并确定其磷酸化位点。通过连续柱层析从新生大鼠脑的膜组分中分别纯化出完整pp60c-src的非神经元形式和神经元形式。这种新型激酶能磷酸化两种形式完整pp60c-src的酪氨酸残基。磷酸化与pp60c-src的自身磷酸化同时发生，并且在两种形式中，最终估计的化学计量与自身磷酸化的化学计量相当相似（约5%）。该酶还能磷酸化其激酶活性已被ATP类似物对氟磺酰苯甲酰5'-腺苷破坏的pp60c-src。通过用甲苯磺酰苯丙氨酰氯甲基酮处理的胰蛋白酶和α-胰凝乳蛋白酶进行连续肽图谱分析，分析了非神经元形式的磷酸化位点。对磷酸化的pp60c-src进行胰蛋白酶消化产生了一种独特的磷酸肽，它能与针对鸡pp60c-src羧基末端序列的特异性抗体发生交叉反应。用胰凝乳蛋白酶消化该磷酸肽产生了一种与对应于鸡pp60c-src羧基末端15个残基的合成磷酸肽迁移率相同的产物。这些结果清楚地表明，大鼠pp60c-src的羧基末端序列与鸡pp60c-src的相同，并且对应于鸡Tyr527的酪氨酸残基是磷酸化位点。这种磷酸化导致pp60c-src的烯醇化酶磷酸化活性降低。动力学实验表明，这种活性降低是由于pp60c-src的Vmax值降低所致。这些发现支持了我们之前的提议，即这种新型酪氨酸激酶在细胞中作为pp60c-src的特异性调节剂发挥作用。
