CHARACTERIZATION OF PP60(C-SRC) TYROSINE KINASE-ACTIVITIES USING A CONTINUOUS ASSAY - AUTOACTIVATION OF THE ENZYME IS AN INTERMOLECULAR AUTOPHOSPHORYLATION PROCESS
CHARACTERIZATION OF PP60(C-SRC) TYROSINE KINASE-ACTIVITIES USING A CONTINUOUS ASSAY - AUTOACTIVATION OF THE ENZYME IS AN INTERMOLECULAR AUTOPHOSPHORYLATION PROCESS
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DOI：
10.1021/bi00045a027
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发表时间：
1995-11-14
期刊：
影响因子：
2.9
通讯作者：
KNIGHT, WB
中科院分区：
文献类型：
--
作者：
BARKER, SC;KASSEL, DB;KNIGHT, WB
开发了pp 60（c-src）酪氨酸激酶（srcTK）的连续测定法。观察到肽RRLIEDAEYAARG的磷酸化滞后，其可以通过与MgATP预孵育来消除。该滞后的诱导时间取决于MgATP和srcTK浓度。当通过[γ-P-32]ATP的P-32掺入监测自磷酸化时，也观察到时间过程的滞后。这些结果表明，自激活是一个分子间的过程。活化前后的酶的电喷雾电离质谱表明，与MgATP孵育后的酶的磷酸化状态的增加。在这些研究中使用的Delta 85-N-末端突变蛋白和全长G2 A pp 60（c-src）突变体（其去除肉豆蔻基化位点）在分离的Y338和Y 530上部分磷酸化。这是第一个关于Y338磷酸化的报道，但这个磷酸化位点的意义尚不清楚。这些磷酸化作用不足以激活酶将MgATP的γ-磷酰基转移至肽。最初存在的未磷酸化的酶在用MgATP处理后转化为单磷酸化的物质。Y-419磷酸化仅在用MgATP处理后明显。这些数据与预测的Y-419上的自磷酸化一致。分子间自磷酸化与srcTK二聚化的能力一致，这类似于受体酪氨酸激酶如EGF受体激酶响应生长因子的活化。这些结果表明，在srcTK的情况下，导致活化的二聚化不需要结合到膜或疏水性N-末端。
A continuous assay for pp60(c-src) tyrosine kinase (srcTK) was developed. A lag in phosphorylation of the peptide RRLIEDAEYAARG was observed that could be eliminated by preincubation with MgATP. The induction time for this lag was dependent upon MgATP and srcTK concentrations. When autophosphorylation was monitored by P-32 incorporation from [gamma-P-32]ATP, a lag in the time course was also observed. These results demonstrate that autoactivation is an intermolecular process. The electrospray ionization mass spectrum of the enzyme before and after activation demonstrated an increase in the phosphorylation state of the enzyme after incubation with MgATP. The Delta 85-N-terminal mutant protein and a full-length G2A pp60(c-src) mutant, which removes the myristylation site, used in these studies were partially phosphorylated on Y338 and Y530 as isolated. This is the first report of phosphorylation on Y338, but the significance of this site of phosphorylation is unknown. These phosphorylations were insufficient to activate the enzyme for transfer of the gamma-phosphoryl of MgATP to the peptides. The unphosphorylated enzyme initially present was converted to a monophosphorylated species upon treatment with MgATP. Y-419 phosphorylation was evident only after treatment with MgATP. These data are consistent with autophosphorylation on Y-419 as predicted. Intermolecular autophosphorylation is consistent with the; ability of srcTK to dimerize, which is analogous to activation of receptor tyrosine kinases such as the EGF receptor kinase in response to growth factors. These results indicate that dimerization leading to activation does not require binding to the membrane or a hydrophobic N-terminus in the case of srcTK.