Exclusion of CD45 inhibits activity of p56lck associated with glycolipid-enriched membrane domains.
Exclusion of CD45 inhibits activity of p56lck associated with glycolipid-enriched membrane domains.
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DOI：
10.1083/jcb.135.6.1515
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发表时间：
1996-12
期刊：
影响因子：
--
通讯作者：
Rose JK
中科院分区：
文献类型：
--
作者：
Rodgers W;Rose JK
p56 lck（Lck）是淋巴特异性Src家族酪氨酸激酶，其对于T细胞发育和活化至关重要。Lck也是一种膜蛋白，大约一半的膜相关Lck与富含糖脂的膜（GEM）部分相关，该部分对Triton X-100（TX-100）的溶解具有抗性。为了比较存在于Jurkat细胞的GEM和TX-100可溶性级分中的膜相关Lck，用磷酸酪氨酸抗体对来自每个级分的Lck进行免疫印迹。发现GEM级分中的Lck在酪氨酸上过度磷酸化，并且这与相对于TX-100可溶性Lck的较低激酶比活性相关。肽图和磷酸酶分析显示，GEM相关Lck的过度磷酸化和较低的激酶活性是由于调节性COOH末端Tyr 505的磷酸化。此外，我们确定，膜结合酪氨酸磷酸酶CD 45是从创业板馏分缺席。缺乏CD 45的细胞在GEM和TX-100可溶性膜中显示出相同的Lck磷酸化。我们提出GEM组分代表T细胞中存在的特定膜结构域，并且GEM相关Lck的酪氨酸过度磷酸化和较低的激酶活性是由于从这些结构域排除CD 45。因此，与GEM结构域相关的Lck可以构成可以容易地被激活的酶的储库。
p56lck (Lck) is a lymphoid-specific Src family tyrosine kinase that is critical for T-cell development and activation. Lck is also a membrane protein, and approximately half of the membrane-associated Lck is associated with a glycolipid-enriched membrane (GEM) fraction that is resistant to solubilization by Triton X-100 (TX-100). To compare the membrane-associated Lck present in the GEM and TX-100-soluble fractions of Jurkat cells, Lck from each fraction was immunoblotted with antibody to phosphotyrosine. Lck in the GEM fraction was found to be hyperphosphorylated on tyrosine, and this correlated with a lower kinase specific activity relative to the TX-100-soluble Lck. Peptide mapping and phosphatase diagests showed that the hyperphosphorylation and lower kinase activity of GEM-associated Lck was due to phosphorylation of the regulatory COOH-terminal Tyr505. In addition, we determined that the membrane-bound tyrosine phosphatase CD45 was absent from the GEM fraction. Cells lacking CD45 showed identical phosphorylation of Lck in GEM and TX-100-soluble membranes. We propose that the GEM fraction represents a specific membrane domain present in T-cells, and that the hyperphosphorylation of tyrosine and lower kinase activity of GEM-associated Lck is due to exclusion of CD45 from these domains. Lck associated with the GEM domains may therefore consitute a reservoir of enzyme that can be readily activated.