Ruzzene M, Songyang Z, Marin O, Donella-Deana A, Brunati A M, Guerra B, Agostinis P, Cantley L C, Pinna L A
Dipartimento di Chimica Biologica, Università di Padova, and Centro di Studio delle Biomembrane del Consiglio Nazionale delle Ricerche, Italy.
Eur J Biochem. 1997 Jun 1;246(2):433-9. doi: 10.1111/j.1432-1033.1997.t01-1-00433.x.
An eicosapeptide encompassing the C-terminal tail of c-Src (Tyr527) which is conserved in most Src-related protein kinases, is phosphorylated by C-terminal Src kinase (CSK) and by the two Src-related protein kinases c-Fgr and Lyn, with similar kinetic constants. Two related peptides reproducing the C-terminal segments of c-Src mutants defective in CSK phosphorylation [MacAuley, A., Okada, M., Nada, S., Nakagawa, H. & Cooper, J. A. (1993) Oncogene 8, 117-124] AFLEDSCTGTEPLYQRGENL (mutant number 28) and AFLEDNFTGTKPQYHPGENL (mutant number 29), proved a better and a much worse substrates, respectively than the wild-type peptide, with either CSK or the two Src kinases. By changing individual residues in the best peptide substrate, it was shown that the main element responsible for its improved phosphorylation is leucine at position -1 (instead of glutamine), while lysine at position -3 (instead of glutamate) has a detrimental effect, possibly accounting for the negligible phosphorylation of peptide derived from mutant number 29. By contrast to most peptide substrates, including the Src C-terminal peptides, which exhibit relatively high K(m) values, a polyoma-virus-middle-T-antigen-(mT)-derived peptide with tyrosine embedded in a highly hydrophobic sequence (EEEPQFEEIPIYLELLP) exhibits with CSK a quite low K(m) value (63 microM). Consistent with this, the optimal sequence selected by CSK in an oriented peptide library is XXXIYMFFF. This is different from sequences selected by Lyn (DEEIYEELX) and c-Fgr (XEEIYGIFF), although they all share a high selection for a hydrophobic residue at n-1. In sharp contrast, TPKIIB/p38syk, related to the catalytic domain of p72syk, selects acidic residues at nearly all positions, n-1 included. These data support the notion that the features determining the specific phosphorylation of the C-terminal tyrosine residue of Src do not reside in the primary structure surrounding the target tyrosine. They also show that this site does not entirely fulfil the optimal consensus sequence recognized by CSK, disclosing the possibility that as yet unrecognized CSK targets structurally unrelated to the C-terminal tyrosine residue of Src kinases may exist.
一种包含c-Src（Tyr527）C末端尾巴的二十碳肽，在大多数Src相关蛋白激酶中是保守的，可被C末端Src激酶（CSK）以及两种Src相关蛋白激酶c-Fgr和Lyn磷酸化，且动力学常数相似。两种相关肽段重现了CSK磷酸化缺陷的c-Src突变体的C末端片段【麦考利，A.，冈田，M.，名田，S.，中川，H. & 库珀，J. A.（1993年）《癌基因》8，117 - 124】AFLEDSCTGTEPLYQRGENL（突变体编号28）和AFLEDNFTGTKPQYHPGENL（突变体编号29），结果证明，与野生型肽相比，它们分别是CSK以及两种Src激酶更好和更差的底物。通过改变最佳肽底物中的单个残基，发现负责其磷酸化改善的主要元素是-1位的亮氨酸（而非谷氨酰胺），而-3位的赖氨酸（而非谷氨酸）有不利影响，这可能解释了源自突变体编号29的肽磷酸化可忽略不计的原因。与大多数肽底物不同，包括Src C末端肽段，它们表现出相对较高的K(m)值，一种多瘤病毒中T抗原（mT）衍生的肽，其酪氨酸嵌入高度疏水序列（EEEPQFEEIPIYLELLP），与CSK反应时表现出相当低的K(m)值（63 microM）。与此一致，CSK在定向肽库中选择的最佳序列是XXXIYMFFF。这与Lyn（DEEIYEELX）和c-Fgr（XEEIYGIFF）选择的序列不同，尽管它们在n - 1位都高度选择疏水残基。与之形成鲜明对比的是，与p72syk催化结构域相关的TPKIIB/p38syk几乎在所有位置，包括n - 1位，都选择酸性残基。这些数据支持这样一种观点，即决定Src C末端酪氨酸残基特异性磷酸化的特征并不存在于靶酪氨酸周围的一级结构中。它们还表明该位点并未完全符合CSK识别的最佳共有序列，这揭示了可能存在与Src激酶C末端酪氨酸残基结构无关的尚未被识别的CSK靶点。
