Couture C, Williams S, Gauthier N, Tailor P, Mustelin T
Department of Medicine, McGill University, Montreal, Canada.
Eur J Biochem. 1997 Jun 1;246(2):447-51. doi: 10.1111/j.1432-1033.1997.00447.x.
The Syk protein-tyrosine kinase is expressed in many hematopoietic cells and is involved in signaling from various receptors for antigen and Fc portions of IgG and IgE. After cross-linking of these receptors, Syk is rapidly phosphorylated on tyrosine residues. We have previously reported that Syk expressed in COS cells is predominantly phosphorylated at both Tyr518 and Tyr519 at its putative autophosphorylation site. In this study, we have examined the role of each of these two residues for the catalytic activity of Syk in vitro and for the Syk-induced phosphorylation of cellular proteins in intact cells. Mutation of either residue had minor effects on the catalytic activity of Syk, and even the double mutant [F518, F519]Syk was about 60% as active as the wild-type enzyme. In intact cells, however, all three mutants consistently failed to induce the extensive tyrosine phosphorylation of cellular proteins typically observed with wild-type Syk. We have recently shown that the doubly phosphorylated Y518/Y519 site is also the site for association of Syk with the SH2 domain of the Lck kinase, which suggests that although phosphates at Y518/Y519 may enhance the catalytic activity of Syk, its interaction with Src family protein-tyrosine kinases is at least equally important for the induction of downstream substrate phosphorylation.
Syk蛋白酪氨酸激酶在许多造血细胞中表达，并参与来自各种抗原受体以及IgG和IgE的Fc部分的信号传导。这些受体交联后，Syk在酪氨酸残基上迅速磷酸化。我们之前报道过，在COS细胞中表达的Syk在其假定的自磷酸化位点主要在Tyr518和Tyr519处磷酸化。在本研究中，我们检测了这两个残基中的每一个对于体外Syk催化活性以及完整细胞中Syk诱导的细胞蛋白磷酸化的作用。任一残基的突变对Syk的催化活性影响较小，甚至双突变体[F518,F519]Syk的活性约为野生型酶的60%。然而，在完整细胞中，所有这三种突变体始终无法诱导通常在野生型Syk中观察到的细胞蛋白广泛酪氨酸磷酸化。我们最近表明，双磷酸化的Y518/Y519位点也是Syk与Lck激酶的SH2结构域结合的位点，这表明尽管Y518/Y519处的磷酸可能增强Syk的催化活性，但其与Src家族蛋白酪氨酸激酶的相互作用对于诱导下游底物磷酸化至少同样重要。
