Law C L, Chandran K A, Sidorenko S P, Clark E A
Department of Microbiology, University of Washington, Seattle, USA.
Mol Cell Biol. 1996 Apr;16(4):1305-15. doi: 10.1128/MCB.16.4.1305.
Antigen receptor ligation on lymphocytes activates protein tyrosine kinases and phospholipase C-gamma (PLC-gamma) isoforms. Glutathione S-transferase fusion proteins containing the C-terminal Src-homology 2 [SH2(C)] domain of PLC-gamma1 bound to tyrosyl phosphorylated Syk. Syk isolated from antigen receptor-activated B cells phosphorylated PLC-gamma1 on Tyr-771 and the key regulatory residue Tyr-783 in vitro, whereas Lyn from the same B cells phosphorylated PLC-gamma1 only on Tyr-771. The ability of Syk to phosphorylate PLC-gamma1 required antigen receptor ligation, while Lyn was constitutively active. An mCD8-Syk cDNA construct could be expressed as a tyrosyl-phosphorylated chimeric protein tyrosine kinase in COS cells, was recognized by PLC-gamma1 SH2(C) in vitro, and induced tyrosyl phosphorylation of endogenous PLC-gamma1 in vivo. Substitution of Tyr-525 and Tyr-526 at the autophosphorylation site of Syk in mCD8-Syk substantially reduced the kinase activity and the binding of this variant chimera to PLC-gamma1 SH2(C) in vitro; it also failed to induce tyrosyl phosphorylation of PLC-gamma1 in vivo. In contrast, substitution of Tyr-348 and Tyr-352 in the linker region of Syk in mCD8-Syk did not affect the kinase activity of this variant chimera but almost completely eliminated its binding to PLC-gamma1 SH(C) and completely eliminated its ability to induce tyrosyl phosphorylation of PLC-gamma1 in vivo. Thus, an optimal kinase activity of Syk and an interaction between the linker region of Syk with PLC-gamma1 are required for the tyrosyl phosphorylation of PLC-gamma1.
淋巴细胞上的抗原受体连接激活蛋白酪氨酸激酶和磷脂酶C-γ（PLC-γ）亚型。含有PLC-γ1 C末端Src同源2 [SH2(C)]结构域的谷胱甘肽S-转移酶融合蛋白与酪氨酸磷酸化的Syk结合。从抗原受体激活的B细胞中分离出的Syk在体外使PLC-γ1的Tyr-771和关键调节残基Tyr-783磷酸化，而来自同一B细胞的Lyn仅使PLC-γ1的Tyr-771磷酸化。Syk使PLC-γ1磷酸化的能力需要抗原受体连接，而Lyn具有组成性活性。mCD8-Syk cDNA构建体可以在COS细胞中表达为酪氨酸磷酸化的嵌合蛋白酪氨酸激酶，在体外被PLC-γ1 SH2(C)识别，并在体内诱导内源性PLC-γ1的酪氨酸磷酸化。在mCD8-Syk中Syk自磷酸化位点的Tyr-525和Tyr-526被取代，在体外大大降低了激酶活性以及该变体嵌合体与PLC-γ1 SH2(C)的结合；它在体内也未能诱导PLC-γ1的酪氨酸磷酸化。相反，在mCD8-Syk中Syk接头区域的Tyr-348和Tyr-352被取代并不影响该变体嵌合体的激酶活性，但几乎完全消除了其与PLC-γ1 SH(C)的结合，并完全消除了其在体内诱导PLC-γ1酪氨酸磷酸化的能力。因此，PLC-γ1的酪氨酸磷酸化需要Syk的最佳激酶活性以及Syk接头区域与PLC-γ1之间的相互作用。
