PHOSPHORYLATION OF THE GRB2-BINDING AND PHOSPHATIDYLINOSITOL-3-KINASEP85-BINDING P36 38 BY SYK IN LCK-NEGATIVE T-CELLS/

Activation of the mitogen-activated protein kinase (MAPK) pathway by t he T-cell antigen receptor (TCR) in T cells involves a positive role f or phosphatidylinositol 3-kinase (PI3K) activity. We recently reported that over-expression of the Syk protein tyrosine kinase in the Lck ne gative JCaM1 cells enabled the TCR to induce a normal activiation of t he Erk2 MAPK and enhanced transcription of a reporter gene driven by t he nuclear factor of activated T cells and AP-1. Because this system a llows us to analyse the targets for Syk in receptor-mediated signallin g, we examined the role of PI3K in signalling events between the TCR-r egulated Syk and the downstream activation of Erk2. We report that inh ibition of PI3K by wortmannin or an inhibitory p85 construct, p85 Delt a iSH2, reduced the TCR-induced Syk-dependent: activation of Erk2, as well as the appearance of phospho-Erk and phospho-Mek. At the same tim e, expression of Syk resulted in the activation-dependent phosphorylat ion of three proteins that bound to the src homology 2 (SH2) domains o f PI3K p85. The strongest of these bands had an apparent molecular mas s of 36-38 kDa on SDS gels, and it was quantitatively removed from the lysates by adsorption to a fusion protein containing the SH2 domain o f Grb2. The appearance of this band was Syk dependent, and it was seen only upon triggering of the TCR complex. Thus, p36/38 was phosphoryla ted by Syk or a Syk-regulated kinase, and this protein may provide a l ink to the recruitment and activation of PI3K, as well as to the Ras-M APK pathway, in TCR-triggered T cells. (C) 1998 Elsevier Science Inc.