Phosphorylation of the Grb2- and phosphatidylinositol 3-kinase p85-binding p36/38 by Syk in Lck-negative T cells.
Activation of the mitogen-activated protein kinase (MAPK) pathway by the T-cell antigen receptor (TCR) in T cells involves a positive role for phosphatidylinositol 3-kinase (PI3K) activity. We recently reported that over-expression of the Syk protein tyrosine kinase in the Lck-negative JCaM1 cells enabled the TCR to induce a normal activation of the Erk2 MAPK and enhanced transcription of a reporter gene driven by the nuclear factor of activated T cells and AP-1. Because this system allows us to analyse the targets for Syk in receptor-mediated signalling, we examined the role of PI3K in signalling events between the TCR-regulated Syk and the downstream activation of Erk2. We report that inhibition of PI3K by wortmannin or an inhibitory p85 construct, p85deltaiSH2, reduced the TCR-induced Syk-dependent activation of Erk2, as well as the appearance of phospho-Erk and phospho-Mek. At the same time, expression of Syk resulted in the activation-dependent phosphorylation of three proteins that bound to the src homology 2 (SH2) domains of PI3K p85. The strongest of these bands had an apparent molecular mass 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 of Grb2. The appearance of this band was Syk dependent, and it was seen only upon triggering of the TCR complex. Thus, p36/38 was phosphorylated by Syk or a Syk-regulated kinase, and this protein may provide a link to the recruitment and activation of PI3K, as well as to the Ras-MAPK pathway, in TCR-triggered T cells.
Division of Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121, USA.
M
M
von Willebrand
S
S
Williams
P
P
Tailor
T
T
Mustelin
D048868:Adaptor Proteins, Signal Transducing; D000730:Androstadienes / Q000494:pharmacology; D017252:CD3 Complex / Q000502:physiology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002460:Cell Line; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004792:Enzyme Precursors / Q000378:metabolism*; D051380:GRB2 Adaptor Protein; D047908:Intracellular Signaling Peptides and Proteins; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000502:physiology; D048728:MAP Kinase Kinase Kinase 1; D019950:Mitogen-Activated Protein Kinase 1; D008970:Molecular Weight; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology; D000072377:Syk Kinase; D013601:T-Lymphocytes / Q000201:enzymology / Q000378:metabolism*; D000077191:Wortmannin; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D000730:Androstadienes; D017252:CD3 Complex; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D051380:GRB2 Adaptor Protein; D047908:Intracellular Signaling Peptides and Proteins; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D048728:MAP Kinase Kinase Kinase 1; D019950:Mitogen-Activated Protein Kinase 1; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D011506:Proteins; D011948:Receptors, Antigen, T-Cell; D000072377:Syk Kinase; D000077191:Wortmannin; C000606896:SYK protein, human; C482576:MAP3K1 protein, human