Direct T cell activation by chimeric single chain Fv-Syk promotes Syk-Cbl association and Cbl phosphorylation.
The protein tyrosine kinase Syk is activated upon engagement of immune recognition receptors. We have focused on the identification of signaling elements immediately downstream to Syk in the pathway leading to T cell activation. To circumvent T cell receptor (TCR). CD3 activation of Src family kinases, we constructed a signaling molecule with an extracellular single chain Fv of an anti-TNP antibody, attached via a transmembrane region to Syk (scFv-Syk). In a murine T cell hybridoma, direct aggregation of chimeric Syk with antigen culminates in interleukin-2 production and target cell lysis. Initially, it causes an increase in the association between scFv-Syk and the cytosolic protein Cbl and subsequently promotes tyrosine phosphorylation of Cbl. Interestingly, although both Cbl and phospholipase C-gamma (PLC-gamma) are phosphorylated in this hybridoma upon TCR.CD3 cross-linking, these two events are uncoupled in scFv-Syk-transfected cells, in which we were unable to detect antigen-driven PLC-gamma phosphorylation. These results support a model in which Syk can initiate and directly activate the T cell's signaling machinery and position Cbl as a primary tyrosine kinase substrate in this pathway. Furthermore, for efficient PLC-gamma phosphorylation to occur in these cells, the combined actions of different tyrosine kinase families may be required.
Fitzer-Attas
C J
CJ
Department of Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel.
Schindler
D G
DG
Waks
T
T
Eshhar
Z
Z
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000378:metabolism*; D007376:Interleukin-2 / Q000096:biosynthesis; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000378:metabolism; D008213:Lymphocyte Activation; D051379:Mice; D050722:Oncogene Protein v-cbl; D051966:Phospholipase C gamma; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015682:Retroviridae Proteins, Oncogenic / Q000378:metabolism*; D000072377:Syk Kinase; D010738:Type C Phospholipases / Q000378:metabolism
D004792:Enzyme Precursors; D007135:Immunoglobulin Variable Region; D007376:Interleukin-2; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D050722:Oncogene Protein v-cbl; D011993:Recombinant Fusion Proteins; D015682:Retroviridae Proteins, Oncogenic; D011505:Protein-Tyrosine Kinases; D000072377:Syk Kinase; C000606897:Syk protein, mouse; D010738:Type C Phospholipases; D051966:Phospholipase C gamma