Efficient CD28 signalling leads to increases in the kinase activities of the TEC family tyrosine kinase EMT/ITK/TSK and the SRC family tyrosine kinase LCK

Abstract

Optimal T cell activation requires crosslinking of the T cell receptor (TCR) concurrently with an accessory receptor, most efficiently CD28. Crosslinking of CD28 leads to increased interleukin 2 (IL2) production, inhibition of anergy and prevention of programmed cell death. Crosslinking of CD28 leads to rapid increases in tyrosine phosphorylation of specific intracellular substrates including CD28 itself. Since CD28 does not encode an intrinsic tyrosine kinase domain, CD28 must activate an intracellular tyrosine kinase(s). Indeed, crosslinking of CD28 increases the activity of the intracellular tyrosine kinases EMT/ITK and LCK. The phosphatidylinositol 3-kinase (PI3K) and GRB2 binding site in CD28 is dispensable for optimal IL2 production in Jurkat T cells. We demonstrate herein that murine Y170 (equivalent to human Y173) in CD28 is also dispensable for activation of the SRC family tyrosine kinase LCK and the TEC family tyrosine kinase EMT/ITK. In contrast, the distal three tyrosines in CD28 are required for optimal IL2 production as well as for optimal activation of the LCK and EMT/ITK tyrosine kinases. The distal three tyrosines of CD28, however, are not required for recruitment of PI3K to CD28. Furthermore, PI3K is recruited to CD28 in JCaM1 cells which lack LCK and in which EMT/ITK is not activated by ligation of CD28. Thus optimal activation of LCK or EMT/ITK is not obligatory for recruitment of PI3K to CD28 and thus is also not required for tyrosine phosphorylation of the YMNM motif in CD28. Taken together the data indicate that the distal three tyrosines in CD28 are integral to the activation of LCK and EMT/ITK and for subsequent IL2 production.

CD28 and ICOS: Similar or separate costimulators of T cells?

“…The phosphatase PP2a associates with the unphosphorylated Tyr170 and is implicated to negatively regulate CD28 signalling [52]. The CD28 COOH terminal PYxPP motif ascertains binding of SRC-family kinase Lck [53], mediating tyrosine phosphorylation of CD28 itself thereby facilitating binding of PI3K, Grb-2 and activation of Itk (IL-2 inducible T cell kinase) [54,55]. Moreover, Itk itself and Tec (Tyrosine kinase expressed in hepatocellular carcinoma) physically associate with the proline motifs in the CD28 intracellular tail, which regulate a variety of downstream events through activation of PLC-␥1 and Erk [56].…”

Autonomous induction of proliferation, JNK and NF-κB activation in primary resting T cells by mobilized CD28

“…An initial step observed after TCR triggering is the activation of the src kinase p56lck (Lck), which phosphorylates multiple substrates of the TCR signaling complex [21]. Furthermore, Lck activation in response to CD28 ligation has previously been reported in other systems and is thought to act as an enhancer of the TCR signal [3,8,12,13,17]. In vitro kinase assays ( Fig.…”

“…Thus, the tyrosine phosphorylation events observed after stimulation of CD28 alone, which appear to be initiated by the src kinase Lck and target downstream kinases of the TEC family as well as adaptor molecules [12][13][14][15][16][17], are insufficient for the induction of proliferation or IL-2 secretion in primary resting T cells or T lymphoma cells, respectively. This view, however, has recently been challenged by experiments employing novel mitogenic CD28-specific mAb [18][19][20] which showed that CD28 is also able to transduce a proliferative signal without occupancy of the TCR.…”

CD28 and Lipid Rafts Coordinate Recruitment of Lck to the Immunological Synapse of Human T Lymphocytes

“…Due to the absence of an intrinsic kinase activity, the CD28-induced increase in tyrosine phosphorylation is probably due to the recruitment and/or activation of tyrosine kinases. Both the thymusexpressed chemokine family tyrosine kinase IL-2-inducible T cell kinase (ITK) 4 (10) and the Src family kinase Lck (11) are activated after CD28 stimulation. ITK association with CD28 after CD28 stimulation is involved in generating both positive and negative signaling (11)(12)(13).…”