Combined spatial and enzymatic regulation of Csk by cAMP and protein kinase A inhibits T cell receptor signaling

Vang T, Abrahamsen H, Myklebust S, Horejesi V and Taskén K
Source: J Biol Chem
Publication Date: (2003)
Issue: 278(20): 17597-17600
Research Area:
Immunotherapy / Hematology
Cells used in publication:
T cell, human peripheral blood unstim.
Species: human
Tissue Origin: blood
Cyclic AMP (cAMP) inhibits TCR-induced T cell activation and thereby exerts important immunoregulatory functions. In this context, Csk, a ubiquitously expressed cytosolic PTK (protein tyrosine kinase) appears to be an important means of negative regulation of TCR signaling. Csk is recruited to rafts via its SH2 domain. Csk contains a catalytic kinase domain and transiently dissociates from lipid rafts upon TCR triggering. This publication is aimed at revealing the role of Csk in TCR-induced signaling by cAMP. Primary T cells were nucleofected with an empty plasmid or a plasmid encoding kinase deficient HA-tagged Csk-SH3-SH2, which has an intact and functional SH3 and SH2 domain but lacks the kinase domain. Cells were treated with PGE2 (prostaglandin E2) to elevate the intracellular cAMP level. The results indicated that the inhibitory effects of PGE2/cAMP on TCR signaling are dependent on the presence of Csk in rafts and/or the ability of recruiting additional amounts of Csk to rafts.
Raft-associated Csk controls signaling through the T cell receptor (TCR) and was mainly anchored to Cbp/PAG (phosphoprotein associated with glycosphingolipid-enriched membrane domains). Treatment of cells with the cAMP-elevating agent prostaglandin E(2) (PGE(2)) augmented the level of Cbp/PAG phosphorylation with a concomitant increase in amounts of Csk bound to Cbp/PAG. While TCR-triggering resulted in transient dissociation of Csk from Cbp/PAG/rafts allowing TCR-induced tyrosine phosphorylation to occur, pretreatment with PGE(2) reduced Csk dissociation upon TCR triggering. This correlated with lowered TCR-induced phosphorylation of CD3 zeta-chain and linker for activation of T cells. Moreover, competition of endogenous Csk from lipid rafts abolished PGE(2)-mediated inhibition of TCR-induced zeta-chain phosphorylation and activation of the nuclear factor of activated T cells (NFAT) activator protein 1 (AP-1). Finally, raft-associated Csk already activated via Cbp/PAG binding, gained additional increase in phosphotransferase activity upon protein kinase A-mediated phosphorylation of Csk. We propose that cAMP regulates Csk via both spatial and enzymatic mechanisms, thereby inhibiting signaling through the TCR.