Csk is present in all human cells as a key regulator of Src kinases 7
. The fact that the presence of Y505 in Lck is essential for the inhibitory effect of cAMP on ζ chain phosphorylation and IL-2 production indicates that the PKA-mediated phosphorylation of Csk may be a major mechanism by which cAMP inhibits TCR-mediated T cell activation (). A two- to fourfold increase in Csk activity by phosphorylation of S364 appears to have similarly distinct effects on T cell function as a two- to threefold Csk overexpression, which abolishes activation through the TCR 6
and downstream IL-2 production ( C). Furthermore, the stoichiometry of Csk phosphorylation by PKA in vitro is 0.3–0.5 mol/mol of Csk under optimal conditions, indicating a single site not fully phosphorylated (quite common with bacterially produced protein). In vivo, we anticipate that a specific pool of Csk may be preferentially phosphorylated by colocalized PKA and reaches a higher stoichiometry and extent of activation. In addition, Jurkat and other leukemic T cell lines have higher levels of tyrosine phosphorylation 34
and Src kinase activities 35
than peripheral T cells. Thus, normal T cells appear to have a more controlled and managed Lck activity that may implicate Csk regulation and a PKA-Csk inhibitory pathway to a larger extent than apparent from, for example transfection studies on Jurkat T cells. Indeed, low level labeling of peptide 1 (representing Csk-S364) was seen in the tryptic peptide mapping of Csk from metabolically labeled unstimulated cells ( C) which increased strongly by treatment with PGE1 alone (data not shown). This indicates that this site is phosphorylated under physiological conditions. The mechanism for Csk activation by S364 phosphorylation is currently under investigation in our laboratory, and data in progress indicate that interaction with the intrachain SH3 domain is implicated in the PKA-mediated activation of Csk.
Figure 10 Model for a molecular mechanism whereby cAMP inhibits T cell function. Activation of adenylyl cyclase (AC), e.g., by binding of ligands such as adrenalin and PGE2 to G protein–coupled receptors (R), turns on a regulatory pathway assembled in lipid (more ...)
We have recently reported that the T cell dysfunction in HIV can be reversed by inhibition of the increased activity of PKA type I 18
, indicating that immunomodulation through cAMP/PKA contributes to the pathogenesis of this immunodeficiency. Inhibition of Lck through activation of Csk provides a molecular mechanism for this effect. Furthermore, PKA-mediated regulation of the activity of various Src kinase family members by phosphorylation of Csk may also provide a molecular mechanism for cAMP-mediated regulation of both B and NK cell activation 3637
. Finally, Csk and Src kinases are expressed in other tissues, including neuronal tissues 38
, and the impact of cAMP regulation of Csk in these tissues will be interesting to pursue. The PKA phosphorylation site in Csk is conserved between vertebrates, suggesting that this site may have been subject to selection pressure, but is only partially conserved (RFS or KFT) in Csk homologous kinase (Chk/Lsk/Hyl/Matk) and Csk-type protein kinase (Ctk/Bhk/Ntk).
In conclusion, we report the mapping of a PKA phosphorylation site on Csk and regulation of Csk activity by cAMP/PKA. Localization of both Csk and PKA type I to lipid rafts supports the notion that this novel inhibitory pathway is assembled in membrane microdomains where it can intersect TCR-induced signaling at a proximal level. The presence of adenylyl cyclase that generates cAMP in lipid rafts of S49 lymphoma cells further supports assembly of the cAMP-PKA type I-Csk inhibitory pathway in lipid rafts (; reference 39
). The constitutive localization of components of this pathway in lipid rafts may indicate that a tonic level of inhibition of T cell activation is imposed on resting T cells. PKA-mediated activation of Csk provides a molecular mechanism for cAMP-dependent inhibition of lymphocyte activation, and Csk-S364 and/or Lck-Y505 may be future targets for immunomodulating therapies. Furthermore, this mechanism may regulate signaling through Src kinases in general.