cAMP inhibits Src-family kinase signalling by PKA (protein kinase A)-mediated phosphorylation and activation of Csk (C-terminal Src kinase). The PKA type I–Csk pathway is assembled and localized in membrane microdomains (lipid rafts) and regulates immune responses activated through the TCR (T-cell receptor). PKA type I is targeted to the TCR–CD3 complex during T-cell activation via an AKAP (A-kinase-anchoring protein) that serves as a scaffold for the cAMP–PKA/Csk pathway in lipid rafts of the plasma membrane during T-cell activation. Displacement of PKA by anchoring disruption peptides prevents cAMP/PKA type I-mediated inhibition of T-cell activation. These findings provide functional evidence that PKA type I regulation of T-cell responses is dependent on AKAP anchoring. Furthermore, we show that upon TCR/CD28 co-ligation, β-arrestin in complex with PDE4 (phosphodiesterase 4) is recruited to lipid rafts. The CD28-mediated recruitment of PDE4 to lipid rafts potentiates T-cell immune responses and counteracts the local, TCR-induced production of cAMP that produces negative feedback in the absence of a co-receptor stimulus. The specific recruitment of PDE4 thus serves to abrogate the negative feedback by cAMP which is elicited in the absence of a co-receptor stimulus.
TCR (T-cell receptor) is a multimeric complex consisting of two functional units, one involved in ligand binding (αβ) and the other in signal transduction (CD3 γ, δ, ϵ and ζ chains). One of the most immediate events taking place in T-cells after engagement of the TCR is activation of the Src-family protein tyrosine kinases, in particular Lck, and the subsequent phosphorylation of the ITAMs (immunoreceptor tyrosine-based activation motifs) present in the CD3 subunits. This in turn recruits the tandem SH2 domain (Src homology 2 domain) containing ZAP-70 (ζ-chain-associated protein kinase of 70 kDa) that becomes fully active upon Lck-mediated phosphorylation. ZAP-70 plays an essential role with respect to further propagation of the signal downstream of the TCR . Tight regulation of Lck is therefore required both for proper T-cell activation to occur and in order to prevent activation-induced cell death. Csk (C-terminal Src kinase) plays an important inhibitory role in proximal T-cell signalling by inhibiting Lck activity [2,3], and the level of inhibition of Csk is again controlled by PKA (protein kinase A)-mediated phosphorylation and activation of Csk . Furthermore, Csk activity is increased upon docking to Cbp (Csk-binding protein)/PAG (protein associated with glycol-sphingolipid-enriched microdomains) , and the contribution of regulation by PKA and docking to Cbp/PAG increases Csk activity 6–8-fold .
Although most signalling components assemble upon stimulation of the TCR, this alone is not enough to induce full T-cell activation in vivo. However, simultaneous triggering of the co-receptor CD28 has been demonstrated to prevent anergy and cell death and to promote interleukin-2 production and clonal expansion . Despite the central function of CD28 in T-cell activation in vivo , relatively little is known about the molecular basis for the augmented signal transduction upon TCR and CD28 co-stimulation. However, important roles for Lck, Itk, phosphoinositide 3-kinase, SLP-76 (SH2-domain-containing leucocyte protein of 76 kDa), Vav-1 and PLCγ (phospholipase Cγ) have been demonstrated .
cAMP modulates immune functions
cAMP, generated by G-protein-mediated activation of AC (adenylate cyclase), is a versatile and common second messenger controlling numerous cellular processes and has been known to inhibit TCR-induced T-cell proliferation for a long time . cAMP activates PKA (reviewed in ), Epac (exchange protein directly activated by cAMP)  and cAMP-regulated ion channels . Effects mediated by cAMP in T-cells are, however, most likely to be elicited by PKA, as neither Epac nor cAMP-gated ion channels seem to be expressed in lymphocytes (T. Bryn and K. Taskén, unpublished work). The only known mechanism for inactivation of cAMP is through degradation by a large family of cAMP-specific PDEs (phosphodiesterases) [14,15]. Compartmentalization of receptors, ACs and PKA by AKAPs (A-kinase-anchoring proteins) , as well as generation of local pools of cAMP within the cell by the action of PDEs , contribute to a high degree of specificity in PKA-mediated signalling despite the broad substrate specificity of PKA. AKAPs contribute specificity by targeting PKA towards specific substrates as well as versatility by assembling multiprotein signal complexes, allowing for signal termination by phosphoprotein phosphatases and cross-talk between different signalling pathways [11,16]. Integrating PDEs into these anchoring complexes adds a further temporal aspect to the spatial regulation of cAMP signals (reviewed in [18,19]).
In the immune system, PGE2 (prostaglandin E2) and other ligands elevating cAMP by binding to GPCRs (G-protein-coupled receptors) inhibit TCR-induced T-cell activation and thereby exert important immunoregulatory functions . Based on studies with selective agonists, activation of PKA type I (RIα2C2) has been shown to be necessary and sufficient to mediate these effects of cAMP . Similarly, PKA type I negatively regulates activation of B-cells through the B-cell antigen receptor  and NK cell (natural killer cell) cytotoxicity elicited through specific NK cell receptors . Although PKA can modulate TCR signalling at multiple levels (reviewed in ), the observed inhibitory effects of cAMP on TCR-induced ζ-chain phosphorylation point towards an important role for Csk, which is the most upstream PKA target reported so far.
A cAMP–PKA–Csk inhibitory pathway in lipid rafts regulates T-cell immune function
Proteins involved in proximal TCR signalling events are localized in lipid rafts, representing small regions of detergent-resistant lipid domains of the membrane [24,25]. Both the cAMP-generating machinery (AC) and the effectors (PKA type I and Csk) are localized in the lipid rafts. The mechanism involved in targeting RIα to lipid rafts has not yet been fully elucidated. However, analyses of lipid raft purifications from normal resting T-cells for the presence of different subunits of PKA revealed that both the catalytic subunit and the regulatory subunit RIα (but no RII subunits) are constitutively associated with the lipid rafts . This suggests that the observed co-localization of PKA type I and TCR in capped T-cells  occurs in lipid rafts and that there are mechanisms for specific targeting of PKA type I to these areas involving interaction with an AKAP in lipid rafts (A. Ruppelt, M. Grönholm, E.M. Aandahl, D. Tobin, C.R. Carlson, H. Abrahamsen, F.W. Herberg, O. Carpén and K. Taskén, unpublished work).
Csk is constitutively localized to lipid rafts in resting T-cells, but is transiently displaced to the cytosol during T-cell activation  in order to allow the activation cascade to proceed. The phosphatase responsible for the dephosphorylation of Cbp/PAG and the release of Csk was recently identified as CD45 . Lck-mediated phosphorylation of Cbp/PAG  leads to re-recruitment of Csk and re-establishment of the inhibitory pathway.
Csk regulates Lck activity by phosphorylation of a C-terminal inhibitory tyrosine residue (Lck-Tyr505). So far, two different mechanisms are reported to regulate Csk activity [4,5]. PKA type I, through phosphorylation of Ser364, increases Csk kinase activity by 2–4-fold, leading to reduced Lck activity and TCR ζ-chain phosphorylation which inhibits T-cell activation (Figure 1) . The other mechanism involves the adaptor molecule Cbp/PAG. Csk is recruited to lipid rafts and the site of action by binding to Tyr314/Tyr317-phosphorylated (rat/human) Cbp/PAG through its SH2 domain , and the interaction between Csk-SH2 and Cbp/PAG increases Csk activity . Addition of either recombinant Cbp/PAG or phosphopeptides corresponding to the Csk-SH2 binding site in Cbp/PAG significantly increased Csk kinase activity towards an Src substrate in vitro. Lastly, the PKA phosphorylation of Csk and its interaction with Cbp/PAG act together in turning on Csk activity in a time- and space-regulated fashion , providing a powerful mechanism for terminating activation through receptors eliciting Src kinase signalling (Figure 1).
cAMP inhibits T-cell activation through a PKA type I–Csk–Lck inhibitory pathway
cAMP levels are increased in lipid rafts upon TCR stimulation
It has previously been demonstrated that stimulation of the TCR results in elevated cAMP levels in the cell . However, since increased cAMP concentrations inhibit T-cell function and proliferation [30,31], it is important that TCR-mediated cAMP production is tightly regulated. The significance of activation-induced cAMP production has been poorly understood to date, as has the location in the cell where cAMP is generated. However, we were recently able to show that upon engagement of the TCR in primary T-cells, cAMP was rapidly produced in lipid rafts, resulting in raft-associated PKA activation . The G-proteins Gi, Gs and AC have been reported to segregate into lipid rafts  and our results indicate that recruitment to lipid rafts of the stimulatory G-protein Gs and dissociation of the inhibitory G-protein Gi play an important role for the cAMP production that occurs upon TCR activation. A local increase in cAMP is therefore generated in T-cell lipid rafts upon activation. In contrast, T-cells activated by TCR and CD28 cross-ligation revealed decreased cAMP levels compared with control cells and an increase in cAMP levels was only observed in the presence of the non-selective PDE inhibitor isobutylmethylxanthine. Furthermore, phosphorylation of PKA substrates in lipid rafts was less abundant when cells were only activated through the TCR compared with concomitant TCR and CD28 stimulation . We therefore hypothesized that TCR-induced cAMP production must be accompanied by cAMP degradation by PDEs to allow full T-cell activation to proceed.
PDE4 is recruited to lipid rafts upon TCR and CD28 co-stimulation
Isoforms from the PDE type 4 subfamily account for most of the cAMP-hydrolysing activity in T-cells [34,35]. In accordance with this, we observed PDE4 activity in lipid rafts upon T-cell activation. In particular, TCR and CD28 co-stimulation resulted in profound raft-associated increase in PDE4 activity . This specific increase in PDE4 activity in lipid raft fractions upon TCR and CD28 engagement indicates that temporal changes in PDE4 activity can play a key role in tuning intracellular activation-induced gradients of cAMP in T-cell lipid rafts, and thereby increase signal propagation upon co-stimulation.
PDE4s are encoded by four genes and give rise to more than 16 isoforms subdivided into PDE4A, PDE4B, PDE4C and PDE4D families, each of which is characterized by a unique N-terminal region . The mechanisms by which PDE4 isoforms are recruited to specific locations upon T-cell activation are now being unravelled [32,36]. It has been described that PDE4B can associate with the TCR complex  and transfected PDE4B relocalizes to the synapse area between the Jurkat T-cell and the antigen-presenting cell upon contact . We have demonstrated that PDE4A4, PDE4B2 and PDE4D1/PDE4D2 are recruited to lipid rafts upon TCR and CD28 co-stimulation in human peripheral T-cells . Members of the PDE4 family have previously been described to associate with the scaffolding protein β-arrestin, and β-arrestin has been shown to be responsible for bringing PDE4 to the plasma membrane of HEK-293 cells (human embryonic kidney cells) and to the activated GPCRs where cAMP production is taking place . Intriguingly, we found that both TCR and CD28 co-stimulation and CD28 stimulation alone caused a clear recruitment of β-arrestin to T-cell lipid raft fractions concurrently with PDE4 . In addition, immunoprecipitation from both unstimulated and co-stimulated cells revealed that β-arrestin and PDE4 pre-exist in a complex prior to stimulation, indicating that they are recruited to rafts together .
Control of cAMP levels is implicated in normal and diseased T-cell functions
PKA is activated upon TCR-induced cAMP production in lipid rafts and inhibits proximal T-cell signalling. However, overexpression of PDE4 isoforms or β-arrestin has been demonstrated to increase T-cell activation, revealing regulatory roles for both proteins in T-cell signalling . Other regulatory roles for β-arrestin in T-cells have also been described. For example, β-arrestin plays a positive regulatory role in chemotaxis  and in migration into the airways during asthma . The activities of both PKA and PDE4 therefore seem to be important for regulation of TCR-induced signalling and T-cell function. We propose a novel role for TCR and CD28 co-stimulation in down-modulation of TCR-induced cAMP-mediated inhibitory signals through the recruitment of β-arrestin and PDE4 to lipid rafts, thus allowing a full T-cell response to occur.
Interestingly, the cAMP inhibitory pathway has also been shown to be implicated in several immune diseases [41–47]. T-cells from HIV-infected patients have elevated levels of cAMP and hyperactivation of PKA. Targeting of the cAMP–PKA type I pathway by selective antagonists reverses T-cell dysfunction in HIV T-cells ex vivo [41,42] and targeting cyclo-oxygenase 2 to reduce PGE2 production lowers cAMP and increases T-cell function in vivo [43,44]. A similar mechanism contributes to the T-cell dysfunction in a subset of patients with common variable immunodeficiency , and to the severe T-cell anergy in a murine immunodeficiency model termed MAIDS (mouse AIDS) [46,47].
Compartmentalization of Cyclic AMP Signalling: Biochemical Society Focused Meeting held at King's College, Cambridge, U.K., 29–30 March 2006. Organized by D. Cooper (Cambridge, U.K.), M. Houslay (Glasgow) and M. Zaccolo (Padua, Italy). Edited by D. Cooper.
C-terminal Src kinase
exchange protein directly activated by cAMP
- NK cell
natural killer cell
protein associated with glycol-sphingolipid-enriched microdomains
protein kinase A
- SH2 domain
Src homology 2 domain
ζ-chain-associated protein kinase of 70 kDa
We are grateful to Dr Einar Martin Aandahl (University of Oslo) for preparation of illustrations. Our work is supported by grants from the Functional Genomics Programme, The Research Council of Norway and The Norwegian Cancer Society.