A cAMPPKACsk Inhibitory Pathway in Lipid Rafts Regulates TCell Immune Function

Proteins involved in proximal TCR signaling events are localized in lipid rafts, representing small regions of detergent-resistant lipid domains of the membrane (Montixi et al. 1998; Xavier et al. 1998). Both the cAMP-generating machinery (adenylyl cyclase) and the effectors (PKA type I and Csk) are localized in lipid rafts. 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 RIa (but not RII subunits) are constitutively associated with the lipid rafts (Vang et al. 2001). This suggests that the observed co-localization of PKA type I and TCR in capped T cells (Skalhegg et al. 1994) 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, which we recently identified as ezrin (Ruppelt et al. 2007).

Csk is constitutively localized to lipid rafts in resting T cells, but is transiently displaced to the cytosol during T-cell activation (Torgersen et al. 2001) in order to allow the activation cascade to proceed. The phosphatase responsible for the dephos-phorylation of Cbp/PAG and the release of Csk was recently identified as CD45 (Davidson et al. 2003). Fyn-mediated phosphorylation of Cbp/PAG (Brdicka et al. 2000; Kawabuchi et al. 2000) 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-Y505). So far, two different mechanisms are reported to regulate Csk activity. PKA type I, through phosphorylation of Ser-364, increases Csk kinase activity two- to four-fold, leading to reduced Lck activity and TCR Z-chain phosphorylation, which inhibits T-cell activation (Fig. 2). The other mechanism involves the adaptor molecule Cbp/PAG. Csk is recruited to lipid rafts and the site of action by binding to Y314/Y317-phosphorylated (rat/human) Cbp/PAG through its SH2 domain (Brdicka et al. 2000; Kawabuchi et al. 2000), and the interaction between Csk-SH2 and Cbp/PAG increases Csk activity

Fig. 2 Modulation of proximal TCR signaling by cAMP and PKA. Generation of local pools of cAMP through G protein-coupled receptors and adenylyl cyclase activity is able to attenuate proximal TCR signaling by activating a PKA-Csk-Lck inhibitory pathway. Ezrin acts as an AKAP and targets PKA type I to lipid rafts where it can phosphorylate and activate Csk. PKA-mediated phosphorylation and targeting to the transmembrane adaptor Cbp/PAG both contribute to increased Csk activity and thereby increased tonic inhibition of Src kinases such as Lck. Engagement of the T-cell receptor leads to dephosphorylation of Cbp/PAG, dissociation of Csk from lipid rafts and increased Lck activity

Fig. 2 Modulation of proximal TCR signaling by cAMP and PKA. Generation of local pools of cAMP through G protein-coupled receptors and adenylyl cyclase activity is able to attenuate proximal TCR signaling by activating a PKA-Csk-Lck inhibitory pathway. Ezrin acts as an AKAP and targets PKA type I to lipid rafts where it can phosphorylate and activate Csk. PKA-mediated phosphorylation and targeting to the transmembrane adaptor Cbp/PAG both contribute to increased Csk activity and thereby increased tonic inhibition of Src kinases such as Lck. Engagement of the T-cell receptor leads to dephosphorylation of Cbp/PAG, dissociation of Csk from lipid rafts and increased Lck activity

(Takeuchi et al. 2000). Addition of either recombinant Cbp/PAG or phos-phopeptides corresponding to the Csk-SH2 binding site in Cbp/PAG significantly increased Csk kinase activity towards a 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 (Vang et al. 2003), providing a powerful mechanism for terminating activation through receptors eliciting Src kinase signaling (Fig. 2).

Immunofluorescense and immunoprecipitation studies of ezrin in normal T cells revealed colocalization with PKA RIa and unveiled a complex including Csk and Cbp/PAG interacting as well as the ezrin-Cbp/PAG linker EBP50 (Ruppelt et al. 2007). Together this demonstrates formation of a lipid raft-associated complex where ezrin brings PKA type I in the proximity of its downstream substrate Csk by forming a supramolecular signaling complex consisting of PKA type I, ezrin, EBP50, Cbp/PAG and Csk. Ezrin is bound via CD43, CD44 or ICAMs to the plasma membrane (Heiska et al. 1998; Tsukita et al. 1994; Yonemura et al. 1998) and via its C-terminus to the actin cytoskeleton (Algrain et al. 1993), and EBP50 links ezrin to Cbp/PAG (Itoh et al. 2002). Thus, ezrin positions PKA type I in proximity of its substrate Csk, which is bound to Cbp/PAG (Fig. 2), leading to PKA phosphorylation of the pool of Csk anchored to Cbp/PAG (Vang et al. 2001, 2003). This, together with the observations that downstream effector functions modulated by the PKA-Csk pathway are released from cAMP inhibition by knockdown of ezrin, argues that the PKA regulation of Csk is discretely coordinated and spatiotemporally restricted to this supramolecular complex (Ruppelt et al. 2007).

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