In T cells, cAMP-dependent protein kinase (PKA) type I colocalizes with the T cell receptor–CD3 complex (TCR/CD3) and inhibits T cell function via a previously unknown proximal target. Here we examine the mechanism for this PKA-mediated immunomodulation. cAMP treatment of Jurkat and normal T cells reduces Lck-mediated tyrosine phosphorylation of the TCR/CD3 ζ chain after T cell activation, and decreases Lck activity. Phosphorylation of residue Y505 in Lck by COOH-terminal Src kinase (Csk), which negatively regulates Lck, is essential for the inhibitory effect of cAMP on ζ chain phosphorylation. PKA phosphorylates Csk at S364 in vitro and in vivo leading to a two- to fourfold increase in Csk activity that is necessary for cAMP-mediated inhibition of TCR-induced interleukin 2 secretion. Both PKA type I and Csk are targeted to lipid rafts where proximal T cell activation occurs, and phosphorylation of raft-associated Lck by Csk is increased in cells treated with forskolin. We propose a mechanism whereby PKA through activation of Csk intersects signaling by Src kinases and inhibits T cell activation.

Two different mammalian genes for the catalytic subunit (C) of cAMP-dependent protein kinase have previously been characterized (C alpha, C beta). In the present study, we report the molecular cloning of a third isoform of C, from a human testis cDNA library, as well as the isolation of human cDNAs for C alpha and C beta. This third form of C, which we will designate C gamma, is clearly derived from a distinct gene and shows a tissue-specific expression. A close evolutionary relation between C gamma and C alpha was suggested by nucleotide homologies (86% inside the open reading frame, 81% in the 3'-untranslated region). Thus, the C gamma cDNA cross-hybridized with the 2.8 kilobase (kb) C alpha mRNA, present at high levels in most human tissues, as well as with a 1.8 kb C gamma-specific mRNA, which was only found at detectable levels in human testis. However, at the amino acid level, C alpha and C beta showed a close relationship (93% homology), whereas C gamma diverged significantly from both C alpha (83%) and C beta (79%). Taken together with the tissue-specific expression of C gamma, this suggests a pressure on C gamma during evolution, acting to modulate it in a functionally specific way. Certain amino acid substitutions make C gamma a distinct member of the cAMP-dependent subfamily of protein kinases, and suggest that C gamma may be distinct in its protein substrate specificity or its interaction with the different regulatory subunits.

Selective activation of cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase type I (cAKI), but not type II, is sufficient to mediate inhibition of T cell replication induced through the antigen-specific T cell receptor-CD3 (TCR-CD3) complex. Immunocytochemistry and immunoprecipitation studies of the molecular mechanism by which cAKI inhibits TCR-CD3-dependent T cell replication demonstrated that regulatory subunit I alpha, along with its associated kinase activity, translocated to and interacted with the TCR-CD3 complex during T cell activation and capping. Regulatory subunit II alpha did not. When stimulated by cAMP, the cAKI localized to the TCR-CD3 complex may release kinase activity that, through phosphorylation, might uncouple the TCR-CD3 complex from intracellular signaling systems.

Cyclic AMP-dependent protein kinase A (PKA) type I has been established as an acute inhibitor of T cell activation. For this reason, we investigated the possible role of PKA type I in HIV-induced T cell dysfunction. T cells from HIV-infected patients have increased levels of cAMP and are more sensitive to inhibition by cAMP analog than are normal T cells. A PKA type I-selective antagonist increases the impaired proliferation of T cells from HIV-infected patients to normal or subnormal levels (up to 2.8-fold). Follow-up of patients after initiation of highly active antiretroviral treatment revealed that a majority of patients have a persistent T cell dysfunction that is normalized by incubation of T cells with Rp-8-Br-cAMPS. These observations imply that increased activation of PKA type I may contribute to the progressive T cell dysfunction in HIV infection and that PKA type I may be a potential target for immunomodulating therapy.

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