In several cases of immunodeficiency and autoimmunity, the dysfunctional immune system is associated with either hypo- or hyperactive T and B cells. In autoimmune conditions such as systemic lupus erythematosus (SLE) and immunodeficiencies such as acquired immunodeficiency syndrome (AIDS), it has been demonstrated that the regulatory effect of the signaling pathway of cyclic 3', 5' adenosine monophosphate (cAMP) and cAMP-dependent protein kinase (PKA) is abrogated. PKA is well-known as a key regulator of immune responses in that it inhibits both early and late phases of antigen induced T and B cell activation. Here we will discuss a potential useful strategy for therapeutic interventions of dysfunctional T cells associated with SLE and HIV by modulation of the cAMP-PKA pathway. Therefore, we will describe the components and architecture of the cAMP-PKA signaling pathway in T cells in order to point out one or several steps which potentially may serve as targets for therapeutic intervention.

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Cyclic AMP‐dependent protein kinase (PKA) is a holoenzyme that consists of a regulatory (R) subunit dimer and two catalytic (C) subunits that are released upon stimulation by cAMP. Immunoblotting and immunoprecipitation of T‐cell protein extracts, immunofluorescence of permeabilized T cells and RT/PCR of T‐cell RNA using C subunit‐specific primers revealed expression of two catalytically active PKA C subunits Cα1 (40 kDa) and Cβ2 (47 kDa) in these cells. Anti‐RIα and Anti‐RIIα immunoprecipitations demonstrated that both Cα1 and Cβ2 associate with RIα and RIIα to form PKAI and PKAII holoenzymes. Moreover, Anti‐Cβ2 immunoprecipitation revealed that Cα1 coimmunoprecipitates with Cβ2. Addition of 8‐CPT‐cAMP which disrupts the PKA holoenzyme, released Cα1 but not Cβ2 from the Anti‐Cβ2 precipitate, indicating that Cβ2 and Cα1 form part of the same holoenzyme. Our results demonstrate for the first time that various C subunits may colocate on the same PKA holoenzyme to form novel cAMP‐responsive enzymes that may mediate specific effects of cAMP.

It is well documented that the β‐gene of the catalytic (C) subunit of protein kinase A encodes a number of splice variants. These splice variants are equipped with a variable N‐terminal end encoded by alternative use of several exons located 5′ to exon 2 in the human, bovine and mouse Cβ gene. In the present study, we demonstrate the expression of six novel human Cβ mRNAs that lack 99 bp due to loss of exon 4. The novel splice variants, designated CβΔ4, were identified in low amounts at the mRNA level in NTera2‐N cells. We developed a method to detect CβΔ4 mRNAs in various cells and demonstrated that these variants were expressed in human and Rhesus monkey brain. Transient expression and characterization of the CβΔ4 variants demonstrated that they are catalytically inactive both in vitro against typical protein kinase A substrates such as kemptide and histone, and in vivo against the cAMP‐responsive element binding protein. Furthermore, co‐expression of CβΔ4 with the regulatory subunit (R) followed by kinase activity assay with increasing concentrations of cAMP and immunoprecipitation with extensive washes with cAMP (1 mm) and immunoblotting demonstrated that the CβΔ4 variants associate with both RI and RII in a cAMP‐independent fashion. Expression of inactive C subunits which associate irreversibly with R may imply that CβΔ4 can modulate local cAMP effects in the brain by permanent association with R subunits even at saturating concentrations of cAMP.

BackgroundTwo main genes encoding the catalytic subunits Cα and Cβ of cyclic AMP dependent protein kinase (PKA) have been identified in all vertebrates examined. The murine, bovine and human Cβ genes encode several splice variants, including the splice variant Cβ2. In mouse Cβ2 has a relative molecular mass of 38 kDa and is only expressed in the brain. In human and bovine Cβ2 has a relative molecular mass of 47 kDa and is mainly expressed in lymphoid tissues.ResultsWe identified a novel 47 kDa splice variant encoded by the mouse Cβ gene that is highly expressed in lymphoid cells. Cloning, expression, and production of a sequence-specific antiserum and characterization of PKA catalytic subunit activities demonstrated the 47 kDa protein to be a catalytically active murine homologue of human and bovine Cβ2. Based on the present results and the existence of a human brain-specifically expressed Cβ splice variant designated Cβ4 that is identical to the former mouse Cβ2 splice variant, the mouse splice variant has now been renamed mouse Cβ4.ConclusionMurine lymphoid tissues express a protein that is a homologue of human and bovine Cβ2. The murine Cβ gene encodes the splice variants Cβ1, Cβ2, Cβ3 and Cβ4, as is the case with the human Cβ gene.

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Different classes of glycans are implicated as mediators of apical protein sorting in the secretory pathway of epithelial cells, but recent research indicates that sorting to the apical and basolateral surfaces may occur before completion of glycan synthesis. We have previously shown that a proteoglycan (PG) core protein can obtain different glycosaminoglycan (GAG) structures in the apical and basolateral secretory routes (Tveit H, Dick G, Skibeli V, Prydz K. 2005. A proteoglycan undergoes different modifications en route to the apical and basolateral surfaces of Madin-Darby canine kidney cells. J Biol Chem. 280:29596-29603) of epithelial Madin-Darby canine kidney (MDCK) cells. We have now also determined the detailed N-glycan structures acquired by a single glycoprotein species in the same apical and basolateral secretory pathways. For this purpose, rat growth hormone (rGH) with two N-glycan sites (rGH-2N) inserted into the rGH portion (NAS and NFT) was fused to green fluorescent protein (GFP) and expressed in MDCK cells. Immunoisolated rGH variants were analyzed for site occupancy and N-glycan structure by mass spectrometry. The extent of NAS and NFT site occupancy was different, but comparable for rGH-2N secreted apically and basolaterally. Microheterogeneity existed for the glycans attached to each N-glycan site, but no major differences were observed in the apical and basolateral pathways. Transfer of the GAG modification domain from the PG serglycin to the fusion site of rGH-2N and GFP allowed polymerization of GAG chains onto the novel protein variant and influenced the microheterogeneity of the N-glycans toward more acidic glycans, but did not alter the relative site occupancy. In conclusion, no major differences were observed for N-glycan structures obtained by the expressed model proteins in the apical and basolateral secretory pathways of epithelial MDCK cells, but insertion of a GAG attachment domain shifted the N-glycans to more acidic structures.