Activation of the COOH-terminal Src kinase (Csk) by cAMP-dependent protein kinase inhibits signaling through the T cell receptor.

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 zeta 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 zeta 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.

Localization of a novel human A-kinase-anchoring protein, hAKAP220, during spermatogenesis.

Using a combination of protein kinase A type II overlay screening, rapid amplification of cDNA ends, and database searches, a contig of 9923 bp was assembled and characterized in which the open reading frame encoded a 1901-amino-acid A-kinase-anchoring protein (AKAP) with an apparent SDS-PAGE mobility of 220 kDa, named human AKAP220 (hAKAP220). The hAKAP220 amino acid sequence revealed high similarity to rat AKAP220 in the 1167 C-terminal residues, but contained 727 residues in the N-terminus not present in the reported rat AKAP220 sequence. The hAKAP220 mRNA was expressed at high levels in human testis and in isolated human pachytene spermatocytes and round spermatids. The hAKAP220 protein was present in human male germ cells and mature sperm. Immunofluorescent labeling with specific antibodies indicated that hAKAP220 was localized in the cytoplasm of premeiotic pachytene spermatocytes and in the centrosome of developing postmeiotic germ cells, while a midpiece/centrosome localization was found in elongating spermatocytes and mature sperm. The hAKAP220 protein together with a fraction of PKA types I and II and protein phosphatase I was resistant to detergent extraction of sperm tails, suggesting an association with cytoskeletal structures. In contrast, S-AKAP84/D-AKAP1, which is also present in the midpiece, was extracted under the same conditions. Anti-hAKAP220 antisera coimmunoprecipitated both type I and type II regulatory subunits of PKA in human testis lysates, indicating that hAKAP220 interacts with both classes of R subunits, either through separate or through a common binding motif(s).

Evidence for independent control at the mRNA and protein levels of cellular retinol binding protein 1 in rat Sertoli cells.

Cellular retinol binding protein 1 (CRBP1) is the cytosolic carrier for retinol. It is expressed in many tissues, but the concentrations vary considerably. In Sertoli cells from immature rat testis, CRBP1 is highly expressed. The results of the present study show that regulation of CRBP1 expression at the protein level appears to be independent of regulation at the mRNA level. In Sertoli cells from prepubertal 19-day-old rats, CRBP1 mRNA is strongly induced for up to 72 h by the presence of serum factors. In contrast, treatment of the cells with cAMP analogues led to a rapid reduction in mRNA to quantities less than 5% of control values. However, the changes in CRBP1 mRNA did not lead to similar changes in the concentration of CRBP1 protein during 72 h of observation. Similarly, treatment of cells from 32- and 44-day-old rats with serum led to increased CRBP1 mRNA, whereas cAMP treatment resulted in a decrease in CRBP1 mRNA. Again, no changes were observed in the concentration of CRBP1 protein. Furthermore, co-incubation of Sertoli cells from 19-day-old rats with purified pachytene spermatocytes or round spermatids resulted in an increase in mRNA for CRBP1. However, comparable changes in CRBP1 protein concentrations were not observed. Neither cAMP nor serum changed the fraction of CRBP1 mRNA that was associated with polysomes. As a possible explanation for some of the results, pulse-chase experiments showed that the rate of CRBP1 degradation in cultured Sertoli cells is decreased by cAMP. It is proposed that these changes at the level of protein turnover contribute to the maintenance of stable concentrations of CRBP1 even when the corresponding mRNA concentrations vary markedly.

Cyclic-AMP-dependent protein kinase (PKA) in testicular cells. Cell specific expression, differential regulation and targeting of subunits of PKA.

LH and FSH regulate via cyclic adenosine 3'5' cyclic monophosphate (cAMP) and cAMP-dependent protein kinase (PKA), steroid biosynthesis is Leydig and Sertoli cells, respectively. Cyclic AMP also regulates a number of different cellular processes such as cell growth and differentiation, ion channel conductivity, synaptic release of neurotransmitters, and gene transcription. The principle intracellular target for cAMP in mammalian cells is the PKA. The fact that this broad specificity protein kinase mediates a number of discrete physiological responses following cAMP engagement, has raised the question of how specificity is maintained in the cAMP/PKA system. Here we describe features of this signaling pathway that may contribute to explain how differential effects of cAMP may occur.

Cyclic-AMP-dependent protein kinase (PKA) in testicular cells. Cell specific expression, differential regulation and targeting of subunits of PKA

LH and FSH regulate via cyclic adenosine 3'5' cyclic monophosphate (cAMP) and cAMP-dependent protein kinase (PKA), steroid biosynthesis is Leydig and Sertoli cells, respectively. Cyclic AMP also regulates a number of different cellular processes such as cell growth and differentiation, ion channel conductivity, synaptic release of neurotransmitters, and gene transcription. The principle intracellular target for cAMP in mammalian cells is the PKA. The fact that this broad specificity protein kinase mediates a number of discrete physiological responses following cAMP engagement, has raised the question of how specificity is maintained in the cAMP/PKA system. Here we describe features of this signaling pathway that may contribute to explain how differential effects of cAMP may occur.

PKAI as a potential target for therapeutic intervention.

We have mapped a molecular mechanism for the impaired T-cell function in HIV infection and common variable immunodeficiency (CVI). Protein kinase A type I (PKAI) has a key role as an inhibitor of immune function in T lymphocytes and is activated following antigen receptor triggering. T cells from patients with HIV infection and CVI have increased activation of PKAI. This inhibits immune function and proliferation of T cells. Selective antagonists that block cAMP action through PKAI improve the immune function of T cells from HIV-infected patients up to 300%. Furthermore, combination of cAMP antagonists with interleukin-2 normalized immune responses of T cells from all patients examined and stimulated immune function of T cells from HIV-infected patients up to 600%. In addition, in vitro experiments indicate that approximately 50% of patients with CVI have a T-cell dysfunction that might benefit from a treatment reversing PKAI hyperactivation. This outlines PKAI as a potentially attractive drug target for immunomodulating therapy in HIV infection, as well as for the treatment of other immunodeficiency disorders such as CVI.

Additive effects of IL-2 and protein kinase A type I antagonist on function of T cells from HIV-infected patients on HAART.

OBJECTIVE: To explore the basis for a possible immunomodulatory combination therapy with IL-2 and agents inhibiting protein kinase A (PKA) type I. DESIGN: Highly active antiretroviral therapy (HAART) has dramatically improved HIV therapy, but fails to eradicate the virus, and the persistence of HIV-associated immunodeficiency demonstrates the need for additional immunomodulating therapies. We have previously shown that hyperactivation of PKA type I inhibits the function of HIV-infected patient T cells. The separate and combined effect of a PKA type I-selective antagonist (Rp-8-Br-cAMPS) and Interleukin (IL)-2 on the function of T cells from HIV-infected patients on HAART was examined. METHODS: The effect of Rp-8-Br-cAMPS on anti-CD3 stimulated proliferation and IL-2 production and the combined effect with exogenous IL-2 was studied in vitro with cells from 13 HIV-infected patients on HAART and six uninfected controls. RESULTS: The PKA type I-selective antagonist improved cell proliferation (median 1.5-fold, maximal 2.8-fold) and IL-2 production (median 1.5-fold, maximal 2.4-fold) in T cells from HIV-infected patients on HAART, but not in controls. The addition of IL-2 enhanced proliferation of T cells from HIV-infected patients (approximately 1.9-fold) and that of controls (approximately 1.4-fold), but IL-2 had no effect at the concentrations produced by treatment with PKA type I antagonist. However, the combined effect of IL-2 and PKA type I antagonist was additive and resulted in a further increase in T-cell proliferation (median 2.5-fold, maximal 5.8-fold), reaching levels comparable with those of uninfected controls in most of the patients. CONCLUSION: Our findings suggest a basis for a novel strategy in treatment of HIV infection by combining IL-2 therapy and treatment modalities counteracting PKA type I activity with HAART.

Cyclic-AMP-dependent protein kinase (PKA) in testicular cells. Cell specific expression, differential regulation and targeting of subunits of PKA.

LH and FSH regulate via cyclic adenosine 3'5' cyclic monophosphate (cAMP) and cAMP-dependent protein kinase (PKA), steroid biosynthesis is Leydig and Sertoli cells, respectively. Cyclic AMP also regulates a number of different cellular processes such as cell growth and differentiation, ion channel conductivity, synaptic release of neurotransmitters, and gene transcription. The principle intracellular target for cAMP in mammalian cells is the PKA. The fact that this broad specificity protein kinase mediates a number of discrete physiological responses following cAMP engagement, has raised the question of how specificity is maintained in the cAMP/PKA system. Here we describe features of this signaling pathway that may contribute to explain how differential effects of cAMP may be contributed to features of the PKA signaling pathway.

Method of transfection affects the cAMP-mediated induction of the RIIbeta subunit of protein kinase A in Sertoli cells: inhibition of response by increase in intracellullar calcium.

mRNA for the regulatory subunit RIIbeta of cAMP-dependent protein kinase is stimulated more than 50-fold by cAMP in primary cultures of rat Sertoli cells. We have previously shown that this induction involves regulation of transcriptional activation as well as mRNA stabilization. The rat RIIbeta gene contains no cAMP response element (CRE), and the induction of RIIbeta mRNA is slow and requires on-going protein synthesis. When a construct containing the 5'-flanking region of the RIIbeta gene upstream of a CAT reporter was transfected into Sertoli cells by the calcium phosphate method, low and variable responses to cAMP (three- to fivefold) were observed, whereas a 15- to 20-fold increase in reporter activity by cAMP was observed after lipofectamine transfection. Interestingly, when a vector containing CRE elements upstream of a reporter gene was transfected into Sertoli cells, the responses to cAMP were similar regardless of the transfection method used. We have also demonstrated that increased intracellular levels of calcium by A23187 and thapsigargin dramatically inhibit cAMP-mediated induction of RIIbeta mRNA, but not the mRNA for the CRE-containing RIalpha gene. Furthermore, decreased cAMP responsiveness of endogenous RIIbetamRNA (but not RIalpha) was also observed in calcium phosphate-transfected Sertoli cells but not in lipofectamine-transfected cells. Thus, calcium-mediated reduction in cAMP response appears to be a gene-specific phenomenon.

Differential localization of protein kinase A type II isozymes in the Golgi-centrosomal area.

Selectivity in the action of cAMP may be mediated by compartmentalized pools of cyclic AMP-dependent protein kinase (PKA). PKA type II is directed to different subcellular loci by interaction of the type II regulatory subunits (RIIalpha, RIIbeta) with A-kinase anchoring proteins. In order to separately investigate the subcellular localization of PKA type II isozymes, monospecific antibodies to human RIIalpha and RIIbeta subunits of PKA were developed. We demonstrate that centrosomes bind both RIIalpha and RIIbeta. Centrosomes were the preferred intracellular anchoring site for RIIbeta. However, centrosomal localization of RIIbeta was observed only in some highly differentiated cells such as keratinocytes, granulosa cells, and macrophages and in all neoplastic cell lines examined. Centrosomal localization of RIIbeta was not observed in normal undifferentiated cells such as fibroblasts, myoblasts, and T and B cells. In contrast, RIIalpha was abundant in the Golgi area and in the trans-Golgi network (TGN). Furthermore, although RIIalpha appeared to colocalize with microtubules in the Golgi/TGN, extractions with nonionic detergent demonstrated that RIIalpha was mainly membrane-associated. In addition, alterations of microtubule dynamics with Nocodazole or Taxol affected the distribution of the detergent-extractable pool of RIIalpha, indicating that RIIalpha may localize with microtubule-associated vesicles. Thus, RIIalpha and RIIbeta clearly localize differently in the Golgi-centrosomal region. This indicates specific roles for PKA isozymes containing either RIIalpha or RIIbeta.

Calcium/phospholipid-dependent protein kinases in rat Sertoli cells: regulation of androgen receptor messenger ribonucleic acid.

The possibility that Sertoli cell responses to testosterone are modulated by the calcium/phospholipid-dependent protein kinase (protein kinase C; PKC) was examined in rat Sertoli cells in culture. Both soluble and particulate cell fractions showed low constitutive phosphotransferase activity. Incubation with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA; 10(-7) M) was associated with a transient induction in both cell fractions of calcium/phosphatidylserine-dependent PKC activity, which was elevated from 15 min to 1 h. Consistent with this, mRNAs for the calcium/phospholipid-dependent isomeric forms of PKC (alpha, beta, and gamma) were detected. The expression levels of mRNAs for PKCalpha and PKCbeta were also up-regulated (2.5- to 3-fold) by TPA (10(-7) M), but these effects were much slower (peaking after 12 h) than those on phosphotransferase activity. In the presence of TPA (10(-7) M), expression of androgen receptor (AR) mRNA showed a transient time-dependent down-regulation ( approximately 70%), in which the nadir was reached after 6 h and baseline expression was again obtained after 12 h. The regulatory effect of PKC activation on AR mRNA was confirmed by the absence of response to a biologically inactive phorbol ester. A concentration-dependent decrease (half-maximal effect at approximately 10(-8) M TPA) of AR mRNA was also observed. These data suggest that Sertoli cell responses to testosterone may be inhibited by a transiently active PKC with a wide intracellular distribution.

Increased activation of protein kinase A type I contributes to the T cell deficiency in common variable immunodeficiency.

The molecular mechanisms underlying the T cell dysfunction often present in common variable immunodeficiency (CVI) are not established. cAMP-dependent protein kinase A type I (PKAI) is an important inhibitor of T cell proliferation after Ag stimulation. We therefore investigated the possibility that activation of PKAI may be involved in the development of T cell dysfunction in CVI. An exogenously added PKAI-selective antagonist (Rp-8-Br-cAMPS) induced a significant increase in anti-CD3-stimulated PBMC proliferation in 20 CVI patients compared with no effect in 15 controls. Purified T cells from 7 CVI patients with strictly defined T cell deficiency had elevated endogenous cAMP levels compared with controls. Treatment of T cells from these CVI patients with Rp-8-bromo-cAMP-phosphorothioate markedly improved anti-CD3-stimulated proliferation (up to 3.7-fold), particularly in CD4+ lymphocytes, reaching proliferation levels comparable to control values. No effect of cAMP antagonist on T cell proliferation was seen in controls. In these CVI patients, cAMP antagonist also increased IL-2 production in anti-CD3-stimulated T cells. However, exogenously added IL-2 at concentrations comparable to the achieved increase in IL-2 levels after addition of cAMP antagonist had no effect on T cell proliferation. Furthermore, the stimulatory effects of exogenously added IL-2 at higher concentrations and cAMP antagonist on T cell proliferation were additive. Our findings indicate that increased PKAI activation may be an important molecular basis for the T cell defect in CVI and suggest that the cAMP/PKAI system may be a potential molecular target for immunomodulating therapy in these patients.

Isozymes of cyclic AMP-dependent protein kinases (PKA) in human lymphoid cell lines: levels of endogenous cAMP influence levels of PKA subunits and growth in lymphoid cell lines.

Activation of the cAMP signaling pathway in lymphoid cells is known to inhibit cell proliferation of T and B cells as well as cytotoxicity of natural killer (NK) cells. In order to find suitable model systems to study cAMP-mediated processes, we have examined the expression of cAMP-dependent protein kinase (PKA), endogenous levels of cAMP, and cell proliferation in eight cell lines of B lineage origin, four cell lines of T lineage origin, and normal human B and T cells. We demonstrated that the expression of mRNA and protein for one of the regulatory (R) subunits of PKA (RIalpha) was present in all the cells investigated, in contrast to the other R subunits (RIbeta, RIIalpha, and RIIbeta). Furthermore, three T cell lines and one B cell line expressed only RIalpha and C, implying these cells to contain solely PKA type I. Moreover, for the RI subunit, we observed an apparent reciprocal relationship between levels of mRNA and protein. Generally, RIalpha protein was low in cell lines where mRNA was elevated and vice versa. This was not the case for the RII subunits, where high levels of mRNA were associated with elevated levels of protein. Interestingly, we demonstrated an inverse correlation between levels of endogenous cAMP and cell growth as determined by [3H]-thymidine incorporation and cell-doubling rate (P < 0.05). Taken together, our results demonstrate great differences in PKA isozyme composition, which should be taken into consideration when using lymphoid cell lines as model system for cAMP/PKA effects in normal lymphocytes.

Kinetic properties of the C-terminal Src kinase, p50csk.

Csk is an important regulator of tyrosine kinases of the Src family. In this paper, we have characterised the kinetics and catalytic properties of a highly active and stable enzyme obtained in milligram amounts by expressing the enzyme as a fusion protein with glutathione-S-transferase (GST) in Escherichia coli. Using the synthetic polyamino acid poly(Glu, Tyr) as substrate, phosphotransferase activity was linear for 7-8 min with Mg2+ and 5 min with Mn2+. With Mg2+ and Mn2+, respectively, K(m) (ATP) was 56.9 +/- 6.2 and 5.4 +/- 0.6 microM and Vmax was 293 +/- 52 and 217 +/- 38 pmol phosphate transferred (microgram Csk)-1 min-1. Optimal concentrations of Mg2+ and Mn2+ were 4-10 mM and 2-3 mM, respectively, and higher concentrations of both cations were inhibitory. The Csk activity was highly sensitive to monovalent (Na+, K+) and divalent (Ca2+) cations, the sensitivity being 2-5-fold higher with Mg2+ than Mn2+. Physiological concentrations of Ca2+ (less than 10 microM) were without effect. Autophosphorylation of Csk was demonstrated in vitro, but did not influence the catalytic activity. Addition of inorganic phosphate above 100 microM strongly inhibited Csk catalytic activity towards poly(Glu, Tyr) in the presence of Mn2+, but not in the presence of Mg2+. Phosphorylation of a physiological substrate (Lck) and autophosphorylation of Csk was not inhibited by phosphate, indicating that the phosphate-dependent inhibition of Csk activity was substrate specific.

Protein kinase A type I antagonist restores immune responses of T cells from HIV-infected patients.

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.

Residual bodies and IL-1alpha stimulate expression of mRNA for IL-1alpha and IL-1 receptor type I in cultured rat Sertoli cells.

The cytokine interleukin (IL)-1alpha may be produced both by Sertoli cells and immature male germ cells from rat and is thought to play a role in autocrine and/or paracrine regulation of the spermatogenesis. The localization of IL-1 receptors in seminiferous tubules is unknown. In this study we found a constitutive expression of IL-1 receptor type I (IL-I RI) mRNA in cultured Sertoli cells and peritubular cells from rat, whereas no such transcripts were observed in immature germ cells (pachytene spermatocytes and round spermatids). An autostimulation of IL-1alpha mRNA synthesis has previously been described in other cell types. Stimulation of Sertoli cells with recombinant IL-1alpha for 0-7 h resulted in a rapid increase in both IL-1alpha and IL-1 RI mRNA. When Sertoli cells were cultured with residual bodies for 0-48 h, mRNA levels for both IL-1alpha and IL-1 RI were increased in a biphasic manner. We suggest that phagocytosis of residual bodies triggers an autocrine IL-1alpha loop in Sertoli cells where both IL-1alpha and one of its receptors are stimulated.

The gene encoding the C gamma catalytic subunit of cAMP-dependent protein kinase is a transcribed retroposon.

Three different catalytic isoforms of cAMP-dependent protein kinase have been identified (C alpha, C beta, and C gamma). We report the cloning and characterization of the human and rhesus monkey genes encoding the testis-specific C gamma subunit. The human C gamma gene is intronless with an open reading frame similar to the previously published cDNA sequence. The 3' and 5' flanking regions share high similarity with the C alpha nontranslated regions (82%) also outside the regions corresponding to the C gamma cDNA. The human gene is flanked by an Alu-related sequence in the 5'-end and there are insertions of two Alu-related sequences in the 3' nontranslated region. The observation that the C gamma gene is intronless and colinear with C alpha mRNA, together with the presence of remnants of a poly(A) tail and flanking direct repeats, indicates that the C gamma gene is a C alpha-derived retroposon. The 5' flanking region of this gene has a high G/C content and a putative TATA box situated at -138 compared to the translation initiation codon. Cloning and sequencing of a partial C gamma rhesus monkey gene demonstrate conservation of the sequence in primates. Northern analysis on isolated and fractionated human germ cells of testes from normal and estrogen-treated individuals demonstrates that the C gamma gene is expressed only in germ cells in the human testis. Our results indicate that the C gamma gene is a retroposon specifically transcribed in primate testicular germ cells.

Characterization of the 5'-flanking region of the gene for the cAMP-inducible protein kinase A subunit, RIIbeta, in Sertoli cells.

Activation of cyclic AMP-dependent protein kinases (protein kinase A, PKA) by gonadotropins and cyclic AMP (cAMP) plays an important role in the regulation of testicular functions. A regulatory subunit, RIIbeta, of PKA is transcriptionally induced in rat Sertoli cells in response to treatment with cAMP. The present study addresses regulatory mechanisms leading to increased transcription of the rat RIIbeta gene. We have localized a footprint which overlaps one of the major transcription initiation sites in the basal promoter (-293 to -123). One of the proteins binding this sequence belongs to the NF-1 family of transcription factors. We also observed binding to a basic helix-loop-helix (bHLH) response element. Furthermore, transfection studies of various 5'-deletions of the rat RIIbeta gene in primary cultures of rat Sertoli cells and in peritubular cells revealed the presence of an upstream region (-723 to -395, cAMP-responsive region) inhibiting basal expression from the rat RIIbeta gene only in Sertoli cells. This region was found to enhance cAMP responsiveness in Sertoli cells but not in peritubular cells. Interactions with downstream elements seemed to be important for the function of the cAMP-responsive region. Although some short stretches reveal homology to the cAMP-responsive regions of other slowly cAMP-responding genes, and an AP-1-like element is present, no strong resemblance to any known regulatory element responsive to cAMP is found.

Selective activation of cAMP-dependent protein kinase type I inhibits rat natural killer cell cytotoxicity.

The present study examines the expression and involvement of cAMP-dependent protein kinase (PKA) isozymes in cAMP-induced inhibition of natural killer (NK) cell-mediated cytotoxicity. Rat interleukin-2-activated NK cells express the PKA alpha-isoforms RIalpha, RIIalpha, and Calpha and contain both PKA type I and type II. Prostaglandin E2, forskolin, and cAMP analogs all inhibit NK cell lysis of major histocompatibility complex class I mismatched allogeneic lymphocytes as well as of standard tumor target cells. Specific involvement of PKA in the cAMP-induced inhibition of NK cell cytotoxicity is demonstrated by the ability of a cAMP antagonist, (Rp)-8-Br-adenosine 3',5'-cyclic monophosphorothioate, to reverse the inhibitory effect of complementary cAMP agonist (Sp)-8-Br-adenosine 3',5'-cyclic monophosphorothioate. Furthermore, the use of cAMP analog pairs selective for either PKA isozyme (PKA type I or PKA type II), shows a preferential involvement of the PKA type I isozyme, indicating that PKA type I is necessary and sufficient to completely abolish killer activatory signaling leading to NK cell cytotoxicity. Finally, combined treatment with phorbol ester and ionomycin maintains NK cell cytotoxicity and eliminates the cAMP-mediated inhibition, demonstrating that protein kinase C and Ca2+-dependent events stimulate the cytolytic activity of NK cells at a site distal to the site of cAMP/PKA action.

The human gene for the regulatory subunit RI alpha of cyclic adenosine 3', 5'-monophosphate-dependent protein kinase: two distinct promoters provide differential regulation of alternately spliced messenger ribonucleic acids.

The present study reports the exon-intron organization of the human RI alpha gene of cAMP-dependent protein kinase and approximately kilobases (kb) of the 5'-flanking region obtained by isolation and sequencing of several phage clones from human genomic libraries. The RI alpha gene is composed of nine coding exons of varying lengths, separated by introns, giving the gene a total length of at least 21 kb. our recent cloning of a processed RI alpha pseudogene with a 5'-noncoding region different from the previously reported RI alpha complementary RNA indicated that the RI alpha gene may have multiple leader exons giving rise to alternately spliced messenger RNAs (mRNAs). Reverse transcription of human testis RNA followed by PCR identified two different RI alpha mRNA species (RI alpha 1a and RI alpha 1b) containing distinct sequences due to alternately splicing the gene. The previously known RI alpha 1b mRNA revealed low constitutive expression in a human B lymphoid cell line (Reh) and was stimulated only 4- to 6-fold by treatment with cAMP. In contrast, very low levels of the novel RI alpha 1a mRNA were present in untreated Reh cells, but were stimulated 40-to 50-fold by cAMP. The 5'-flanking sequence of the RI alpha gene was G/C rich and did not contain any TATA box. Several putative transcription initiation sites were identified in front of each leader exon (exons 1a and 1b) by the 5'-rapid amplification of complementary DNA ends technique. To determine whether the sequences 5' of both leader exons had promoter activities, the 5'-flanking sequences of exons 1a and 1b were inserted in front of a chloramphenicol acetyltransferase reporter gene, and their ability to direct transcription were examined. Transfection of these constructs into rat GH4C1 cells demonstrated that both constructs had promoter activities, as evidenced by high levels of chloramphenicol acetyltransferase activity.