Báo cáo khoa học: Identification and characterization of novel PKA holoenzymes in human T lymphocytes pdf

Identification and characterization of novel PKA holoenzymes in human T lymphocytes Sigurd Ørstavik 1 , Ane Funderud 1 , Tilahun Tolesa Hafte 1 , Sissel Eikvar 1,2 , Tore Jahnsen 2 and Bjørn Steen Ska ˚ lhegg 1 1 Department of Nutrition, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Norway 2 Department of Biochemistry, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Norway In the absence of cAMP, cAMP-dependent protein kinase (PKA) is a holoenzyme consisting of two regu- latory (R) subunits bound together in a dimer, with one catalytic (C) subunit bound to each R-subunit. Binding of two cAMP molecules to each R subunit results in a conformational change that promotes release of active C subunits. In mammals, four genes encode different isoforms of the R-subunits, RIa,RIb, RIIa and RIIb, and three different genes encode three C isoforms, Ca,Cb and PRKX [1]. The Ca and Cb isoforms are closely related in protein sequence while the PRKX is more different from Ca and Cb. In pri- mates, a transcribed retroposon Cc has been identified. However, this gene has never been shown to express a protein sequence in vivo making the function of Cc unclear [2]. Several splice variants have been identified for Ca and Cb. In the case of Ca, two active isoforms have been identified and designated Ca1 and Cas. Ca1 is expressed in most tissues while Cas is restricted to sperm cells [3,4]. So far, 10 different splice variants of Cb have been identified, as a result of alternative spli- cing of seven different exons encoding the N-terminal part of the Cb protein [5]. Whereas the majority of the Cb splice-variants are expressed in a brain-specific manner [6] the Cb2 splice variant is highly expressed in lymphoid tissues. Most C isoforms, including Ca1 and Cb1, have a calculated molecular mass of  40 kDa, and with a lack of isoform-specific antibodies they may be difficult to distinguish. In contrast, the Cb2 isoform has a calculated molecular mass of 47 kDa [6] making it more easy to distinguish from other C-sub- units by SDS ⁄ PAGE analysis. The high level of Cb2 mRNA observed in lymphoid tissues led us to investigate whether Cb2 protein could be present in T cells. Previously, T cells have been shown to contain mainly RIa (80%) and some RIIa Keywords antibodies; cAMP; lymphocytes; protein kinase A Correspondence B. Steen Ska ˚ lhegg, Department of Nutrition, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, PO Box 1046, Blindern, N-0316 Oslo, Norway Fax: +47 2285 1347 Tel: +47 2285 1547 E-mail: bjorn.skalhegg@medisin.uio.no Website: http://www.uio.no (Received 2 November 2004, revised 22 December 2004, accepted 13 January 2005) doi:10.1111/j.1742-4658.2005.04568.x 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 immunoprecipita- tion 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 Ca1 (40 kDa) and Cb2 (47 kDa) in these cells. Anti-RIa and Anti-RIIa immunoprecipitations demonstrated that both Ca1 and Cb2 associate with RIa and RIIa to form PKAI and PKAII holoenzymes. Moreover, Anti-Cb2 immunoprecipitation revealed that Ca1 coimmunoprecipitates with Cb2. Addition of 8-CPT- cAMP which disrupts the PKA holoenzyme, released Ca1 but not Cb2 from the Anti-Cb2 precipitate, indicating that Cb2 and C a1 form part of the same holoenzyme. Our results demonstrate for the first time that various C sub- units may colocate on the same PKA holoenzyme to form novel cAMP- responsive enzymes that may mediate specific effects of cAMP. Abbreviations C, catalytic subunit of PKA; FITC, fluoresceinisothiocyanate; PKA, protein kinase A; PKI, protein kinase inhibitor; PLC-c1 ⁄ 2, phospholipase C c1 ⁄ 2; PVDF, poly(vinylidene difluoride); R, regulatory subunit of PKA; TRITC, tetramethyl-rhodamineisothiocyanate. FEBS Journal 272 (2005) 1559–1567 ª 2005 FEBS 1559 (10–20%) as regulatory subunits, and Ca and Cb mRNA [7]. In the present study we demonstrated that T lymphocytes express significant amounts of active Cb2 protein which together with Ca1 associate with both RIa and RIIa to form PKAI and PKAII holo- enzymes in T cells. Results and Discussion Cb2 is expressed in human T cells We have previously shown that human T cells express Ca and Cb mRNA [7] and that human immune tissues such as spleen and thymus express Cb1 and Cb2 mRNA [6]. Total RNA was isolated from human T cells, the T-cell line Jurkat and NT-2 cells, and amplified by RT ⁄ PCR using Cb1 and Cb2-specific primers (Fig. 1A). This demonstrated the presence of Cb1 and Cb2 in all cell types examined. The Cb2 cDNA encodes a protein with an approximate mole- cular mass of 47 kDa [6,8,9]. To investigate if Cb2 protein is expressed in human T cells we used various anti-C antibodies on lysates of resting T cells and Jur- kat cells. As shown in Fig. 1B, three commercially available polyclonal antibodies (anti-PKAacat, anti- PKAbcat and anti-PKAccat) and one monoclonal antibody (anti-Cmono) were immunoreactive to two protein bands of 40 and 47 kDa, respectively (Fig. 1B). All of these antibodies were raised against regions in the C subunit that are conserved and therefore do not differ significantly between the C isoforms (Fig. 1C). Based on this we suggested that both the 47 and 40 kDa forms represented C subunit variants. It has been well documented that the C subunits Ca1 and Cb1 are expressed as 40 kDa proteins [10]. According to the immunoreactivity and theoretical molecular mass the 47 kDa protein may therefore correspond to the Cb2 splice variant. To confirm this, we generated two different antibodies: anti-Cb2(SNO103) was raised against peptide sequences found in the splice variant- specific part of Cb2 and should therefore only react with this isoform; anti-Cb(SNO157) was generated by immunizing a rabbit using a peptide common to all Cb splice variants, but with only weak homology to the Ca-sequence. This antiserum should therefore in the- ory recognize all Cb splice variants and not cross-react with Ca. (Fig. 1C and Experimental procedures). Anti- Cb2(SNO103) recognized the 47 kDa protein band in T cells and Jurkat cells, while anti-Cb(SNO157) recog- nized a 47 kDa band in T cells and Jurkat cells and a 40 kDa protein band in Jurkat cells. As no 40 kDa band could be detected using the anti-Cb(SNO157) in A C T-cell Jurkat NT2 B Fig. 1. Identification of PKA catalytic subunits in human T cells. (A) Human T cells express both Cb1andCb2 mRNA. Total RNA isolated from human T-cells, the T cell line Jurkat and human NT-2 cells were reverse-transcribed (+) and amplified using primers specific for the Cb1 mRNA (upper panel) and Cb2 mRNA (lower panel). Parallel reactions were performed without reverse transcriptase (–) to demonstrate specificity. The amplified fragments were separated by agarose gel-electrophoresis. Both Cb1andCb2 were detected in all three cell types tested, but a significantly weaker signal of Cb1 was detected in T cells, while a significantly weaker signal of Cb2 was detected in NT-2 cells. (B) Lysates of human peripheral blood T cells (T) and the Jurkat T cells (J) were subjected to SDS ⁄ PAGE in 12.5% gels, transferred to PVDF membranes and immunoblotted using three commercially available polyclonal antibodies: anti-PKAacat (i), anti-PKAccat (ii) and anti- PKAbcat (iii), and a commercial monoclonal antibody, anti-Cmono (iv). These antibodies all recognized several different protein bands, but two bands of 40 and 47 kDa were common to all. The Cb2-specific antibody anti-Cb2(SNO103) (v) recognized the 47 kDa band in T cells. A Cb-specific antibody anti-Cb(SNO157) (vi) recognized the 47 kDa band in T-cells, and the 40- and 47 kDa protein bands in Jurkat cells. (C) Lin- ear depiction of Ca1, Cb1 and Cb2 amino acid sequence and the localization of the domains ⁄ antigens used to generate the different anti-C antibodies used in (B). Novel PKA holoenzymes in human T lymphocytes S. Ørstavik et al. 1560 FEBS Journal 272 (2005) 1559–1567 ª 2005 FEBS T cells, it suggested that Cb1 is expressed at nondetect- able levels in human T cells applying the panel of anti- bodies used here (Table 1). This is consistent with the fact that a significantly weaker Cb1 signal was ampli- fied by RT⁄ PCR from T cells when compared to the signals amplified from Jurkat RNA. Based on this, we concluded that the two major isoforms of the PKA C subunit in T cells are Ca1 (40 kDa) and Cb2 (47 kDa), respectively. Cb2 cDNA encodes an active kinase recognized specifically by anti-Cb2 Previously it has been demonstrated that a cDNA encoding the bovine Cb2 was not active when trans- fected into CHO 10260 cells [9]. This prompted us to express the human Cb2 splice variant in eukaryotic cells. Expression vectors encoding native full-length Ca1 (pEFDEST51Ca1) and Cb2 (pEFDEST51Cb2) under a mammalian promoter were constructed (see Experimental procedures) and transiently transfected into 293T cells. Post-transfection the cells were lysed, subjected to SDS⁄ PAGE in 12.5% gels and immuno- blotted using anti-Cmono. Fig. 2A (upper panel) depicts that transfection with pEFDEST51Ca1 yielded a 40 kDa anti-Cmono immunoreactive protein while transfection using pEFDEST51Cb2 resulted in a 47 kDa immunoreactive protein. The mock-transfected cells expressed only a 40 kDa C. This demonstrated that human Cb2 cDNA encodes a 47 kDa protein which is immunoreactive to anti-Cmono. To verify the specificity of the anti-Cb2(SNO103) antibody the ly- sates were immunoblotted using the anti-Cb2(SNO103) antibody, as shown in Fig. 2A (middle panel). The anti-Cb2 antibody recognized a 47 kDa band only pre- sent in the Cb2-transfected cells and not in the Ca1or mock transfected cells. This demonstrated the specifici- ty of anti-Cb2(SNO103) in immunoblots because the 40 kDa protein band of cells overexpressing Ca1 and the 40 kDa protein band of endogenous C were not detected. Furthermore, by monitoring PKA-specific kinase activity of the various cell lysates using Kemptide as a substrate a 3.9- and 3.4-fold higher activ- ity was revealed in the extracts of Ca1 and Cb2 trans- fected cells, respectively, as compared to the mock transfected cells. This demonstrated that human Cb2 cDNA encodes an active protein kinase capable of phosphorylating a typical PKA substrate. Next we immunoprecipitated from the Ca1, Cb 2 and mock transfected cell lysates using anti-Cb2(SNO103) in the presence of 1 mm 8-CPT-cAMP. The cAMP analogue was included to avoid potential binding of non-Cb2 C-subuints to the R-subunits. The resulting precipitates were analysed by SDS ⁄ PAGE and immunoblotting using anti-Cmono (Fig. 2B) and a PKA-specific kinase assay. Anti-Cb2(SNO103) immunoprecipitated a 47 kDa protein only in the Cb2 transfected cells which also demonstrated the specificity of anti-Cb2(SNO103) in immunoprecipitation assays. The kinase activity in the precipitate from Cb2 transfected cells were 15-fold higher than the activity in the precipitate from the Ca1 transfected cells demonstrating that the Cb2 antibody in combination with PKA kinase assay could be used to detect specifically Cb2 activity. To finally conclude that anti-Cb2(SNO103) is specific for Cb2, the Cb2 and mock transfected 293T cells were subjected to immuno- fluorescence. We used anti-Cb2(SNO103) and fluoresce- ine isothiocyanate (FITC)-conjugated secondary antibody to detect Cb2 (Fig. 2C, upper and lower left panels) and anti-c-tubulin and TRITC-conjugated sec- ondary antibody to detect the centrosome (Fig. 2C, upper and lower middle panels). This demonstrated that cells transfected with Cb2 have increased levels of pro- teins immunoreactive to anti-Cb2(SNO103). In addi- tion, this revealed that Cb2 was distributed in the cytosol as well as enriched in the centrosome as judged by the anti-c tubulin staining when merged with the anti-Cb2 staining and compared to mock transfected cells (Fig. 2C, upper and lower right panels). Taken together our results from immunoblotting, immunopre- cipitation, immunofluorescence in addition to measur- ing enzyme activity, enable us to conclude that anti- Cb2(SNO103) specifically recognizes Cb2, and that Cb2 cDNA encode an active protein kinase. Table 1. Antibodies used to identify PKA subunits in T cells at the protein level. Name Source Antigen Specificity Species Anti-PKAacat Santa-Cruz biotechnology C-terminal peptide Ca Cross reacts with Cb Rabbit Anti-PKAbcat Santa-Cruz biotechnology C-terminal peptide Cb Cross reacts with Ca and Rabbit Anti-PKAccat Santa-Cruz biotechnology C-terminal peptide Cc Cross reacts with Ca and Cb Rabbit Anti-Cmono BD bioscience Ca protein Cross reacts with Cb Mouse Anti Cb2(SNO103) Custom made N-terminal part of Cb2 No cross-reaction with Ca1orCb1 Rabbit Anti Cb(SNO157) Custom made Cb peptide Recognizes Cb1andCb2, no cross-reaction with Ca Rabbit S. Ørstavik et al. Novel PKA holoenzymes in human T lymphocytes FEBS Journal 272 (2005) 1559–1567 ª 2005 FEBS 1561 Cb2 in T cells associates with RIa and RIIa Human T cells express the R subunits, RIa and RIIa [7]. RIa is considered the major R subunit in T cells making up more than 80% of the total R subunit activity implying that RIIa constitutes only 10–20% of the activity. To compare the subcellular localization of Cb2, RIa and RIIa, immunofluorescence was per- formed using anti-Cb2(SNO103) in combination with anti-RIa (Fig. 3A, top panel) and anti-RIIa (Fig. 3A, lower panel). As previously described [11] the RIa is located at the membrane ⁄ cytosolic area, while the RIIa A 32 ±5 474 ±179 10 ±6 50 37 Mw Cβ2 Catalytic activity (pmol ATP/mL/min) Anti-Cmono Transfection: Cβ2Cα1 Mock B Anti-Cβ2 Merge Merge Anti-γ-tubulin Anti-Cβ2 Anti-γ-tubulin Transfection Cβ2 Mock C Fig. 2. The human Cb2 cDNA encodes an active protein kinase and the Cb2 peptide antibody is specific for human Cb2. (A) Human 293T cells were transiently transfected using plasmids encoding Ca1(Ca1) and Cb2(Cb2) or the cells were mock transfected (Mock). Cells were lysed and protein extracts subjected to SDS ⁄ PAGE, transferred to PVDF membranes and immunoblotted using anti-Cmono (anti-Cmono, upper panel) and anti-Cb2(SNO103) (middle panel). Lysates were also analysed for PKA-specific kinase activity (lower panel). (B) The lysates from transfected 293T cells were subjected to immunoprecipitation using anti-Cb2, followed by SDS ⁄ PAGE transferred to PVDF membranes and immunoblotted using anti-Cmono (upper panel, B). The same immunoprecipitates were analysed for PKA-specific kinase activity (lower panel, B). (C) 293T cells were either transfected with C b 2 (upper panels, Cb2) or mock transfected (lower panels, Mock) and subjected to immunofluorescence (IF) using anti-Cb2 (SNO103) (left upper and lower panels) and anti-c-tubulin (middle upper and lower panels). The ima- ges were merged (right upper and lower panels) and showed colocalization of Cb2andc-tubulin in Cb2 transfected cells. Novel PKA holoenzymes in human T lymphocytes S. Ørstavik et al. 1562 FEBS Journal 272 (2005) 1559–1567 ª 2005 FEBS is concentrated around the Golgi–centrosomal area. The Cb2 staining demonstrated that Cb2 was colocal- ized with both RIa and RIIa. Based on this, we asked if Cb2 is associated with both RIa and RIIa by anti- Cb2(SNO103) immunoprecipitation of T-cell extracts. After washing, the precipitates were extracted with (+) or without (–) 1 mm 8-CPT-cAMP. In this modified immunoprecipitation procedure the R subunits associ- ated with the antibody-immobilized Cb2 isoform would remain in the pellet in the absence of 8-CPT-cAMP while they would be released to the supernatant in the presence of 8-CPT-cAMP. The precipitates and supern- atants were analysed by SDS ⁄ PAGE and immunoblot- ting using anti-RIIa and anti-RIa (Fig. 3B, upper panel and lower panels). This demonstrated that both RIa and RIIa are immunoprecipitated by anti- Cb2(SNO103). To confirm this interaction, the same lysates were immunoprecipitated with anti-RIa and anti-RIIa. The precipitates were extracted with 8-CPT- cAMP as described in Fig. 3B. The resulting samples were subjected to immunoblotting and incubated with anti-Cmono (Fig. 3C) or anti-PKAbcat (Fig. 3D). Immunoreactive bands of 40 and 47 kDa which were released by 8-CPT-cAMP were detected in both immu- noprecipitates implying that Ca1 and Cb2 are both asso- ciated with RIa and RIIa in human T cells. This Anti-Cβ2 Anti-RIIα Merge Merge Anti-RIα Anti-Cβ2 A Anti-Cβ2IP: cAMP - IR IgG PS SPP ++ 50 50 Anti-RIIαIP: cAMP - IR IgG P -+ 50 SPSPS +++ RIα Cα1 RIIα Cβ2 B C Anti-RIIαAnti-RIα Anti-Cmono Anti-RIαIP: cAMP ++ IR IgG PPSS -+ 50 S + Cα1 Cβ2 D Anti-PKAβcat Fig. 3. Cb2 colocalizes and associates with both RIa and RIIa to form PKAI and PKAII in T cells. (A) Confocal laser immunofluorescence of human T cells using anti-Cb2(SNO103) in combination with anti-RIa and anti-RIIa. Human T cells were incubated with anti-Cb2 and FITC-con- jugated secondary antibody (green, upper and lower left panels), anti-RIa and TRITC secondary antibody (red, upper middle panel) and anti- RIIa and TRITC secondary antibody (red, lower middle panel). Right upper and lower panels show merges of Cb2 and RIa and Cb2 and RIIa, respectively. (B) Human T-cell lysates were immunoprecipitated using anti-C b2(SNO103). The precipitated proteins were washed three times, then extracted using either buffer with (+) or without (–) 8-CPT-cAMP. Irrelevant rabbit IgG was used as a control (IR IgG). The result- ing pellets (P) and supernatants (S) were subjected to SDS ⁄ PAGE and immunoblotted using anti-RIIa (upper) or anti-RIa (lower). (C) Human T-cell lysates were immunoprecipitated using anti-RII a, washed and subsequently extracted using either buffer (–) or buffer containing 8-CPT-cAMP (+). Irrelevant rabbit IgG was used as a control (IR IgG). The resulting pellets (P) and supernatants (S) were subjected to SDS ⁄ PAGE and immunoblotted using monoclonal anti-C. (D) Human T-cell extracts were immunoprecipitated using anti-RIa. The precipitate was extracted first with buffer, then with buffer containing 8-CPT-cAMP. Irrelevant mouse IgG (IR IgG) was used as a control. The resulting samples were subjected to SDS ⁄ PAGE and immunoblotted using anti-PKAbcat. S. Ørstavik et al. Novel PKA holoenzymes in human T lymphocytes FEBS Journal 272 (2005) 1559–1567 ª 2005 FEBS 1563 observation prompted us to ask whether Ca1 and Cb2 may be associated on the same holoenzyme. Cb2 and Ca1 may form part of the same holoenzyme Lysates of human T-cells were immunoprecipitated using anti-Cb2(SNO103), and the resulting precipitates were extracted with (+) or without (–) 8-CPT-cAMP and the resulting samples analysed by SDS ⁄ PAGE and immunoblotting using anti-Cmono. In the absence of 8-CPT-cAMP anti-Cb2(SNO103) precipitated both a 47- and a 40 kDa C. When the precipitate was extrac- ted with 8-CPT-cAMP, the 47 kDa Cb2 remained in the precipitate while the 40 kDa Ca1 was released into the extract (Fig. 4A). This demonstrated that Cb2 was directly immunoprecipitated using anti-Cb2(SNO103), while immunoprecipitation of Ca1 was dependent on an intact R–C interaction, thereby demonstrating association of Cb2toCa1 through an R subunit. This is consistent with what was demonstrated in Fig. 2B, that anti-Cb2(SNO103) specifically immunoprecipitates free Cb2 and not free Ca1. The most likely explan- ation for these results is the presence of holoenzymes containing both Ca1 and Cb2. However, the coimmu- noprecipitation could also be the result of pull-down of larger cell structures, protein clusters or A-kinase anchoring proteins that have the ability to associate with more than one PKA holoenzyme at the time. If so, anti-Cb2-dependent pull-downs of holoenzymes both made up of R subunits associated with two Cb2 or two Ca1 subunits may occur. We find this explanation less likely, as coimmunoprecipitation was observed also in the presence of RIPA-buffer (contain- ing 0.1% SDS). In addition, pull-down was also dem- onstrated in the presence of Ht-31, a peptide known to disrupt the R to AKAP interaction (results not shown). Finally, the precipitates were analysed for PKA-specific kinase activity, as shown in Fig. 4B. This demonstrated that precipitates extracted with 8-CPT- cAMP contained cAMP-inducible PKA activity that could be inhibited by protein kinase inhibitor (PKI). In the presence of cAMP approximately one-third of the PKI-inhibited activity was released into the extract, indi- cating that the precipitated Cb2 colocalized with Ca1on RIa and RIIa in a ratio of 2 : 1. Of the total PKA kin- ase activity,  5% could be immunoprecipitated using anti-Cb2(SNO103) (data not shown). However, this im- munoprecipitation was not complete, as flow-through still contained Cb2 as judged by immunoblot analysis and only 20–30% of Cb2 activity in Cb2-transfected 293T cells was precipitated. Taken together it is there- fore reasonable to assume that Cb2 activity may consti- tute more than 20% of total PKA activity in T-cells. It has been demonstrated that Ca and Cb when combined with RIIa will form PKA holoenzymes (RIIa 2 Ca1 2 and RIIa 2 Cb1 2 ) with different relative association constants (Ka) for cAMP [12]. This implies that C subunit isoforms influence R subunit cAMP- binding features which may have biological implica- tions for cAMP effects conveyed by PKA in vivo. Furthermore, it has been shown that cAMP is formed and degraded during perturbation of the T-cell antigen receptor complex in conjunction with the anti-CD28 marker [7,11,13,14]. During this process, PKAI is translocated to the antigen receptor complex and acti- vated to phosphorylate several substrates important for regulating antigen-dependent activation of T cells to proliferation and clonal expansion. These substrates include PLC-c1 ⁄ 2 [15], p50 csk [16] and Raf-1 [17] in the early phase of the stimulatory process and regula- tion of interleukin-2 production through phosphoryla- tion of the nuclear factor of activated T cells [18] and nuclear factor jB [19]. The fact that human T cells express two distinct C subunits (Ca1 and Cb2) which may be associate on the same R subunits RIa and RIIa suggest the existence of novel PKA holoenzymes with unique properties in T cells. Such properties may be important for PKA holoenzyme features such as localization and substrate preferences. Anti-Cβ2 IP: cAMP ++ - +-+ IR IgG PPPSSS Anti-Cmono dH 2 O 512 ±225 nd nd nd nd nd cAMP 943 ±96 50.9 ±11 817 ±214 313 ±87.7 50.7 ±35.9 7.5 ±5.0 PKI 35.7 ±2.5 nd 12.7 ±4.09 9.1 ±7.7 nd nd Kinase activity (pmol ATP/mL/min) Cα1 Cβ2 50 37 B A Fig. 4. Colocalization of Cb2 and Ca1 on the same PKA holoen- zyme. (A) Human T cells were lysed and immunoprecipitated using anti-Cb2(SNO103). The precipitates were extracted with (+) or with- out (–) 8-CPT-cAMP and the pellet (P) and supernatants (S) analysed for immunoreactive C subunits by SDS ⁄ PAGE and immu- noblotting using monoclonal anti-C. Immunoprecipitation using irre- levant rabbit IgG (IR IgG) was used as a control. (B) Analysis of kinase activity in immunoprecipitates from (A) using a PKA-specific assay. Cyclic AMP (5 lm) was added to samples not extracted with 8-CPT-cAMP, and PKI was included to detect background kinase activity. Novel PKA holoenzymes in human T lymphocytes S. Ørstavik et al. 1564 FEBS Journal 272 (2005) 1559–1567 ª 2005 FEBS Experimental procedures Antibodies In total, six different anti-C Igs were used (Table 1). These comprised three different rabbit polyclonal commercial anti-C Igs with limited isoform specificity (anti-PKAacat, anti-PKAbcat and anti-PKAccat; catalogue numbers sc903, sc904, sc905; Santa Cruz Biotechnology, Santa Cruz, CA, USA); a monoclonal anti-C antibody (anti-Cmono; cata- logue number 610980, BD Biosciences); a rabbit was immunized using two Cb2-specific peptides, NH 2 -MAY REPPCNQYTGTTTALQ-CONH 2 and NH 2 -CFHRHSKG TAHDQKTALEND-CONH 2 generating a splice-variant- specific antibody designated anti-Cb2(SNO103; a Cb-speci- fic antibody (expected to recognize all Cb splice variants but not to cross-react with Ca) was generated by immun- izing a rabbit using the synthetic peptide NH 2 -QNNA GLEDFERK-CONH 2 and designated as anti-Cb(SNO157). Peptide synthesis and rabbit immunizations were performed by Eurogentec SA (Seraing, Belgium). IgG was purified from anti-Cb2(SNO103) anti-Cb(SNO157) antibodies using protein A sepharose (Amersham Biosciences, Oslo, Nor- way, catalogue number 17-0780-01) as described by the manufacturer. A previously described mouse monoclonal anti-RIa antibody [7] was used for immunoprecipitation and immunoblotting of RIa. For detection of RIIa by im- munoblotting, a mouse mAb (anti-RIIa; catalogue number 612243; BD Biosciences, Erembodegem, Belgium) was used, and for immunoprecipitation of RIIa a rabbit anti-peptide Ig previously described was used [20]. Anti-c-tubulin, mouse monoclonal IgG (Sigma Aldrich, Oslo, Norway, catalogue number T6557) was used for labelling centro- somes. Purification of T cells Human T cells were purified from healthy blood donors using negative selection. Written approval was obtained from all donors of blood for use in research. Briefly, mono- nuclear cells were isolated from human blood (Ullevaal University Hospital Blood Centre, Oslo, Norway) using density gradient centrifugation (Lymphoprep; Nycomed, Oslo, Norway). The cells were enriched for T cells by neg- ative selection using anti-CD14 and anti-CD19 Ig-coated magnetic beads (Dynal, Oslo, Norway). Routinely, anti- CD3 labelling demonstrated > 90% CD3 positive cells by flow cytometry. Construction of expression vectors and culture and transfection of 293T cells Human cDNAs encoding full-length Ca1 and Cb2 were amplified from NT-2 cell mRNA using the Promega Reverse Transcription system and the Pfu-ultra amplifica- tion system (Stratagene, La Jolla, CA, USA) as described by the manufacturers. The amplified products were cloned into the mammalian expression vector pEF-DEST51 (Invi- trogen, catalogue number 12285-011), and verified to encode full-length native Ca1 and Cb2 by sequencing (Medigenomix Gmbh, Martinsried, Germany). The human kidney 293T cells were grown in RPMI-1640 medium sup- plemented with 5% fetal calf serum. Semi-confluent cells were transfected using LipofectAMINE2000 (Invitrogen, Carlsbad, CA, USA) as described by the manufacturer. Lysis of cells, immunoprecipitation, immunoblotting and PKA enzymatic assay Cells were lysed either in RIPA buffer [10 mm Tris ⁄ HCl pH 7.5, 1 mm EDTA, 1% (v ⁄ v) Triton X-100, 0.1% (w ⁄ v) SDS, 0.1% (w ⁄ v) Na-deoxycholate and 100 mm NaCl] con- taining 1 mm dithiothreitol, 1 mm phenlymethylsulfonyl fluoride and protease inhibitor cocktail (Roche Diagnostics, Oslo, Norway) for nonenzymatic assays, or in 1% Triton buffer [25 mm Mes, pH 6.5, 100 mm NaCl, 5 mm EDTA, 1.0% (v ⁄ v) Triton X-100 with 1 mm sodium orthovanadate, 1mm phenlymethylsulfonyl fluoride, 10 mm sodium pyro- phosphate, and 50 mm sodium fluoride] for enzymatic assays. Lysates were cleared by centrifugation at 15000 g, 30 min, 4 °C, and subsequently incubated with primary antibody [anti-Cb2(SNO103) 320 lgÆmL )1 , mouse anti-RIa 2.5 lgÆmL )1 , rabbit anti-RIIa serum diluted 1 : 100], for 2 h to overnight. Antibody–antigen complexes were precipi- tated using either Dynabeads protein G (Dynal, catalogue number 100.04), anti-mouse agarose beads or anti-rabbit agarose beads (Sigma, catalogue number A6531, A1027), washed three times using appropriate buffer and extracted with buffer in the presence or absence of 1 mm 8-CPT- cAMP. For immunoblotting, proteins were separated by SDS ⁄ PAGE and transferred to poly(vinylidene difluoride) (PVDF) membranes by electroblotting. Membranes were blocked in 5% (w ⁄ v) skimmed milk powder in Tris-buffered saline containing 0.1% (v ⁄ v) Tween-20 (TBST) for 1 h at room temperature, and then incubated for 1 h at room temperature or overnight at 4 °C with the appropriate pri- mary antibodies diluted in TBST. Membranes were washed for about 1 h in TBST and further incubated with horse- radish peroxidase-conjugated secondary antibodies (MP Biomedicals, Irvine, CA, USA, catalogue number 55689, 55563). Membranes were washed and finally developed using SuperSignalÒ West Pico Chemiluminescent (Pierce Biotechnology, Rockford, IL, USA). cAMP-dependent pro- tein kinase activity was determined as described previously [21], using either untreated lysates or immunoprecipitates. Indirect immunofluorescence Resting human T cells were allowed to attach to poly(l- lysine) coated cover slips for 30 min at room temperature, S. Ørstavik et al. Novel PKA holoenzymes in human T lymphocytes FEBS Journal 272 (2005) 1559–1567 ª 2005 FEBS 1565 followed by fixation in 3% (v ⁄ v) paraformaldehyde. Cells were permeabilized using 0.1% (v ⁄ v) Triton X-100 in NaCl ⁄ P i (PBST), followed by blocking with 2% (w ⁄ v) BSA in PBST. Cells were incubated with primary antibody in PBST ⁄ BSA for 30 min, washed three times in PBST before incubation with fluorochrome-conjugated secondary anti- bodies for 30 min; FITC-conjugated goat antirabbit or Tetramethylrhodamin-isothiocyanate-conjugated goat anti- mouse (Sigma, catalogue number F0382, T5393) diluted 1 : 500 in PBST ⁄ BSA. Finally, cells were washed four time for 5 min in PBST–BSA and the samples were mounted using the Dako fluorescent mounting medium (Dakocytomation, Oslo, Norway; catalogue number S3023). Cells were exam- ined with a Nikon Labophot microscope (Nikon Instruments Europe, Badhoevedorp, Netherlands) equipped with an epifluorescence attachment and a Bio-Rad (Bio-Rad Labora- tories Ltd, Hemel Hempstead, UK) MRC 600 confocal laser scan unit with a krypton ⁄ argon laser, a K1 double dichroic excitation filter block, and a K2 dichroic emission filter block (Bio-Rad). 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