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Identification and Characterization of Myeloid Translocation Gene 16b as a Novel A Kinase Anchoring Protein in T Lymphocytes¹

Robynn V. Schillace^*,, Sarah F. Andrews^*, Greg A. Liberty^*, Michael P. Davey^*, and Daniel W. Carr^2,*,

^* Veterans Affairs Medical Center and Department of Medicine, Oregon Health and Sciences University, Portland, OR 97201

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Increased levels of intracellular cAMP inhibit T cell activation and proliferation. One mechanism is via activation of the cAMP-dependent protein kinase (PKA). PKA is a broad specificity serine/threonine kinase whose fidelity in signaling is maintained through interactions with A kinase anchoring proteins (AKAPs). AKAPs are adaptor/scaffolding molecules that convey spatial and temporal localization to PKA and other signaling molecules. To determine whether T lymphocytes contain AKAPs that could influence the inflammatory response, PBMCs and Jurkat cells were analyzed for the presence of AKAPs. RII overlay and cAMP pull down assays detected at least six AKAPs. Western blot analyses identified four known AKAPs: AKAP79, AKAP95, AKAP149, and WAVE. Screening of a PMA-stimulated Jurkat cell library identified two additional known AKAPs, AKAP220 and AKAP-KL, and one novel AKAP, myeloid translocation gene 16 (MTG16b). Mutational analysis identified the RII binding domain in MTG16b as residues 399–420, and coimmunoprecipitation assays provide strong evidence that MTG16b is an AKAP in vivo. Immunofluorescence and confocal microscopy illustrate distinct subcellular locations of AKAP79, AKAP95, and AKAP149 and suggest colocalization of MTG and RII in the Golgi. These experiments represent the first report of AKAPs in T cells and suggest that MTG16b is a novel AKAP that targets PKA to the Golgi of T lymphocytes.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
For over 30 years cAMP has been known to be a potent inhibitor of T cell activation (1). Transcription factors NFAT and NF-B exhibit decreased activity in the presence of cAMP, resulting in reduced levels of IL-2 transcription (2, 3). The expression of several cell surface molecules, such as CD69, ICAM-1, and LFA, is also diminished, contributing to the ensuing absence of cell proliferation and inflammation (4, 5). Thus, in understanding the mechanism by which cAMP inhibits T cell activation we may learn how to regulate signaling pathways involved in the inflammatory response.

One of the most well-studied effectors of cAMP is the cAMP-dependent protein kinase (PKA)³ (6). PKA is a ubiquitously expressed, broad specificity Ser/Thr protein kinase. The catalytic activity of PKA is controlled via interactions between two catalytic subunits and a dimer of regulatory subunits. When bound to the regulatory subunits, the catalytic subunits are inactive. Binding of cAMP by the regulatory subunits initiates release and thereby activation of the catalytic subunits. PKA has four regulatory subunit isoforms: RI and , and RII and . PKA containing RI isoforms is referred to as type I PKA, while PKA with RII isoforms is type II PKA.

The spatial and temporal regulation of type II PKA activity is believed to occur through interactions with A kinase anchoring proteins, (AKAPs) (7). AKAPs have been shown to localize PKA near cellular substrates, facilitating signal transduction at these sites. The compartmentalized pools of PKA are maintained within the vicinity of activating elements, such as G proteins and transmembrane receptors, and in close proximity to substrates such as ion channels, mitochondria, cytoskeletal components, and cytoplasmic enzymes (8, 9, 10). This molecular organization ensures selectivity of the cAMP response in mammalian cells. Over 40 AKAPs have been identified to date, but as yet none has been identified in T lymphocytes.

In an effort to explore the mechanism of cAMP-mediated inhibition of T cell activation, we have begun to identify AKAPs in T cells. In this study, we report the presence of several AKAPs in T cells. Using Western blot analysis and library screening, we identify six known AKAPs and one novel AKAP, myeloid translocation gene 16 (MTG16b). The Western blot analyses were used to identify AKAP79, AKAP149, and WAVE. AKAP79 is one of the most well-studied AKAPs. The association of AKAP79 with calcineurin and regulation of NFAT activity (11) makes its identification in T cells particularly interesting. AKAP149 is one member of a family of AKAPs generated by alternative splicing (12). These family members are able to interact with both the RI and RII regulatory subunits of PKA (13). WAVE, which stands for Wiskott-Aldrich verpolin domain, is a member of the Wiskott-Aldrich syndrome family of proteins (14). WAVE has recently been identified and characterized as an AKAP with the ability to bind the Abelson tyrosine kinase (15). Immunofluorescent and biochemical experiments characterize the known AKAPs in T cells.

The novel AKAP, MTG16b, was identified through a library screen for RII interacting proteins. MTG16b was originally identified in patients with acute myeloid leukemia, but the normal physiological function of this protein has not been reported (16). We present experiments characterizing MTG16b as an AKAP in T cells. Molecular and biochemical methods delineate the PKA binding site in MTG16b to residues 399–420. Coimmunoprecipitation provides evidence that MTG16b can function as an AKAP in vivo, and immunofluorescent confocal microscopy experiments suggest that MTG16b functions as an AKAP in Golgi. These data suggest that PKA anchoring may be important in T cell biology and that a novel function of MTG16b may be to target PKA to Golgi.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Isolation of T cell-enriched PBMCs

Human PBMCs were isolated from collar blood obtained from the Portland Red Cross (approval VACARR). Eighty to 100 ml of residual leukocytes were drained from the collar and diluted 1/1 with sterile PBS. PBMCs were separated from whole blood using a Ficoll gradient; 25 ml of diluted blood was layered on top of 12.5 ml of Ficoll-Paque-Plus (Amersham Biosciences, Arlington Heights, IL), and centrifuged for 30 min at 1100 x g at room temperature with no brake. The interface was collected and washed twice with RPMI 1640 and antibiotics (penicillin/streptomycin; Life Technologies, Gaithersburg, MD). Cells were pelleted by centrifugation at 250 x g for 10 min. T lymphocytes were enriched for using adherence separation. Cells were plated in RPMI 1640 and antibiotics for 90 min at 37°C. Suspension cells were collected; washed with RPMI 1640, 1% FCS, and antibiotics; and replated overnight at 37°C. Suspension cells were again collected and washed with RPMI 1640, 10% FCS, and antibiotics. The T cell-enriched population thus obtained was analyzed for purity by flow cytometry and contained >90% CD3-positive cells, <3% CD19-positive cells, and 1% CD14-positive cells. Throughout this report, the T cell-enriched population will be referred to as T cells.

T cells for Western blot analysis and RII overlay

Cells were collected by centrifugation at 250 x g for 10 min, washed twice with ice-cold PBS, and lysed with boiling SDS gel-loading sample buffer. Samples were subjected to SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA). WAVE Abs were provided by the laboratory of Dr. John D. Scott (Howard Hughes Medical Institute, Vollum Institute, Portland, OR). AKAP79, AKAP149, and AKAP95 Abs were purchased from Transduction Laboratories (Lexington, KY). Secondary Abs, goat anti-rabbit, and goat anti-mouse HRP conjugates were purchased from Sigma-Aldrich (St. Louis, MO), and NEN Renaissance chemiluminescence (Boston, MA) was used for detection. RII overlays were performed as previously described (17).

cAMP pull down analysis and immunoprecipitation

For cAMP pull down, extracts were prepared from 150 million T cell-enriched PBMCs by resuspending them in 2 ml of lysis buffer (20 mM HEPES (pH. 7.4), 250 mM sucrose, 0.5 mM EGTA, 0.1% Triton X-100, 1/100 protease inhibitor mixture (Sigma-Aldrich), 1 mM benzamidine, and 10 µg/ml soybean trypsin inhibitor) sonicating 1x 10 bursts at 30% power (Branson Sonifier 450 Branson, Danbury, CT), incubating on ice for 10 min, spinning at 13,000 x g for 5 min at 4°C, and removing the supernatant. One milliliter of extract was incubated with rotation overnight at 4°C with 100 µl of cAMP-agarose beads, which had been washed four times with lysis buffer. Where indicated, 75 mM cAMP was added to the pull down to specifically prevent association of cAMP binding proteins from the beads. The beads were spun at 1000 x g, the supernatant was discarded, and the beads were washed twice with PBS, twice with PBS/0.5 M NaCl, and once more with PBS. The beads were then boiled with SDS-sample buffer, and the resulting proteins in the sample buffer were analyzed with SDS-PAGE and Western blot analysis. For immunoprecipitation, 50 million Jurkat cells were resuspended in 1.5 ml of lysis buffer (1% Triton X-100, 150 mM NaCl, 20 mM Tris-HCl (pH 7.5), 2 mM EDTA, 2 mM EGTA, 1/100 protease inhibitor mixture (Sigma-Aldrich), 1 mM benzamidine, and 10 µg/ml soybean trypsin inhibitor) and incubated with rotation at 4°C for 30 min. After spinning at 13,000 x g for 15 min at 4°C, the supernatant was precleared for 30 min at 4°C with 20 µl of 50% protein A-Sepharose slurry made in lysis buffer. The precleared supernatant (0.5 ml) was incubated for 2 h with rotation at 4°C with 5 µg of human IgG or anti-PKA catalytic subunit (Santa Cruz Biotechnology, Santa Cruz, CA). After adding 20 µl of 50% protein A-Sepharose slurry, the immunoprecipitations were incubated for another 1.5 h at 4°C with rotation. The beads were then spun down, washed twice with cold lysis buffer, and boiled for 5 min with SDS-sample buffer. The resulting eluates were analyzed with SDS-PAGE and Western blot analysis.

Library screen

A PMA-stimulated Jurkat cell cDNA library (ZAP express vector; Stratagene, La Jolla, CA) was titrated and plated following the manufacturer’s protocol. Plaques were transferred to PVDF membranes and incubated with 2 mM isopropyl--D-thiogalactopyranoside to induce protein expression. The filters were then probed with radiolabeled RII, washed, and exposed to x-ray film. Positive plaques were isolated to single plaque purity, and DNA was excised and sequenced using the Big-Dye Terminator Sequencing kit (Applied Biosystems, Foster City, CA).

Cloning and mutagenesis

Full-length MTG16b was isolated from Jurkat cell cDNA by PCR using the forward primer 5'-CCCGAATTCCCCCCAGTGGACAGGAAAG-3' and the reverse primer 5'-AGGTCGACCAGTAGCAGCAGTGACTAATTG, which attach EcoRI and SalI restriction sites, respectively, to the full-length clone. MTG16b was then subcloned into pET30a and pGEX-5X-1, using the EcoRI and SalI sites, for protein expression and pull down analysis. MTG16b fragments 160–344 and 344–432 (amino acid numbering) were generated by restriction enzyme digestion of the full-length clone with SacI. The two fragments were gel purified and subclonec into pET30a. The fragments were sequenced to confirm the orientation of the clone. The DNA was transformed in BL21 DE3 pLysS competent bacteria for protein expression and purification using the His tag and fast protein liquid chromatography (FPLC) as described previously (18). Site-directed mutagenesis was performed on MTG16b in pGEX-5X-1 following the QuickChange 1-day site-directed mutagenesis protocol (Stratagene) with the following primers: valine 408 to proline (forward primer, 5'-TGAAGAGGCCCCGAATGAGGTGAA-3'; reverse primer, 5'-TTCACCTCATTCGGGGCTCTTCA-3'), and valine 408 to alanine (forward primer, 5'-TGAAGAGGCCGCGAATCAGGTGAA-3'; reverse primer, 5'-TTCACCTCATTCGCGGCCTCTTCA-3'). The resulting mutated DNA was transformed into Escherichia coli Super Competent JM109 cells (Promega, Madison, WI) and grown on antibiotic resistant Luria broth agar plates. Colonies were screened by sequence analysis using vector-specific primers. Constructs containing the mutations were sequenced in full to identify any additional unwanted PCR-induced mutations; none were found. DNA was transformed into BL21 cells, grown, and induced for protein expression using isopropyl--D-thiogalactopyranoside. Cellular extracts were separated on 10% SDS-PAGE. Recombinant protein was detected using anti-GST-HRP conjugate (Sigma-Aldrich).

In vitro binding assay of MTG16b and MTG16bpro and the PKA regulatory subunit RII

E. coli transformed with pET30 plasmid expressing full-length MTG16b or MTG16b proline was grown to mid-logarithm phase at 37°C in 1 liter Luria broth medium. Cells were cultured for an additional 2 h at 37°C in the presence of 0.2 mM isopropyl--D-thiogalactopyranoside to induce synthesis of the fusion protein. Crude extracts were prepared by sonicating the bacteria in 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, and a 1/100 dilution of protease inhibitor mixture (Sigma P8340), 10 µg/ml soybean trypsin inhibitor, and 1 mM benzamidine. The extract was centrifuged at 13,000 x g for 10 min at 4°C. The supernatant was incubated for 30 min at room temperature with 500 µl of S protein-agarose beads, followed by extensive washing in PBS to remove nonspecifically bound proteins.

Before the addition of RII, 200 µl of the S-tag MTG16b or MTG16bpro beads were incubated with 1 ml of Blotto for 30 min at room temperature. One microgram of RII was then added to the Blotto and rotated at room temperature for 2 h. After washing twice with PBS, twice with PBS with 0.5 M NaCl, and finally with PBS, proteins were eluted by boiling in SDS gel-loading buffer, and the eluates were separated by 10% SDS-PAGE. Regulatory subunit RII was detected by Western blot analysis using rabbit antisera against RII and secondary goat anti-rabbit HRP conjugate (Sigma-Aldrich). The recombinant expressed MTG16b or MTG16bpro protein was detected with S protein-HRP conjugate (Novagen, Madison, WI).

MTG Ab production and characterization

Ab against the MTG family of proteins was made by Zymed (South San Francisco, CA) using peptide 83–108 Cys-GARQLSKLKRFLTTLQQFGSDISPE as Ag. The cysteine-linked peptide Ab from two different rabbits was affinity purified using sulfolink columns according to the manufacturer’s instructions (Pierce, Rockford, IL). Western blot analysis was conducted on PBMC and Jurkat cell extracts using the Ab at 1/2000 dilution in the presence and absence of 1 µM blocking peptide, using goat anti-rabbit HRP-conjugated secondary Ab and NEN Renaissance chemiluminescence.

Immunofluorescence and confocal microscopy

Jurkat cells obtained from American Type Culture Collection (Manassas, VA), clone E6-1, were centrifuged (5 min at 100 x g) onto poly-L-lysine-coated coverslips. The cells were fixed for 15 min at room temperature with 3.7% paraformaldehyde and 120 mM sucrose in PBS. After washing twice with PBS/0.1% BSA, the cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature. Permeabilized cells were incubated with 10 µg of primary Ab for 1 h at room temperature, washed twice with PBS/0.1% BSA, and incubated with FITC-conjugated or Texas Red-conjugated secondary Ab for 1 h at room temperature (1/100 dilution; Jackson ImmunoResearch Laboratories, West Grove, PA). AKAP79, AKAP149, and AKAP95, and RII Abs were purchased from Transduction Laboratories. MTG Ab is described above and in Fig. 6A. Hoechst stain (Molecular Probes, Eugene, OR) was included with secondary Ab at 5 µg/ml. After rinsing twice with PBS/0.1% BSA and once with distilled water, the coverslips were mounted onto slides using Slowfade mounting medium (Molecular Probes) and sealed with nail polish. Epifluorescence microscopy and digital imaging were conducted on an Olympus IX70 imaging system (New Hyde Park, NY) using a x40 SLCplanFl objective (Olympus, Melville, NY) and Magnafire image acquisition software (Optronics, Goleta, CA). Confocal microscopy images were taken using a Leica TCS-NT confocal imaging system (Deerfield, IL) with a x100 Pl apo oil immersion objective (Zeiss, Thornwood, NY).

View larger version (79K): [in this window] [in a new window]   FIGURE 6. Immunofluorescent and confocal analysis of AKAPs in Jurkat cells. Jurkat cells were centrifuged onto poly-L-lysine-coated coverslips, fixed with 3.7% paraformaldehyde and 120 mM sucrose in PBS, permeabilized with 0.1% Triton X-100 in PBS, blocked with 0.1% BSA in PBS, and stained with 2.5 µg primary Ab in 0.1% BSA in PBS. FITC-conjugated secondary Ab (green) detects the primary Ab, and Hoechst DNA stain (blue) was used to delineate the nucleus. A–D, AKAP95 staining. A and B, Whole cell staining; B is a merged image of Hoechst staining and AKAP95 staining. C, Confocal image of AKAP95 staining. D, Phase contrast image of the cells shown in C. E–H, AKAP79 staining. E and F, Whole cell staining, where F is a merged image of Hoechst staining and AKAP79 staining. G, Confocal image of AKAP79 staining. H, Phase contrast image of the cells shown in G. I–L, AKAP149 staining. I and J, Whole cell staining, where J is a merged image of Hoechst staining and AKAP149 staining. K, Confocal image of AKAP149 staining. L, Phase contrast image of the cells shown in K. Whole cell stain images were recorded with an Olympus IX70 imaging system using a x40 SLCplanFl objective and Magnafile image acquisition software. The confocal images were acquired using a Leica TCS-NT confocal imaging system with a x100 Pl apo oil immersion objective. The scale bar shown in C is the same for G and K; the sections are 1.0 µm thick. Images shown are a representative view of cells from three independent experiments.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

AKAPs target type II PKA in the cell through interactions with the regulatory subunit (RII). These interactions will also occur in vitro in a solid-phase assay known as an RII overlay (19). AKAPs, or cell extracts containing AKAPs, are subjected to SDS-PAGE and transferred to PVDF membranes. The membranes are then incubated with RII that has been phosphorylated with ³²P catalyzed by PKA. Washed membranes are exposed to x-ray film, and the resulting bands represent AKAPs. Fig. 1A illustrates that human peripheral blood-enriched T cells contain several AKAPs detectable by the overlay assay (left panel). AKAPs and RII interact through an amphipathic helix in the AKAP and hydrophobic residues in the N terminus of RII (20, 21). This interaction can be effectively blocked by the addition of a peptide containing an amphipathic helix that competes with the AKAP for binding to RII (22). As illustrated in the right panel of Fig. 1A, addition of the anchoring inhibitor peptide, Ht31, is able to block RII binding to the T cell AKAPs.

View larger version (25K): [in this window] [in a new window]   FIGURE 1. RII overlay and cAMP pull down assays illustrate the presence of AKAPs in T cells. A, T cell-enriched PBMC extracts were separated by SDS-PAGE and transferred to a PVDF membrane. The membranes were then incubated with radiolabeled RII in the absence (left panel) or the presence (right panel) of 10 µM of the anchoring inhibitor peptide Ht31, washed, and exposed to x-ray film. B, Schematic of the isolation of AKAPs with the RII subunit using cAMP agarose. The small circles with a cA in the middle represent cAMP. Exogenous cAMP will block the association of AKAPs with the agarose. C, cAMP pull down from Jurkat cell lysate. Jurkat cell lysates (lane 1) were incubated with cAMP agarose in the absence (lane 2) or presence (lane 3) of 75 mM exogenous cAMP. After extensive washing the agarose was boiled with SDS-sample buffer, and the eluates were subjected to SDS-PAGE, transferred to PVDF membrane, and incubated with radiolabeled RII. Shown is one representative result of more than three independent experiments.

Western blot analysis of AKAPs in T lymphocytes

Although a cAMP pull down will indicate the presence of AKAPs in a cell, the identity of these AKAPs is not revealed by this technique. Furthermore, several AKAPs have been characterized that are not readily detectable by RII overlay of a cell extract. Therefore, we used Western blot analysis to identify four known AKAPs in T cells. AKAP79, AKAP149, and AKAP95 are present as single bands of the same size reported in other tissues (Fig. 2, A–C). A family of WAVE proteins appears to exist in peripheral blood T lymphocytes. In contrast to a single band of 84 kDa detected in rat brain (Fig. 2D, lane 1) (15), PBMC T cell extracts contain four bands of approximate MW 116, 84, 58, and 38 kDa (Fig. 2D, lane 2). To confirm that these proteins also function to localize PKA in T cells, cAMP pull down and Western blot analyses were conducted. AKAP79 and AKAP149 both form complexes with the regulatory subunit of PKA in T cells (Fig. 2, E and F, lane 2). Control experiments demonstrate that the interaction is cAMP specific (Fig. 2, E and F, lane 3).

View larger version (28K): [in this window] [in a new window]   FIGURE 2. Identification of AKAPs by Western blot analysis. T cell-enriched PBMC extracts were separated by SDS-PAGE and transferred to PVDF membrane. The membranes were then probed with Abs for various AKAP proteins. A, AKAP79; B, AKAP149; C, AKAP95. D contains rat brain extract in lane 1 and T cell extract in lane 2 to demonstrate a family of WAVE proteins present in T cells compared with the single band detected in the brain. E and F, cAMP pull down from Jurkat cell lysate. Jurkat cell lysates (lane 1) were incubated with cAMP agarose in the absence (lane 2) or the presence (lane 3) of 75 mM exogenous cAMP. After extensive washing the agarose was boiled with SDS-sample buffer, and the eluates were subjected to SDS-PAGE, transferred to PVDF membrane, and incubated with anti-AKAP79 (E) or anti-AKAP149 (F). Shown is one representative result for each of more than three independent experiments.

To identify additional known AKAPs as well as to look for novel T cell-specific AKAPs, we conducted a screen of an expression library using ³²P-labeled RII as a probe. Preliminary evidence suggested that the expression of several AKAPs was induced upon T cell stimulation; therefore, we chose to screen a PMA-stimulated Jurkat cell library. Nine positive colonies were identified, purified, and sequenced from this screen (Table I). Six of the sequences matched either AKAP95 or AKAP149 (or its splice variants d-AKAP1, and s-AKAP-84) both of which had been previously identified using Western blot analysis. Two of the sequences matched AKAPs, AKAP220 and AKAP-KL, which had not been previously detected.

In addition to identifying four known AKAPs in the T cell library screen, we isolated one novel AKAP, MTG16b. MTG16b was initially characterized from patients with acute myelogenous leukemia (AML) (16). A gene translocation between chromosomes 16 and 21 produces a fusion product with the AML protein. Hence, the protein encoded by the piece of DNA from chromosome 16 was named MTG16, for myeloid translocation gene on chromosome 16. The fusion protein is thought to inhibit gene transcription, inducing hemopoietic cell transformation. Northern blot analysis detects MTG16b in several different tissues, providing evidence that MTG16b exists in the absence of a gene translocation event (16). Two isoforms of this protein have been described, MTG16a and MTG16b, and they appear to be part of a larger family of proteins. MTG8 was the first family member characterized and has since been shown to form a complex with the phosphoprotein MTGR1 (MTG-related protein) (23, 24). The following studies represent an initial characterization of MTG16b as an AKAP in T cells.

Compared with the published sequence of MTG16b, only a fragment of MTG16b had been isolated in the library screen. We used PCR with gene-specific primers to isolate full-length MTG16b from Jurkat cell cDNA and subclone it into the pGEX bacterial expression vector. Full-length recombinant GST-tagged MTG16b binds to RII in an overlay assay (Fig. 3A). Addition of the Ht31 peptide to the RII overlay blocked the RII/MTG16b interaction, indicating that MTG16b binds to RII in a manner similar to other known AKAPs (Fig. 3B).

View larger version (41K): [in this window] [in a new window]   FIGURE 3. In vitro interactions of MTG16b and RII; identification of the RII binding site. Recombinantly expressed MTG16b was subjected to SDS-PAGE and transferred to a PVDF membrane. The membrane was then incubated with radiolabeled RII in the absence (A) or the presence (B) of 10 µM of the anchoring inhibitor peptide Ht31, washed, and exposed to x-ray film. C, Cartoon of fragments designed to identify the RII binding domain in MTG16b. The white box indicates the region predicted by computer alignment as the putative RII binding domain. The fragments were expressed in bacteria, purified via the His tag and FPLC, subjected to SDS-PAGE, and transferred to a PVDF membrane. Duplicate membranes were then incubated with radiolabeled RII (D) or S protein-HRP, which recognizes the protein expression tag (E). F, Sequence alignment of MTG family members illustrating a high degree of conservation with each other and with the conserved residues of the AKAP consensus RII binding site, indicated in bold. Amino acid target of site-directed mutagenesis is depicted with an arrow. Site-directed mutations were introduced using PCR (see Materials and Methods). The mutated constructs were expressed in bacteria, and extracts were subjected to SDS-PAGE and transfer to a PVDF membrane. The membranes were subjected to RII overlay (G) or Western blot analysis using an Ab against the fusion protein tag, anti-GST (H). I and J, Solution interaction of MTG16b and RII. Recombinantly expressed S-tagged full-length MTG16b or MTG16b proline (V408P) was coupled to S protein agarose beads and incubated with RII. After extensive washing the beads were boiled with SDS-sample buffer, subjected to SDS-PAGE, and transferred to a PVDF membrane. RII binding to MTG was detected using a polyclonal anti-RII Ab (I). The amount of MTG or MTG proline coupled to the beads was assayed by detection with S protein HRP conjugate (J). As indicated, lane 1 is the incubation of MTG16b with RII, lane 2 is the incubation of MTG16b proline with RII, and lane 3 is the incubation of S protein beads with RII. The lower band is nonspecific cross-reaction of the Ab. For each panel one representative result of more than three independent experiments is presented.

A computer alignment with the amphipathic helix regions of 20 other AKAP sequences predicted residues 399–420 of MTG16b as a putative RII binding site. Using restriction enzyme digests we generated two constructs in pET30 that would produce fusion proteins with S protein. The expressed proteins were then purified via the His tag using FPLC. One construct was a fragment containing the predicted RII binding site of MTG16b, 344–432, while a second fragment, 160–344, did not contain the predicted RII binding site (Fig. 3C). As illustrated in Fig. 3D, fragment 344–432 binds to RII in the overlay assay, while fragment 160–344 does not bind. Control experiments demonstrated that the proteins were loaded in equal amounts (Fig. 3E). This experiment illustrates that the RII binding site of MTG16b is contained within amino acids 344–432.

Sequence alignment of the MTG family of proteins revealed considerable homology within the putative RII binding region (Fig. 3F). Introduction of site-directed mutations into key positions within the amphipathic helix of an AKAP has been shown to disrupt RII binding (20). We therefore used PCR to introduce either a proline or an alanine into position 408 of MTG16b. As demonstrated in Fig. 3G, both the proline and alanine mutations disrupted MTG16b/RII interactions in an RII overlay assay. Control experiments again demonstrate that equal amounts of each protein were loaded (Fig. 3H). These data suggest that position 408 in MTG16b is critical for mediating an interaction with RII.

MTG16b/RII interact in solution

To provide evidence that MTG16b could form a stable complex with RII in solution, MTG16b was expressed as a fusion with S protein to conduct pull down experiments. MTG16b and RII were able to form a complex in solution (Fig. 3I, lane 1). When the same experiment was repeated with MTG16b containing a proline mutation in the amphipathic helix (V408P), a stable complex could no longer be detected (Fig. 3I, lane 2). The lower band in these lanes was the result of nonspecific Ab binding. Control experiments illustrate the lack of nonspecific binding to the S protein beads (Fig. 3I, lane 3), and that the absence of a complex with MTG16b V408P occurs in the presence of a large excess of the mutated protein (Fig. 3J, lane 2). These results demonstrate that MTG16b and RII are able to interact when both proteins are in their folded state, and that this interaction is mediated via the RII binding domain located between residues 399–420 of MTG16b.

In vivo studies on the interaction of MTG16b and RII

A polyclonal peptide Ab generated against a conserved region of the MTG family of proteins (aa 83–108) detects three bands in Jurkat cell and PBMC T cell extracts (Fig. 4A). Control experiments indicate that recognition of all three bands is specific, as the signal is blocked when the antigenic peptide is included in the Western blot incubation (Fig. 4B). An endogenous complex between MTG and PKA was isolated by coimmunoprecipitation from Jurkat cells, as illustrated in Fig. 4C. A polyclonal PKA catalytic subunit Ab was used in immunoprecipitation from Jurkat cell lysates. The eluted complexes were subjected to SDS-PAGE and probed for MTG, RII, and catalytic subunit by Western blot analysis (Fig. 4C, upper, middle, and lower panels, respectively). Control experiments include immunoprecipitation with nonspecific IgG and nonspecific binding to the protein A-Sepharose (PC). Confocal analysis of Jurkat cells further suggests that MTG16b and RII form a complex in intact cells. Jurkat cells were stained with anti-MTG (green) and anti-RII (red; Fig. 4, D and E, respectively). Overlapping staining of the 0.5-µm sections is shown in Fig. 4F; yellow indicates regions where the MTG and RII staining overlap. Note that only a subset of MTG staining overlaps with the RII staining. Fig. 4G is a combination of the overlapping staining pattern and phase images of the cells. Fig. 4, H and I, are control experiments demonstrating that the MTG staining is eliminated when the antigenic peptide is included in the Ab incubation.

View larger version (67K): [in this window] [in a new window]   FIGURE 4. In vivo interactions of MTG16b and RII. A, Western blot analysis of Jurkat cell (J), and peripheral blood T cell (P) lysates using a peptide Ab generated against amino acid residues 83–108 of MTG16b (Zymed). Cell extract was subjected to SDS-PAGE and transferred to a PVDF membrane. The membrane was probed with a 1/2000 dilution of affinity-purified anti-MTG Ab, followed by incubation with goat anti-rabbit HRP-conjugated secondary Ab and chemiluminescence detection. B, Western blot analysis with 1 µM MTG83–108 peptide included in the primary Ab incubation. J, Jurkat cell lysate; P, peripheral blood T cell lysate. C, Coimmunoprecipitation of MTG with the PKA holoenzyme. Jurkat cell lysates were incubated with anti-PKA catalytic subunit Ab (Santa Cruz Biotechnology) and protein A-Sepharose beads, control IgG and beads, or beads alone (PC). After washing, the beads were boiled with SDS-sample buffer, and the eluted proteins were subjected to SDS-PAGE and Western blot analysis. I, Input lysate; PC, precleared beads; IgG, control; IP, PKA catalytic subunit. The upper panel was probed with anti-MTG, the middle panel was probed with anti-RII, and the lower panel was probed with anti-PKA catalytic subunit Ab. One representative blot of four independent experiments is shown. D–G, Confocal microscopy of Jurkat cells stained with anti-MTG Ab and FITC-conjugated donkey anti-rabbit secondary Ab (D), and anti-RII Ab and Texas Red-conjugated donkey anti-mouse secondary Ab (E). F, Overlay of stained 0.5-µm sections illustrating overlapping staining patterns of MTG and RII. G, Phase contrast image of cell with overlay staining pattern. H, Jurkat cells stained as in D with peptide included in primary Ab incubation step. I, Phase contrast image of these cells. The images were acquired using a Leica TCS-NT confocal imaging system with a x100 Pl apo oil immersion objective. One representative view of cells from more than three independent experiments is shown.

The region of overlap between MTG and RII is suggestive of Golgi staining. The MTG staining pattern seen in Jurkat cells was also seen in peripheral blood T cells (R. Shillace, unpublished observation). To confirm the possibility of Golgi staining, the Jurkat cells were costained for MTG and GM130, a Golgi marker. As seen with RII, there was a distinct region of overlapping staining (Fig. 5A, anti-MTG; B, anti-GM130; C, overlay). The formation of the Golgi complex is known to be brefeldin A sensitive. To test whether MTG staining was also brefeldin A sensitive, the cells were treated with brefeldin A to disrupt the Golgi and stained for MTG. In brefeldin A-treated cells, the MTG staining pattern was clearly disrupted (Fig. 5D). The GM130 staining pattern was not as dramatically disrupted (Fig. 5E), but was significantly different from that in the control cells (Fig. 5B), suggesting that MTG and GM130 may be occupying distinct, yet overlapping, regions of the Golgi. Interestingly, the region of MTG and GM130 overlap is completely disrupted in the brefeldin A-treated cells (Fig. 5F). However, when the cells were treated with nocodazole to disrupt microtubule-associated organization, MTG and GM130 staining patterns were disrupted, but the overlay still showed regions of overlapping staining (Fig. 5G, anti-MTG; H, anti-GM130; I, overlay). Interestingly, the MTG and RII overlap was also brefeldin A sensitive. Fig. 5, J–L, illustrate staining patterns for MTG, RII, and the overlay for this experiment. In comparison, Fig. 5M shows that MTG staining was disrupted when the cells were treated with brefeldin A. The effects of brefeldin A on the RII staining were less obvious; however, a careful examination of the cells revealed that the RII staining around the centrosome was less intense (arrowheads point to centrosomes, Fig. 5N). In addition, the overlapping staining pattern between MTG and RII was disrupted in the presence of brefeldin A (Fig. 5O). Similar to the GM130 staining, when the cells were treated with nocodazole, the organization of MTG staining was disrupted (Fig. 5P); again, this was less obvious for RII staining but still present (Fig. 5Q). Also similar to GM130, the overlapping staining between MTG and RII remained in the absence of the organized structure (Fig. 5R). These data illustrate that MTG and RII have regions of distinct and overlapping staining. The brefeldin A and GM130 staining experiments suggest that the overlapping pattern is associated with the Golgi.

View larger version (42K): [in this window] [in a new window]   FIGURE 5. Overlapping staining patterns are localized to Golgi and are brefeldin A sensitive. Confocal analysis of Jurkat cells treated with DMSO (control), 10 ng/ml brefeldin A, or 10 µM nocodazole. After 1-h incubation at 37°C, the cells were fixed, permeabilized, and stained with anti-MTG and FITC-conjugated donkey anti-rabbit secondary Ab (A, D, G, J, M, and P), either anti-GM130 (Golgi marker; B, E, and H) or anti-RII Ab (K, N, and Q), and Texas Red-conjugated donkey anti-mouse secondary Ab. Arrowheads indicate centrosomes. C, F, I, L, O, and R, Overlay images of the panels above them. The data indicate that MTG staining and overlap with RII are Golgi associated and brefeldin A sensitive. The images were acquired using a Leica TCS-NT confocal imaging system with a x100 Pl apo oil immersion objective. Images shown are a representative view of cells from three independent experiments.

Consistent with a hypothesis in which AKAPs localize PKA to unique subcellular locations, immunofluorescent and confocal analyses illustrate that AKAP95, AKAP79, and AKAP149 also exhibit different subcellular locations in Jurkat cells. Fig. 6 illustrates both whole cell staining (upper six panels) and confocal analysis (lower six panels) of Jurkat cells stained with Abs to AKAP95, AKAP79, and AKAP149. AKAP staining is shown in green, Hoechst DNA stain is blue, and phase contrast images, used to delineate the outline of the cells, are gray scale. Control experiments were conducted to confirm that the staining was not due to secondary Ab alone (data not shown). Although AKAP95 staining overlapped with the DNA stain (Fig. 6, A and B), AKAP79 and AKAP149 were excluded from the nucleus (Fig. 6, E and F, and I and J, respectively). These interpretations were confirmed by confocal analysis of the cells. AKAP95 nuclear staining is presented in C with the phase images of these cells in D. Confocal analysis and phase contrast images of AKAP79 and AKAP149 are shown in G and H, and K and L, respectively. Although overexpression of AKAP79 in many cell types (25), including Jurkat cells (A. L. Asirvatham and D. W. Carr, unpublished observation), is membrane localized, endogenous expression of AKAP79 appears to be mainly cytoplasmic in Jurkat cells (Fig. 6, E–H). This staining pattern is in accordance with the endogenous staining pattern seen in other cell types, and with the idea that AKAP79 may be positioning calcineurin to regulate T cell activation via NFAT activation (see Discussion). The endogenous staining pattern of AKAP149 appears cytoplasmic, but additional experiments will need to be conducted to confirm mitochondrial association.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In an effort to understand the mechanism by which cAMP, a potent inhibitor of T cell activation, affects signal transduction in T lymphocytes, we have begun to characterize the presence of AKAPs in T cells. Using an in vitro method known as an RII overlay we were able to detect the presence of several AKAPs in T cells. Biochemical analyses were used to confirm that the known AKAPs, AKAP79, AKAP95, AKAP149, and WAVE are expressed in T cells and that AKAP79 and AKAP149 form complexes with the RII regulatory subunit of PKA. Library screening identified two additional known AKAPs: AKAP220 and AKAP-KL.

The finding that AKAP79 is expressed in T cells is very exciting in light of research conducted on AKAP79 in other cells types. AKAP79 has the ability to interact with several signaling molecules (26), but it is the interaction of AKAP79 with the protein phosphatase, calcineurin, that is the most interesting with respect to T lymphocyte activation. Calcineurin has been shown to be a key regulator of the immune response through dephosphorylation of NFAT (27). Dephosphorylation of NFAT facilitates its translocation into the nucleus, where it activates transcription of IL-2. AKAP79 has been shown to inhibit calcineurin activity in vitro, and transfection studies in the kidney cell line HEK293 have demonstrated that AKAP79 is able to inhibit calcineurin-dependent dephosphorylation of NFAT, thereby inhibiting NFAT activity in cells (11). Thus, in T cells the localization of calcineurin with AKAP79 may function to inhibit IL-2 transcription, suggesting a role for anchoring of signaling molecules via AKAP79 in regulating T cell activation.

The demonstration that a family of proteins is recognized by the anti-WAVE Ab is also intriguing. WAVE-1 is a member of the Wiskott-Aldrich syndrome protein family of scaffolding proteins that coordinate actin reorganization by coupling Rho-related small molecular weight GTPases to the mobilization of the ARP2/3 complex. WAVE-1 has recently been identified as an AKAP, which also binds to the Abelson tyrosine kinase (15). These studies suggest that the interaction of WAVE-1 with actin may be regulated by interactions with PKA and may be influenced by exposure to growth factors.

AKAP95 was initially identified in rats in association with the nuclear matrix (28). The human homologue has now been cloned and implicated as a targeting protein for the human chromosome-associated protein (29). AKAP95 redistributes from the nuclear matrix to chromatin, recruiting human chromosome-associated protein and condensin to chromosomes and thereby promoting chromosome condensation and resolution.

AKAP220 anchors both PKA and the protein phosphatase, PP1. PP1 activity, like PKA activity, is inhibited when bound to AKAP220, but inhibition of PP1 appears to be regulated by interactions between AKAP220 and PKA (30). AKAP-KL is a family of six isoforms generated as a result of alternative splicing (31). The isoforms are expressed differentially in tissue. In transfected cells the AKAP-KL2A and B isoforms are enriched just below the plasma membrane and interact with and modulate the actin cytoskeleton.

AKAP149 is a member of the s-AKAP84, AKAP121, and d-AKAP-1 family of AKAPs. Although AKAP149 was initially identified as a mitochondria-associated protein, later reports suggest that another function of AKAP149 may be to tether PKA and the type 1 Ser/Thr protein phosphatase, PP1, to the nuclear lamina. These associations were reported to be essential for nuclear reassembly at the end of mitosis (32). In addition, the splice variant of AKAP149, d-AKAP-1, has been demonstrated to anchor both type I and type II isoforms of PKA (13). Skalhegg and Kammer and their respective colleagues (33, 34, 35, 36) have published elegant studies identifying roles for type I PKA in T cell activation, but at this point the identities of the AKAPs involved, if any, are unknown. Their experiments make the discovery of AKAP149 in T cells even more intriguing.

Through the library screen we also identified a novel AKAP, MTG16b and proceeded to characterize the interaction between MTG16b and RII. We have defined the RII binding site in MTG16b to residues 399–420 and demonstrate that MTG16b and RII can interact in solution. Coimmunoprecipitation and confocal microscopy further suggest that MTG16b and RII form a complex in T cells and that this complex is associated with the Golgi. A proposed role for family member MTG8 is in regulating transcription, and the current model for MTG/AML hemopoietic transformation includes active repression of AML-mediated transcriptional activation in addition to interfering with the normal functions of MTG family members (as reviewed in Ref. 37). However, the physiological functions of MTG8 and MTG16b have not been reported. Sequence analysis of MTG family members suggests that all the family members may be AKAPs. Indeed, while this study was being reviewed, Fukuyama et al. (38) reported that MTG8 is also an AKAP in T cells, confirming this theory that MTG16b is one of a family of AKAPs. In agreement with postulated roles for MTG8 and experiments with transfected MTG8, overexpression of MTG16b is nuclear (data not shown), yet the endogenous expression pattern appears to be cytoplasmic and Golgi associated. It will be interesting to investigate the physiological relevance of the PKA/MTG16b interaction, in addition to determining whether the MTG16b/AML fusion proteins retain the ability to bind the regulatory subunit of PKA.

A common theme in the field of signal transduction is the localization of broad specificity signaling molecules to discrete subcellular locations through interactions with scaffolding or targeting proteins (39). It is through these interactions that spatial and temporal regulation of signaling events is believed to occur. We have identified the presence of several known and one novel AKAPs in T lymphocytes and demonstrate different subcellular distributions of these molecules, thereby suggesting that subcellular targeting of PKA in T cells may play a role in mediating the inhibitory effects of cAMP on inflammation.

	Acknowledgments

 
We thank Shayne Bagnall for technical assistance, and Neal Alto and Dario Diviani for intellectual contributions.

	Footnotes

 
¹ This work was supported by a Veterans Affairs Merit Grant (to D.W.C.) and National Institutes of Health Fellowship AI10520 (to R.V.S.).

² Address correspondence and reprint requests to Dr. Daniel W. Carr, Veterans Affairs Medical Center, R&D 8, 3710 SW U.S. Veterans Hospital Road, Portland, OR 97201. E-mail address:carrd{at}ohsu.edu

³ Abbreviations used in this paper: PKA, cAMP-dependent protein kinase; MTG, myeloid translocation gene; AKAP, A kinase anchoring protein; AML, acute myelogenous leukemia; FPLC, fast protein liquid chromatography; PVDF, polyvinylidene difluoride; R, protein kinase A regulatory subunit.

Received for publication September 28, 2001. Accepted for publication December 5, 2001.

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