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The Journal of Immunology, 1999, 162: 2016-2023.
Copyright © 1999 by The American Association of Immunologists

Specific CD3{varepsilon} Association of a Phosphodiesterase 4B Isoform Determines Its Selective Tyrosine Phosphorylation After CD3 Ligation1

Miren L. Baroja*, Lenora B. Cieslinski{ddagger}, Theodore J. Torphy{ddagger}, Ronald L. Wange§ and Joaquín Madrenas2,*,{dagger}

* Transplantation and Immunobiology Group, John P. Robarts Research Institute, and {dagger} Departments of Microbiology and Immunology, and Medicine, University of Western Ontario, London, Ontario, Canada; {ddagger} Division of Pharmacological Sciences, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406; and § Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
cAMP-specific phosphodiesterases (PDE) comprise an extensive family of enzymes that control intracellular levels of cAMP and thus regulate T cell responses. It is not known how the function of these enzymes is altered by TCR engagement. We have examined this issue by studying one of the PDE isozymes (PDE4B). PDE4B RNA and protein were detected in resting PBLs, and the levels of PDE4B protein increased with cell cycling. In peripheral blood T cells, two previously reported PDE4B isoforms could be detected: one was 75–80 kDa (PDE4B1) and the other was 65–67 kDa (PDE4B2). These two isoforms differed in their N-terminal sequence, with the presence of four potential myristylation sites in the PDE4B2 that are absent in PDE4B1. Consequently, only PDE4B2 was found in association with the CD3{varepsilon} chain of the TCR. In addition, although both isoforms were phosphorylated in tyrosines in pervanadate-stimulated T cells, only the TCR-associated PDE4B2 was tyrosine-phosphorylated following CD3 ligation. The kinetics of phosphorylation of TCR-associated PDE4B2 correlated with changes in cAMP levels, suggesting that tyrosine phosphorylation of the TCR-associated PDE4B isoform upon engagement of this receptor may be an important regulatory step in PDE4B function. Our results reveal that selectivity of PDE4B activation can be achieved by differential receptor association and phosphorylation of the alternatively spliced forms of this PDE.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Ligation of the TCR causes an early, transitory increase in cAMP generation that is rapidly followed by a return to baseline levels within minutes of TCR engagement (1, 2). This tight regulation of the intracellular concentration of cAMP is essential for T cell activation since sustained high levels of cAMP correlate with inhibition of T cell responses such as intracellular signaling (3), IL-2 R expression, IL-2 production, and T cell proliferation (4, 5). Therefore, it is logical to assume that TCR-mediated signaling will balance the increase in cAMP levels with activation of negative regulators of cAMP levels. However, it is not known how this occurs.

The levels of intracellular cAMP and cGMP are regulated by a superfamily of enzymes known as phosphodiesterases (PDEs)3 whose main function is to degrade these second messengers into their inactive metabolites, 5' AMP and 5' GMP. These enzymes are classified into at least seven genetically distinct families of PDEs that differ with respect to their substrate specificity, sequence homology, and susceptibility to the action of selective inhibitors (6, 7). Three of these families, PDE3, PDE4, and PDE7, are highly selective for cAMP and are likely to be the most physiologically relevant cAMP-metabolizing enzymes. A complex pattern of PDE isoform expression can be generated by tissue-specific and cell lineage-specific gene expression and by alternative splicing of mRNA for the different types of PDEs (8, 9). All three cAMP-selective isozymes (PDE3, PDE4, and PDE7) are expressed in lymphocytes (10, 11, 12, 13, 14) and could be involved in the regulation of cAMP levels.

Little is known about the distribution and function of specific subtypes and splice variants of PDE3 and PDE4 in T cells. There are at least two genes coding for two subtypes of PDE3, known as PDE3A and PDE3B. Only PDE3B is expressed on T cell clones (15), whereas PDE3A is found on platelets-megakaryocytes (16, 17). Four different PDE4 genes have been reported. They code for four different subtypes of PDE4: PDE4A, PDE4B, PDE4C, and PDE4D. These subtypes are expressed in leukocytes; however, not all of them are expressed in any given cell type (18, 19). RNA coding for PDE4A, PDE4B, and PDE4D has been shown in CD4+ peripheral T cells (11), CD8+ peripheral T cells (20), and T cell clones (15). In contrast, only PDE4A and PDE4D (RNA and protein) have been reported in the Jurkat T cell line (21). An increase in the total PDE4 activity is detected after TCR/CD3 ligation in murine thymocytes (22). However, it is not known how the function of specific PDE4 isoforms upon TCR ligation is regulated and, more importantly, how their activity is coordinated with TCR-mediated signaling.

It has been previously demonstrated that some PDE4 subtypes can be serine-phosphorylated following cell activation (23, 24, 25, 26). For example, protein kinase A (PKA)-dependent phosphorylation of PDE4D3 at serine residues occurs in rat thyroid cells and in a human promonocytic cell line (24, 26). This phosphorylation is associated with an increase in PDE catalytic activity. In addition, an alternatively spliced form of PDE4B (PDE4B2B) can be serine phosphorylated in vitro by mitogen-activated protein kinase (MAPK) (25). The biological relevance of this phosphorylation is not clear. Phosphorylation at tyrosine residues or serine/threonine residues may play an important role in the regulation of PDE activity by inducing conformational changes required for enzymatic activation (26, 27). Alternatively, phosphorylation may determine specific intracellular redistribution of a given PDE isoform and facilitate specific protein-protein interaction.

We hypothesized that the link between TCR-mediated signaling and the associated transient increase in cAMP content could involve PDE regulation in the form of activation-dependent phosphorylation of a TCR-related compartment of PDE. To examine this hypothesis, we looked at changes in the association and phosphorylation of one of the PDE4 subtypes (PDE4B) in PBMC and in cycling T cells following TCR/CD3 ligation. Here, we report that PDE4B protein is expressed in peripheral blood T cells in at least two different variants: one of 75–80 kDa, and another of 65–67 kDa. However, only the 65- to 67-kDa variant is associated with CD3{varepsilon}, and this translates into its unique tyrosine phosphorylation after TCR/CD3 ligation. Changes in phosphorylation of TCR-associated PDE4B correlated with changes in cAMP levels. Our data reveal that the link between TCR-mediated signaling and the resulting down-regulation of cAMP levels may involve selective tyrosine phosphorylation-dependent activation of a TCR-associated PDE pool.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Cells

PBMC were isolated from heparinized blood on Ficoll-Hypaque (Pharmacia Biotech, Uppsala, Sweden) gradient. Cells were resuspended at 1 x 106/ml after three washings in complete culture RPMI 1640 medium containing 2 mM L-glutamine, penicillin (100 U/ml), streptomycin (100 µg/ml), 1 mM sodium pyruvate, 10 mM HEPES, and 10% FBS (Life Technologies, Grand Island, NY). T cell blasts were generated by culturing PBMC with PHA (5 µg/ml) (Sigma, St. Louis, MO) and IL-2 (10 U/ml) (Boehringer Mannheim, Laval, Quebec, Canada) for 72 h at 37°C, 5% CO2. In some experiments, pure T cells were used after monocyte depletion by incubation in plastic petri dishes (Falcon, Becton Dickinson, Franklin Lakes, NJ) at 37°C for 1 h, followed by passing through T cell enrichment columns (R&D Systems, Minneapolis, MN). The resulting population contained >95% CD3+ cells. The U937 promyelocytic cell line and the EBV-transformed B cell line (GM4672) were cultured in complete culture medium at 37°C, 5% CO2.

Reagents

The following mAbs were used in these experiments: 12F6, a mouse IgG2a Ab against the human CD3{varepsilon} chain, kindly provided by Dr. A. Lazarovits (Robarts Research Institute, London, Ontario, Canada) (28); 64.1 (mouse IgG2a) against the human CD3{varepsilon} chain (obtained from Oncogen Science, Uniondale, NY); UCHT-1 (mouse IgG1) and an irrelevant mouse IgG2a mAb (purchased from PharMingen, San Diego, CA); 4G10, a mouse IgG2b mAb against phosphotyrosine (kindly provided by Dr. B. Druker, Oregon Health Sciences University, Portland, OR) (29); 387, a rabbit antiserum to TCR {zeta} chain (kindly provided by Dr. L. E. Samelson, National Institute of Child Health and Human Deveopment, National Institutes of Health, Bethesda, MD); normal rabbit serum as a negative control; and M4-3, a rabbit polyclonal antiserum raised against the peptide DIDIATEDKSPVDT, corresponding to residues 551–564, from the unique carboxyl terminus of PDE4B (30, 31). Pervanadate was prepared by dissolving 100 µl of 0.1 M sodium orthovanadate (Sigma) into 900 µl of double-distilled water and 3.3 µl of 30% hydrogen peroxide and was kept at room temperature for 15 min before use.

Extraction and quantification of cAMP levels was carried out using a commercial ELISA kit (cAMP/low pH immunoassay) according to the manufacturer’s indications (R&D Systems).

T cell stimulation

Nonactivated PBMC or T cell blasts (10 x 106/100 µl) were stimulated with the anti-CD3 mAbs 12F6 or UCHT-1, or with pervanadate for different times at 37°C. Cells were washed in cold PBS containing sodium orthovanadate (0.4 mM) and EDTA (0.4 mM) and lysed in 500 µl 1x lysis buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris (pH 7.6), 5 mM EDTA, 1 mM sodium orthovanadate, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 25 µM p-nitrophenyl-p-guanidinobenzoate) on ice for 30 min. Cell lysates were obtained after removal of cell debris by centrifugation at 14,000 rpm for 10 min at 4°C.

Immunoprecipitations and Western blotting

Detection of PDE4B proteins was performed by SDS-PAGE of T cell lysates (600,000 cell equivalents per group) on a 10% gel transferred to polyvinylidene difluoride membranes, and immunoblotted with the rabbit antiserum against PDE4B. Signal detection was performed by chemiluminescence (Boehringer Mannheim). For blocked Ab experiments, the antiserum against PDE4B was preabsorbed with the peptide (100 µg/ml) used as immunogen. Specific protein immunoprecipitations (from 10 x 106 cells/group) were prepared using Abs against PDE4B, CD3{varepsilon}, or TCR {zeta} as described previously (32). Immunoprecipitates were then immunoblotted for phosphotyrosine or PDE4B. Signal detection was performed by chemiluminescence, and intensity was quantitated using an imaging densitometer (model GS 700, Bio-Rad, Hercules, CA) and the molecular Analyst Software (version 1.0, 1994, Bio-Rad).

Reverse transcription and PCR amplification

RT-PCR (33) was carried out using a kit according to the manufacturer’s instructions (Perkin Elmer, Foster City, CA). First-strand cDNA was generated from 1 µg total RNA using oligo(dT)16 to prime the reverse transcription and was directly amplified by PCR after the addition of specific primers and Amplitaq DNA polymerase. Oligonucleotide primers were as follows (31): PDE4A, 5'-AACAGCCTGAACAACTCTAAC-3' and 3'-TCAGAGTCCACCAAAATAAC-5', defining a 907-bp product containing a XhoII (34) site; PDE4B, 5'-AGCTCATGACCCAGATAAGTG-3' and 3'-CTGTGAGTCCTTCTACCAATA-5', defining a 625-bp product containing a SalI (35, 36) site; PDE4C, 5'-CTTTGCCCAGGTCCTGGCCAGT-3' and 3'-GCGAGGCCCTTGGTCCACAGG-5', defining a 315-bp product containing an AvrII (34) site; PDE4D, 5'-CGGAGATGACTTGATTGTGAC-3' and 3'-CGTGTGGTAAAAAGTCCTTGC-5', defining a 641-bp product containing a StuI (34, 37) site; and PDE7, 5'-ATAATGGACAAGCCAAGTGT-3' and 3'-CGACTTATTTCGGTCGACCT-5', defining a 936-bp product containing a StyI (38) site. Normalization of mRNA was achieved by RT-PCR of a constitutive marker, human glyceraldehyde 3-phosphate dehydrogenase, using a commercially available primer set (Clontech, Palo Alto, CA). For each RNA sample, controls lacking reverse transcriptase were included for all PCR reactions. Reactions were performed with an initial holding step at 95°C for 105 s, followed with 95°C for 15 s (melt) and 60°C for 30 s (anneal-extend) for 35 cycles,and a final holding step at 72°C for 7 min as recommended by Perkin Elmer. Under these conditions, all PCR reactions were on the linear portion of the time/product curve. PCR products, along with m.w. markers (100-bp DNA ladder, Life Technologies), were electrophoresed on 2% agarose gels and visualized by ethidium bromide.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Initially, we investigated the presence of PDE4B mRNA in purified T cells cultured in medium alone or after blast transformation with PHA plus IL-2, to detect possible differences in the expression of PDE4B after induction of cell cycling. We used RT-PCR with PDE4B isoform-specific primers. As previously reported (31), these primers yield a 625-bp PCR product that is digested by SalI into two products of 284 bp and 341 bp. Fig. 1A shows that PDE4B isoform-specific RNA was amplified by RT-PCR in purified resting and blast T cells. DNA contamination was excluded, because these products were not detected in the absence of the reverse transcriptase step. Furthermore, SalI digestion confirmed amplification of only the RNA coding for the correct PDE4B isoform. As positive control, we used RNA from the U937 cell line, for which the expression of PDEs has been extensively characterized in previous reports (24, 30, 39). In the same experiment, we confirmed the expression of other PDE families in PBLs. As shown in Fig. 1B, we detected PDE4A and PDE7 RNA but were unable to detect the expression of PDE4C transcripts. These findings are consistent with previous reports (11, 15, 20, 40). However, in contrast with previous reports in T cell clones (15) or CD4+ peripheral T cells (11, 20), we were not able to detect significant amounts of PDE4D RNA in blast T cells.

Next, we examined PDE4B protein expression in PBMC and blasts by Western blotting of cell lysates using a previously reported PDE4B-specific antiserum (31). As noted by Manning et al. (31) and as shown in Fig. 1C, PDE4B runs as a pair of doublets: a short form of ~65–67 kDa, which corresponds to PDE4B2, and a long form that runs as a broad band ad 75–80 kDa, which corresponds to PDE4B1. These two forms of PDE4B correspond to alternatively spliced RNAs from the same gene (35). Both forms of PDE4B were present in very low amounts in PBMC. PDE4B protein levels did not change after short (10 min) or long (3 h) TCR ligation with an anti-CD3{varepsilon} mAb (data not shown). However, upon cell entry into cycling, the levels of both forms of PDE4B increased significantly. The specificity of these findings was further strengthened by two additional experiments. First, we used the EBV-transformed B cell line as a negative control and the U937 monocytic cell line as a positive control for PDE4B expression. The U937 cell line has been extensively characterized at the gene and protein levels for expression and function of PDEs (24, 30, 39). Second, we repeated blotting for PDE4B after preabsorption of the antiserum with the peptide used as immunogen to raise the antiserum against PDE4B. In these experiments, disappearance of the PDE4B2-corresponding 65- to 67-kDa band occurred when blotting was performed in these conditions (Fig. 1C), whereas only a slight decrease in the 75- to 80-kDa band was observed. It is of interest to point out that we detected an additional immunoreactive protein of approximately 50 kDa in T cells and in U937 cells (data not shown). It is not known whether this protein represents another alternative spliced isoform of PDE4B (30) or merely represents a cross-reactive protein. However, we did not consistently observe a 100-kDa band compatible with a recently described form of PDE4B (PDE4B3) (41). Thus, the antiserum against PDE4B shows the expected reactivity in T cells (31).

Previous studies have suggested that PDE4 isozymes play a role in T cell activation, because selective PDE4 inhibitors can suppress cytokine production and T cell proliferation (11, 15, 20, 42, 43, 44). However, the role of individual cAMP-specific PDEs in T cell activation and the link between these enzymes and TCR/CD3-mediated signaling have not been established. We hypothesized that there may be a fraction of PDE4B that is associated to the TCR/CD3 complex as shown for other regulatory enzymes (45). We tested this hypothesis by blotting CD3{varepsilon} immunoprecipitates from both PBMC and blast T cells for PDE4B. As shown in Fig. 2Go, only a 65- to 67-kDa band, compatible with the short form of PDE4B, was detected in CD3{varepsilon} immunoprecipitates from both PBMC and blasts when blotted with an antiserum against PDE4B. The level of this protein in CD3{varepsilon} immunoprecipitates from blasts was higher than in the same immunoprecipitates from PBMC. TCR ligation did not induce further increases of this band. The possibility of the band being an artifact due to recognition of the immunoprecipitating Ab by the secondary Ab used in the immunoblots was ruled out by its absence when blotting the immunoprecipitating Ab alone without cell lysates and by its absence in immunoprecipitates with an irrelevant IgG2a isotype mAb (isotype-matched to mAb 64.1). Furthermore, a similar 65- to 67-kDa band was obtained when another anti-CD3{varepsilon} Ab (UCHT-1) was used for immunoprecipitation (Fig. 2Go).



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  		FIGURE 2. PDE4B2 association with CD3{varepsilon}. Cell lysates from nonstimulated (-) PBMC or blasts (10 x 106 per lane) or lysates from cells stimulated with the anti-CD3 mAb 12F6 (10 µg/ml) for 10 min (+), were prepared and immunoprecipitated with different anti-CD3{varepsilon} mAbs (64.1 or UCHT-1), and subsequently blotted with a specific antiserum against PDE4B. Immunoprecipitations with an irrelevant IgG2a Ab and the mAb used for immunoprecipitation without cell lysate were included as controls. CL, cell lysates; Ip, immunoprecipitating Ab; H chain, heavy chain.

 
The previous result suggests that the short isoform of PDE4B (PDE4B2), but not PDE4B1, is associated with the TCR. To corroborate this finding, we investigated the presence of CD3{varepsilon} chain in PDE4B immunoprecipitates. Given the lack of an adequate blotting Ab against CD3{varepsilon}, we examined the association of CD3{varepsilon} chain with PDE4B indirectly, by looking at tyrosine phosphorylated TCR subunits in PDE4B immunoprecipitates. Stimulation with anti-CD3 mAb of either PBMC or blasts resulted in tyrosine phosphorylation of both CD3{varepsilon} and TCR {zeta} as described previously (46, 47) (Fig. 3Go). In PBMC, a significant amount of phospho-CD3{varepsilon} and phospho-TCR {zeta} can be detected in nonstimulated T cells upon long exposure of the blotted membranes (46). In PDE4B immunoprecipitates from anti-CD3-stimulated PBMC and blasts (Fig. 3Go), we detected a 23- to 25-kDa tyrosine-phosphorylated protein that was compatible with phospho-CD3{varepsilon} chain, further supporting our finding of PDE4B association with the CD3{varepsilon} chain. This association was more intense in cycling T cells than in resting T cells. Similar results were obtained using ZAP-70 immunoprecipitates in addition to CD3{varepsilon} immunoprecipitates (data not shown).



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  		FIGURE 3. Phospho-CD3{varepsilon} association with PDE4B2 after TCR ligation. PBMC or blasts (10 x 106 per lane) that were left unstimulated (-) or stimulated with the anti-CD3 mAb 12F6 (10 µg/ml) for 10 min (+) were lysed; the lysates were immunoprecipitated with a specific antiserum against PDE4B, with a mAb against CD3{varepsilon}, with a specific antiserum against TCR {zeta}, or with beads alone. Immunoprecipitates were immunoblotted for phosphotyrosine. Ip, immunoprecipitating Ab; L chain, light chain of the mouse anti-human CD3{varepsilon} mAb.

 
Our results indicate that T cells have a fraction of PDE4B that is constitutively associated, directly, or indirectly, with the TCR/CD3 complex. Differential cytosolic or membrane targeting of PDE isoforms has been reported for PDE4A and PDE4D, although the mechanism for this compartmentalization is unknown (48, 49, 50). One possibility to explain the association of PDE4B2 to the TCR is that this isoform is selectively targeted to the cell membrane. This was examined by sequence analysis of the N terminus of the PDE4B2 isoform and comparing it with the other two known PDE4B isoforms. We observed that the CD3{varepsilon} chain-associated form of PDE4B (PDE4B2) had four potential myristylation sites at residues 6, 7, 16, and 17 in its N-terminal region. These sites were absent in PDE4B1 and PDE4B3. These myristylation sites likely correlate with predominant membrane targeting of PDE4B2. The sequence of events leading to its association with CD3{varepsilon} remains to be established.

The functional significance of multiple localization patterns of cAMP-specific PDEs is not completely understood, but may be related to compartmentalization of cAMP signaling. Hence, spatial gradients of intracellular cAMP levels could be modulated by the specific site of PDE activity within the cell. It is plausible that the short PDE4B form associated with the CD3{varepsilon} chain could be more effective in decreasing intracellular cAMP levels than other forms located in the cytosol in response to TCR-mediated signaling. In this regard, a recent report has shown that membrane localization of the nonreceptor tyrosine kinase ZAP-70 in a particular configuration is required for its biological activity, suggesting that spatial orientation of membrane-associated signaling complexes could be an important feature for their function (51).

PDE4B2 is constitutively associated with the TCR complex in resting T cells, and the level of TCR-associated PDE4B2 does not change in response to TCR engagement. Therefore TCR-mediated regulation of PDE4B2 activity must require some mechanism other than TCR association. One potential mechanism may be tyrosine phosphorylation in response to TCR-mediated signaling. Three pieces of evidence support this idea. First, it is known that TCR-mediated signaling involves tyrosine phosphorylation of many signaling molecules that determines the formation of signaling complexes (reviewed in Ref. 52). Second, Stringfield and Moriomoto have shown that tyrosine kinase inhibitors cause a decrease in phosphodiesterase activity in neural cells (53). Third, a comprehensive sequence analysis of PDE4B revealed the presence of a possible tyrosine phosphorylation site at residue 523 in the C terminus region of this protein. This represents a unique feature of the PDE4B that is not present in the other PDE4 isozymes. Therefore, we examined if tyrosine phosphorylation of PDE4B could occur upon TCR/CD3 engagement.

PBMC or T cell blasts were stimulated with an anti-CD3 mAb, and phosphotyrosine immunoblotting was carried out in anti-PDE4B immunoprecipitates from cell lysates. As shown in Fig. 4A, there was no detectable tyrosine-phosphorylated PDE4B in either unstimulated PBMC or blasts. Stimulation with anti-CD3 mAb for 10 min induced tyrosine phosphorylation of PDE4B2. However, TCR ligation did not induce tyrosine phosphorylation of PDE4B1. It is important to note that we have detected interindividual variations in the level of PDE4B tyrosine phosphorylation upon TCR ligation. In this particular experiment, there was a high level of tyrosine phosphorylation of PDE4B2 in PBMC after a 10-min stimulation. However, we have detected tyrosine-phosphorylated PDE4B at earlier times (see Fig. 5B). The possibility that the tyrosine-phosphorylated 65- to 70-kDa band was an artifact of the immunoprecipitating antiserum was excluded by its absence in immunoprecipitations without cell lysate (lane 7, Fig. 4A). In addition, this band was not a product of the stimulating anti-CD3 Ab because it was not recovered in anti-CD3-stimulated PBMC or blast cell lysates treated with protein A-agarose beads without any immunoprecipitating serum (essentially an immunoprecipitation with the stimulating Ab) (Fig. 4A, lanes 3 and 6). Furthermore, normal rabbit serum immunoprecipitates from unstimulated and stimulated PBMC did not precipitate any tyrosine-phosphorylated band with this m.w. range (Fig. 4A). Also, the 65- to 67-kDa band and the 75- to 80-kDa band were also present when pervanadate (a nonspecific phosphatase inhibitor) was used instead of anti-CD3{varepsilon} Abs to activate purified blast T cells (see below).

To confirm the association of tyrosine-phosphorylated PDE4B2 with the CD3{varepsilon} chain, we carried out depletion experiments. As shown in the upper blot of Fig. 4B, the tyrosine-phosphorylated 65- to 67-kDa band present in PDE4B immunoprecipitates from stimulated blast T cells was depleted after three sequential rounds of immunoprecipitations (ips) with an anti-CD3 mAb (post-CD3 ips lane) or after two sequential rounds of immunoprecipitation with the PDE4B-specific antiserum. Consistent with this result and further supporting the association between PDE4B2 and CD3-{varepsilon}, the band corresponding to PDE4B2 in CD3{varepsilon} immunoprecipitates was depleted after three sequential immunoprecipitations with the PDE4B antiserum (post-PDE4B ips lane) or after two sequential CD3{varepsilon} immunoprecipitations (lower blot of Fig. 4B). Overall, these results indicate that the same PDE4B isoform that associates with the CD3{varepsilon} chain (Fig. 2Go) can also be tyrosine-phosphorylated after TCR/CD3 ligation.

Although both PDE4B1 and PDE4B2 isoforms contain a potential tyrosine phosphorylation site in the C terminus, only PDE4B2 (the isoform associated with CD3{varepsilon}) is tyrosine-phosphorylated after TCR ligation. However, nonspecific induction of protein phosphorylation by inhibition of phosphatase activity with pervanadate induced tyrosine phosphorylation of both PDE4B isoforms (Fig. 4C). This finding supports the concept that selective association of PDE4B2 to the TCR complex correlates with its selective tyrosine phosphorylation.

Next, we correlated tyrosine phosphorylation of PDE4B2 with changes in PDE4 activity and cAMP levels. We were not able to detect consistent changes in PDE4 activity using PBMC or T cell blasts. In addition, measurement of PDE4 activity in CD3 or PDE4B immunoprecipitates was not possible due to technical problems related to the binding/detachment of PDE4B in an active form, followed by ion exchange chromatography. Therefore, we focused on measurement of cAMP levels as an indicator of PDE4 activity. As shown in Fig. 5A, T cell blast TCR ligation with an anti-CD3 mAb (UCHT-1) increased cAMP levels at 2 min and reached a peak at 5 min. The cAMP levels declined thereafter by 10 min and 20 min. A similar cAMP kinetic profile was obtained in resting PBMC (data not shown). These results on cAMP levels correlated with a rapid appearance (1 min) of tyrosine-phosphorylated PDE4B2 after CD3 ligation that was sustained for 20 min and disappeared after 60 min of stimulation (Fig. 5B). This suggests that tyrosine phosphorylation may be involved in the regulation of PDE4B2 activity, because PDE4 activation correlates with decreased cAMP levels. This correlation may be oversimplistic, because it does not take into account the role of other PDEs such as PDE3 and PDE7 in the regulation of cAMP levels following TCR-mediated activation (10, 11, 13, 14). In addition, T cell activation can be enhanced by coengagement of TCR and coreceptor (CD4/CD8) or costimulatory molecules (CD28), and this may further regulate cAMP levels through differential activation of PDEs. Previous studies have shown that cross-linking CD4 or CD8 induces accumulation of cAMP, whereas CD28 ligation does not affect cAMP levels (1, 2).

We have provided evidence that one of the PDE4B isoforms, PDE4B2, is selectively associated with the CD3{varepsilon} chain of the TCR in basal conditions and is tyrosine-phosphorylated after TCR/CD3 ligation. The functional consequences of PDE4B tyrosine phosphorylation are not established. However, our data are compatible with tyrosine phosphorylation of PDE4B2 resulting in increased activity of this isoform, similar to the effect of PKA-mediated serine phosphorylation of the PDE4D3 isoform (24, 26). This mechanism may not be operational for other PDE types, because MAPK-mediated serine phosphorylation of PDE4B2B does not correlate with an increase in its activity (25). In support of this hypothesis, previous studies have shown that the use of tyrosine kinase inhibitors results in inhibition of PDE activity and increased cAMP levels, implying that a tyrosine kinase is regulating PDE activity and increasing cAMP degradation (53, 54). This simple scheme does not exclude the involvement of other kinases in the regulation of a fine balance between cAMP generation and cAMP degradation. Alternatively, tyrosine phosphorylation of PDE4B may not impact its activity but rather induce a conformational change in PDE4B2 that increases its stability. A similar possibility has been suggested by the finding that MAPK phosphorylation of a specific PDE4B renders it less susceptible to proteolysis than in the nonphosphorylated state (25). Finally, a third possibility involves the regulation of PDE4B interactions with other proteins containing SH2 domains, by phosphorylation of the phosphotyrosine motifs shown at the C terminus of PDE4B forms.

Understanding how specificity is achieved in signal transduction is a major issue in the field of cell activation. Cell compartmentalization has been proposed as a basic mechanism to explain it. Our data indicate that this may apply to the PDE4B family, and are consistent with the observation that TCR/CD3 ligation promotes the translocation of cAMP-dependent protein kinase type I to the TCR/CD3 complex (45). Taken together, these results suggest that ligation of the TCR triggers recruitment and/or activation of key elements of the cAMP/PKA pathway into a discreet microenvironment of the T cell. Such compartmentalization may contribute to the ability of cAMP to tightly regulate TCR signaling. Further studies will define the physiological role of PDE4B2 tyrosine phosphorylation, and the protein tyrosine kinases involved in this phosphorylation. The demonstration of PDE4B2 phosphorylation provided by our studies in primary T lymphocytes justifies a detailed structure-function analysis of this PDE isoform, using a transfection system to establish the functional link between PDE4B2 phosphorylation status and cAMP generation.



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  		FIGURE 1. Expression of PDE4B RNA and protein. A, RT-PCR analysis of PDE4B from PBMC and blast T cells. Two controls are shown: in the middle panel, the same PCR reactions were performed without previous reverse transcription; in the bottom panel, amplified products were digested with SalI. B, Expression of PDE4 isoforms in blast T cells and in the U937 promyelocytic cell line. C, Western blot analysis for PDE4B from cell lysates of PBMC, blast T cells, U937 cells, and the EBV-transformed B cell line (GM4672). Nonstimulated cells were lysed, and the lysates (6 x 105 cell equivalents) were run in a 10% SDS-PAGE and immunoblotted with a specific antiserum against PDE4B or with the same antiserum after preabsorption with the peptide used for raising such antiserum.

 


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  		FIGURE 4. TCR ligation induces tyrosine phosphorylation of PDE4B2. A, PBMC or blasts (10 x 106 per lane) were stimulated with the anti-CD3 mAb 12F6 (10 µg/ml) for 10 min. Cells were lysed, and the lysates were immunoprecipitated with a specific antiserum against PDE4B, with normal rabbit serum, or with beads alone. Immunoprecipitates were blotted for phosphotyrosine. B, Depletion of PDE4B after sequential immunoprecipitations with a PDE4B antiserum or with an anti-CD3-{varepsilon} mAb (UCTH-1). Four groups of blast T cells (10 x 106/group) were stimulated with the anti-CD3 mAb 12F6 (10 µg/ml) for 10 min. In the PDE4B immunoprecipitation antibody (ip) section, one group of cell lysates underwent three rounds of immunoprecipitations with the PDE4B antiserum; the other group was sequentially immunoprecipitated with three rounds of an anti-CD3{varepsilon} mAb followed by immunoprecipitation with the PDE4B antiserum. Immunoprecipitates were blotted for phosphotyrosine content. In the CD3 ip section, one group of all cell lysates underwent two rounds of immunoprecipitations with an anti-CD3{varepsilon} mAb; the other group was sequentially immunoprecipitated with three rounds of the PDE4B antiserum followed by immunoprecipitation with an anti-CD3-{varepsilon} mAb. Immunoprecipitations were blotted with the PDE4B antiserum. C, Pervanadate induces tyrosine phosphorylation of PDE4B1 and PDE4B2. Pure T cells (10 x 106 per lane) isolated from PBMC (blasts) were stimulated with pervanadate (PV) (10 µl) for 10 min. Cells were lysed, and the lysates were immunoprecipitated with the PDE4B antiserum. Immunoprecipitates were blotted for phosphotyrosine. Ip, immunoprecipitating Ab; NRS, normal rabbit serum; H chains, heavy chains of the immunoprecipitating PDE4B antiserum and the anti-CD3-stimulating mAb.

 


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  		FIGURE 5. Tyrosine phosphorylation of PDE4B2 correlates with cAMP levels. A, T cell blasts (2 x 106 cells) were stimulated with 10 µg/ml of an anti-CD3 mAb (UCHT-1) for the indicated times at 37°C. Reactions were stopped by adding 0.1 N HCL followed by centrifugation at 10,000 rpm for 7 min. cAMP levels were measured in the supernatants using an ELISA kit. Results are expressed as the percentage of the control (basal level: 2.2 pmol/2 x 106 cells) and are representative of two experiments. B, T cell blasts (10 x 106 per lane) were stimulated with an anti-CD3 mAb (UCHT-1: 10 µg/ml) at the indicated times at 37°C. Cells were lysed, and the lysates were immunoprecipitated with the PDE4B antiserum. Immunoprecipitates were blotted for phosphotyrosine.

 

   Acknowledgments
 
We thank the members of the Madrenas laboratory, especially Hamza Suria, for helpful suggestions and comments and Carol Manning for her assistance.


   Footnotes
 
1 This work was supported by grants from the Medical Research Council of Canada, the Kidney Foundation of Canada, the Leukemia Research Fund of Canada, and the Multi-organ Transplant Service of the London Health Sciences Centre. M.L.B. was supported by the Council for Scientific and Humanistic Development of the Central University of Venezuela. Back

2 Address correspondence and reprint requests to Dr. J. Madrenas, John P. Robarts Research Institute, Room 2-05, P.O. Box 5015, 100 Perth Drive, London, Ontario, Canada N6A 5K8. E-mail address: Back

3 Abbreviations used in this paper: PDE, phosphodiesterase; MAPK, mitogen-activated protein kinase; PKA, protein kinase A. Back

Received for publication September 4, 1998. Accepted for publication November 5, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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