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* Institute for Molecular and Cellular Biology, Porto, Portugal; and
Department of Medicine, Stanford University School of Medicine, California
Correspondence: Alexandre M. Carmo, Institute for Molecular and Cellular Biology, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal. E-mail: acarmo{at}ibmc.up.pt
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: rodent • T lymphocytes • antibodies • cell-surface molecules • signal transduction
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Costimulatory [3
4
5
] and inhibitory [6
7
8
] roles have been proposed for CD5, and these may correlate with the activity or nature of the effectors, which upon stimulation, bind to phosphorylated tyrosine residues of CD5. In T cells, Lck is the major kinase responsible for the phosphorylation of CD5 at residues Y429 and Y463 [9
], the former also being the principal site of docking of Lck itself [10
]. ZAP-70 and CD3-
have also been shown to bind to CD5, although the exact mode of interaction is not fully understood: ZAP-70 does not bind directly to the pseudo-immunoreceptor tyrosine-based activation motif (ITAM) sequence containing residues Y429 and Y441 [11
, 12
], and moreover, there is no evidence of direct phosphorylation of CD5 by this kinase. Additionally, CD5 signaling activates a pathway involving phosphatidylinositol 3-kinase, whose two SH2 domains bind to phosphorylated Y441 and Y463 [5
, 11
].
Regarding the inhibitory effect of CD5, the molecular mechanism remains to be elucidated. Two of the tyrosine residues, Y378 and Y441, are within putative immunoreceptor tyrosine-based inhibitory motifs, but the evidence of involvement of these residues in inhibitory effects is very slim. So far, no repressors have been shown to bind to Y441, and only in Jurkat T cells does the phosphatase SHP-1 seems to dock to Y378 [7 ]. No evidence of such binding was found in B cells [8 ]. Curiously, it is residue Y429 that harbors molecules that down-modulate cellular activation, such as Ras-GAP and Cbl in thymocytes [8 , 13 ].
The exact mode of CD5 phosphorylation also remains to be fully deciphered. CD5 is rapidly phosphorylated following T cell receptor (TCR) stimulation [14 ]. However, as the TCR/CD3 complex does not include Lck, the interaction between CD5 and Lck is possibly promoted in assembled signaling micro-domains, namely lipid rafts. Indeed, cross-linking CD5 with the TCR targets both molecules to these specialized domains [15 ]. Paradoxically, stimulation of T cells with antibodies against CD2, a membrane antigen that does associate with Lck [16 , 17 ] and CD5 [18 , 19 ], seems not to induce CD5 phosphorylation but rather its dephosphorylation [18 ].
Given the complexity of the signaling mechanisms involving CD5 and the apparent contradiction between the outcomes of stimulation through the TCR versus CD2, we looked for alternative molecules and kinases that might regulate CD5 phosphorylation. Here, we report on the rat T cell protein, OX52, which appears to have a role in mediating the phosphorylation of CD5, making use of its capacity to associate with tyrosine kinases of different families.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cells and cell lines
Cervical lymph node cells were from 12-week-old Lewis male rats (Charles River Laboratories, Barcelona). Cell lines included: W/FU(C58NT) [17
], a rat thymoma, from Adreas Conzelmann (University of Fribourg, Switzerland), and COS7 [19
], obtained from the American Type Culture Collection (Manassas, VA). Cell lines were maintained in RPMI with 10% fetal calf serum (FCS), 1 mM sodium pyruvate, 2 mM L-glutamine, penicillin G (50 U/ml), and streptomycin (50 µg/ml).
Cell stimulation and detection of tyrosine phosphorylation
Approximately 1 x 107 rat lymph node cells were used per sample. R73 and/or OX52 were added at 10 µg/ml to cells and incubated on ice for 15 min. Cells were washed with phosphate-buffered saline (PBS), resuspended with 20 µg/ml RAM, and incubated for 2 min at 37°C. Cells were lysed by the addition of Nonidet P-40 (NP-40) lysis buffer [10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 1% (v/v) NP-40] for 30 min on ice. The nuclear pellet was removed by centrifugation at 12,000 g for 10 min at 4°C. Cell lysates were denatured in 2 x sodium dodecyl sulfate (SDS) buffer and were subjected to SDS-polyacrylamide gel electrophoresis (PAGE). Immunoblotting and detection of tyrosine phosphorylation were performed as previously [18
], using 4G10-HRP.
Cloning rat CD6 cDNA
Total RNA was isolated from Lewis male rat spleen using Trizol (Life Technologies, Barcelona). cDNA was obtained using the ThermoScript reverse transcriptase-polymerase chain reaction (RT-PCR) system (Life Technologies) from total RNA primed with oligo(dT). Rat Cd6 was amplified with primers designed based on the reported sequence of mouse Cd6 (GenBank U37543). The following primers were used: forward primer from the 5' untranslated region (UTR) of mouse Cd6 5'-GCACTAAGCTTGGGCAGGCGTGAGTAGCAGT-3' and reverse primer containing the stop codon sequence of mouse Cd6 5'-TACGATGACATCGGTGCAGCCTAGCCTCTAGACCCG-3'. The 2.0-Kb fragment obtained in the PCR reaction was then gel-purified and cloned into TOPO 2.1 vector (Invitrogen, Groningen). The sequence was confirmed by de-deoxy sequencing. The sequence of the 3' UTR was obtained by using 3' rapid amplification of cDNA ends (RACE, Life Technologies) following the manufacturer’s instructions. Based on sequence information from the previous PCR, the primers designed were: forward 5'-GCTCCCAGAGACATCCCA-3' (within exon 6) and reverse universal adaptor primer. The first strand for this reaction was obtained using oligo dT/adaptor primer to obtain the 3' end of the cDNA.
Transfection of COS7 cells
A truncated form of rat CD6 (M6.1) lacking part of the cytoplasmic domain (residues 524–665) was used for transient transfection of COS7 cells and was obtained as follows: Rat CD6 cDNA cloned in TOPO 2.1 vector was used as a template for the amplification PCR reaction using as forward primer a sequence derived from the 5' UTR of mouse CD6 5'-GCACTAAGCTTGGGCAGGCGTGAGTAGCAGT-3' and a reverse primer designed within CD6 cytoplasmic tail 5'-CTCTTCAAGTCATTCCTCCAAGGGTGGCATCCG-3', containing a stop codon. The PCR product obtained was blunt-end subcloned into the expression vector pEF-BOS [24
]. The orientation of the insert was confirmed by colony-PCR. Transient transfection in COS7 cells was performed as follows: Cells were grown in 80 cm2 flasks in CO2 incubator, 37°C, 5% CO2, until they were 50–75% confluent. Following washing with RPMI, a transfection mix containing 0.4 mg/ml diethylaminoethyl-Dextran, 100 µM chloroquine, and 1 µg/ml DNA in RPMI was added to the cells for 2 h at 37°C, 5% CO2. Cells were washed with PBS and incubated in dimethyl sulfoxide, 10% in PBS, for 2 min followed by two washings with PBS. Cells expanded for 48 h in RPMI/10% FCS + antibiotics at 37°C in 5% CO2. COS7 cells were detached from flasks 24-h post-transfection with Trypsin-EDTA solution (Sigma-Aldrich, Sintra), washed with PBS, and replated in complete RPMI medium.
Flow cytometry
Forty-eight hours after transfection, COS7 cells were removed from flasks with PBS/EDTA (0.2 g/L) and were resuspended in PBS containing 0.2% bovine serum albumin (BSA) and 0.1% NaN3 (PBS/BSA/NaN3) at a concentration of 1 x 106 cells/ml.
Staining was performed by incubation of 2 x 105 cells/well with mAb (20 µg/ml) for 30 min on ice in 96-well round-bottom plates (Greiner, Nürtingen). Cytometric analysis was as described previously [18 ].
Cell-surface biotinylation and immunoprecipitations
Cell-surface biotinylation, immunoprecipitations, and reprecipitations were performed as described previously [18
].
Immune complex kinase assays
Kinase assay buffer (30 µl) containing 10 mM MnCl2, 1 mM Na3V04, 1 mM NaF, and 50 µCi (185 KBq) of [
-32P]-adenosine 5'-triphosphate (ATP; >5000 Ci/mmol; Amersham, Little Chalfont, UK) was added to the beads containing the immune complexes, and in vitro kinase reactions were allowed to occur for 15 min at 25°C. When indicated, a biotinylated peptide containing the CD5 pseudo-ITAM sequence (Biot-AASHVDNEYSQPPRNSRLSAYPALE-OH) was also included in the reaction mix at a final concentration of 0.5 µg/µl and in this case, incubated at 30°C for 10 min. Reactions were stopped with 30 µl 2 x SDS buffer and boiling for 5 min, and the products were separated by SDS-PAGE. Gels were dried and exposed at -70°C to Biomax MR-1 films. Biotin-labeled CD5 peptide was recovered using avidin beads (Pierce, Rockford, IL), and the incorporated [-32P]-ATP was measured in a Beckman liquid scintillation counter. Results are expressed in counts per minute (cpm).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ß TCR chains, CD5, and CD28 could easily be detected at their expected sizes in the respective lanes. In the lane of OX52, a broad smear was detected above 100 kDa. Common to all lanes, except those of CD28 and OX21, was a protein of 66 kDa, presumed to be CD5. To prove that this protein was CD5, we disrupted the immune complexes and performed reprecipitations using polyclonal anti-CD5. The amount of CD5 precipitating with OX52 was comparable with that associating with CD2 or with the TCR (Fig. 2B)
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It is interesting that although CD5 did coprecipitate with CD2 and TCR, the fraction of CD5 associating with CD2 or the TCR was not highly phosphorylated. In fact, phosphorylated CD5 could hardly be detected in CD2 immunoprecipitations. These data mean that in our experimental conditions, CD2, TCR, and OX52 coprecipitate CD5 equivalently, but only the fraction of CD5 associating with OX52 is highly phosphorylated.
Based on the molecular weight and tissue distribution of OX52 [20 ], we hypothesized that the OX52 antigen was the rat homologue of CD6. Using a polyclonal anti-mouse CD6 serum, we attempted to reprecipitate CD6 from the immune complexes. Reprecipitation of CD6 yielded a phosphoprotein of 110 kDa present in OX52 immune complexes and to a lesser extent, in CD5 immune complexes but not in those of CD2, TCR, or CD28 (Fig. 2E) .
PCR cloning of the rat homologue of CD6
For the purpose of cloning rat Cd6, we obtained cDNA by RT-PCR from RNA isolated from splenocytes of Lewis male rats. Primers were designed based on the cDNA sequence of mouse Cd6 (GenBank U37543). The 5' primer is 17 bases upstream of the start codon, and the 3' primer overlaps with the stop codon. The PCR amplified DNA fragments ranging from 1.4 to 2.0 Kb, probably corresponding to different CD6 isoforms that result from alternative splicing. We gel-purified the heaviest DNA product and subcloned it into the vector TOPO 2.1. Sequencing of this product revealed an open reading frame of 1998 bases. With this sequence information, the gene-specific primer for 3'-RACE was designed (see Materials and Methods). The 3'-RACE reaction produced a fragment that included a further 893 bp from the stop codon until the poly(A) tail.
The coding sequence of rat Cd6 (GenBank accession no. AF488727) corresponds to a 665 amino acid long polypeptide (Fig. 3 ). It has 89% identity with mouse CD6 and 71% identity with the human molecule (GenBank U34625). Domain 2 shares the highest identity, 95%, with the mouse homologue when compared with domains 1 and 3, which have 54% and 88% identity, respectively. The highest level of identity with the human sequence (85%) is found for domain 3.
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OX52 is the rat homologue of CD6
The proof required to assign OX52 as rat CD6 was that this antibody specifically recognizes expressed CD6, and we performed these experiments in a nonlymphoid system. As the OX52 epitope lies within the extracellular domain, we produced a truncated form of rat CD6, which removes part of the cytoplasmic domain (see Materials and Methods). We transfected the CD6 cDNA into COS7 cells, and 48 h post-transfection, cells were surface-biotinylated, lysed with Brij 96, and subjected to immunoprecipitations using the OX52 mAb. As control, we used COS7 cells transfected with the empty vector. OX52 mAb was able to precipitate a labeled protein expressed only in the CD6-transfected cells and not in the control cells (Fig. 4A
). Moreover, we used a polyclonal antibody raised against the extracellular domain of mouse CD6 to immunoprecipitate the expressed rat CD6. Given the high homology between the rat and mouse molecules, it was not surprising that this antiserum precipitated a protein with the same mass as that precipitated by OX52.
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Rat CD6 associates with protein kinases of different families
The high phosphorylation detected in kinase assays in OX52 immune complexes possibly meant that rat CD6 associates with proteins with kinase activity. From immune complexes of CD2, CD5, CD28, TCR, and CD6 obtained from Brij 96-prepared C58 lysates and subjected to in vitro kinase assays, we attempted to identify kinases responsible for the kinase activities. We used antibodies against the Src-family kinases Lck and Fyn, against the Tec-family kinases Tec, Itk, and Txk, and against the Syk-family kinase ZAP-70. Reprecipitations where we detected specific signals are shown in Figure 5
. CD2 coprecipitated the most Lck and Fyn, although the amount coprecipitated by TCR and CD6 was also significant. ZAP-70 could be seen in association with CD5, and to a lesser extent with the TCR and CD6. Finally, Itk associated specifically with CD6 and was not detected in CD2, TCR, CD5, or CD28 immune complexes. The associations between the surface proteins and the kinases, except ZAP-70, were also detected using other detergents such as NP-40 and Triton X-100 (data not shown). CD6 could coprecipitate Lck, Fyn, and Itk in all detergents, whereas ZAP-70 associations were lost when using detergents other than Brij 96.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Surface receptors other than the TCR may also exert some of their activities through CD5. CD2 falls in this category, first, because stimulation via CD2 induces the dephosphorylation of CD5, possibly through the induction of SHP-1 activity [18 ], and second, because CD2 synergizes with CD5 in the regulation of thymocyte selection. CD2 null mice also display enhanced thymocyte reactivity, an effect highly augmented in the CD2/CD5 double-knockout, suggesting that CD2 and CD5 are functionally associated in restraining thymocyte activation [26 ]. It is possible that engagement of different T cell antigens to the counter-receptors on antigen-presenting cells originates different signals that may converge at the level of CD5 and thus regulates CD5 use in signaling pathways. We propose that a third T cell surface protein, CD6, is involved in the regulation of CD5 phosphorylation.
The mAb MRC OX-52 was described in 1986 by Mason and co-workers [20 ] and was found to label an antigen mainly confined to the T cell areas of the lymphoid organs. It has since been considered a marker for the T cell population in rat tissues or cell suspensions. We proved by cloning, cellular expression, cytometric analysis, and molecular characterization that this protein is the rat homologue of CD6.
Having established OX52 as rat CD6, we focused on the role of CD6 in the regulation of CD5 phosphorylation. In our assays, equal amounts of CD5 were obtained in immunoprecipitations of CD2, TCR, and OX52. CD6, conversely, could only be coprecipitated with CD5 but not with CD2 or with the TCR, suggesting a close interaction between CD5 and CD6. It is interesting that whereas CD5 in OX52 immunoprecipitates could be highly phosphorylated in kinase assays, the same amount in CD2 precipitates could hardly be detected (Fig. 2) .
The obvious explanation that CD2 did not associate with kinases capable of phosphorylating CD5 had to be discarded, as CD2 precipitated the largest amount of Lck. The hypothesis that Lck is not responsible for CD5 phosphorylation is equally improbable, given the evidence for this activity collected in the past [9 , 10 , 27 ]. Nevertheless, we tested this hypothesis by incubating Lck directly with a synthetic peptide containing the site of phosphorylation in the cytoplasmic domain of CD5, Y429, preferentially used by Lck. As expected, Lck efficiently phosphorylated the peptide (Fig. 6) .
We have used tyrosine phosphatase inhibitors throughout our experiments. Nevertheless, it cannot be excluded that phosphatases may play a role in the inhibition of phosphorylation of the fraction of CD5 that is coupled to CD2. Most of the phosphatase activity associated with CD2 derives from CD45 [28 , 29 ]. CD45 could dephosphorylate CD5 directly or alternatively, dephosphorylate Lck and thus render it inactive [30 ]. Additionally, SHP-1 may as well dephosphorylate Lck at its activating residue Tyr-394 [31 ].
Although it remains unclear why CD2-associated CD5 is not readily phosphorylated, it appears easier to explain the strong phosphorylation observed in the fraction of CD5 that associates with OX52/CD6. Our studies show that tyrosine kinases of different families bind to CD6. In fact, rat CD6 coprecipitated Lck, Fyn, ZAP-70, and Itk, something not generally observed for the other cell-surface proteins studied, CD2, TCR, CD5, and CD28. It is conceivable that the capacity of CD6 to induce the phosphorylation of CD5 may derive from the fact that it associates with different kinases, which may increase each other’s activities. Indeed, Lck and Fyn synergize with ZAP-70 to produce sustained responses [32 ], and a functional Lck is required for Itk phosphorylation and activation to occur [33 ]. Previous studies, where the levels of CD5 phosphorylation observed in Lck-deficient cells were significantly lower when compared with Lck+ cells, clearly indicated a major role for Lck but nevertheless, suggested that other kinases whose activity may depend on Lck potentially have a role in CD5 tyrosine phosphorylation [10 ].
In individual terms, Lck ranked as the most efficient kinase phosphorylating a CD5 peptide, as can be depicted from Figure 6 . Fyn is significantly less efficient, and Itk and ZAP-70 phosphorylate the CD5 peptide only residually. However, when we used pairs of kinases, the combination of Lck with Fyn was not the most effective. Rather, Lck in conjunction with Itk gave the best incorporation of radioactive ATP in the CD5 peptide. Moreover, we noticed that the Itk kinase was phosphorylated only in the presence of Lck but not in the presence of Fyn or ZAP-70 (not shown). Therefore, Lck and Itk may have a combined role in promoting the efficient phosphorylation of CD5 and thus explain the exceptional capacity of CD6 in supporting the high level of CD5 phosphorylation observed. Alternatively, an independent, inhibitory effect of Fyn and ZAP-70 on Lck, possibly through the phosphorylation of the C-terminal inhibitory tyrosine residue of Lck, may exist, thus explaining why CD5 is less susceptible to tyrosine phosphorylation in the presence of the TCR or CD2.
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ACKNOWLEDGEMENTS |
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Received September 6, 2002; accepted October 9, 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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molecules in human CD3+ thymocytes J. Immunol. 159,3739-3747[Abstract]
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