©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Lck Unique Domain Influences Lck Specificity and Biological Function (*)

(Received for publication, September 1, 1994; and in revised form, October 24, 1994)

Ana C. Carrera (1) (2)(§) Helene Paradis (2)(¶) Luis R. Borlado (1) Thomas M. Roberts (2) Carlos Martinez-A (1)

From the  (1)Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Cientificas, Universidad Autonoma Campus de Cantoblanco, Cantoblanco, 28049 Madrid, Spain and the (2)Department of Cellular and Molecular Biology, Dana Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Src-family tyrosine kinases share structural and amino acid sequence homology, particularly in the catalytic domain as well as in the SH2 and SH3 domains of the regulatory region. However, each src-family member also contains a unique domain which is specific to and characteristic of each individual tyrosine kinase. These unique or specific domains may contribute to the functional specificity of each src-family kinase. To address this possibility, we analyzed the kinase activities and substrate specificities of the lymphoid src-kinase, pp56, and a mutant of pp56 lacking its specific domain. Our data show that both the wild type enzyme and the specific domain-deleted mutant displayed similar affinities for ATP and the non-physiological substrate denatured enolase. However, the specific domain-deleted mutant failed to phosphorylate a number of physiological substrates of pp56. In addition, the ability of pp56 to mediate induction of the interleukin-2 promoter was strongly impaired upon deletion of its specific domain. Thus, the unique domain is not required for the intrinsic kinase activity of pp56, however, it influences substrate preference and contributes to the unique physiological function of this src-family tyrosine kinase.


INTRODUCTION

The src-family of non-receptor tyrosine kinases act as signaling molecules in a wide array of cellular activation processes (for review, see (1) and (2) ). The existence of the various src-family kinases reflects the requirement for different cell-surface receptors to transmit cell type-specific intracellular signals(3, 4, 5, 6) .

The structural basis for the functional specificity of src-kinases is being actively investigated. Src-kinases share a general common organization: an NH(2)-terminal myristoyl group(7) , followed by a non-homologous region (unique or specific domain), two regulatory regions (SH2 and SH3 domains), the conserved catalytic domain (SH1) and a COOH-terminal regulatory region involved in modulating the enzyme activity (reviewed in (1) ). Regarding the involvement of any of these regions in determining the enzyme specificity, SH2 and SH3 are good candidates since these regions have the abilities to bind different signaling molecules(8, 9, 10) . However, the contribution of SH2 and SH3 domains may not be sufficient to determine src-kinases specificity, since SH2 and SH3 domains of src-kinases share more than differ in their agonists(11, 12) . Another region that is likely to confer specificity is the unique domain since it has a different amino acid sequence for each member. In this regard, we and others have already shown that the unique domains of pp56, pp55 and pp59 are implicated in specific receptor interaction(13, 14, 15, 16, 17) . Thus, the interaction of the kinase with a specific transmembrane receptor could be a mechanism to confer specificity. However, this mechanism does not explain all the cases in which src-kinases are essential. For instance, pp56 binds specifically to CD4 and CD8(13, 14, 15) , and shares with pp59the ability to bind to the TcR(^1)(16, 18) . However, pp56 is essential for TcR-mediated IL-2 production in Jurkat cells and for the induction of differentiation of early thymocytes(3, 4, 19) , processes in which neither CD4 nor CD8 seem to participate. In these and other processes, the unique domain could be involved in the specific function of src-kinases through a novel mechanism different from specific transmembrane receptor binding. To test this hypothesis, we have analyzed the function of the specific domain of pp56.

pp56 is a src-kinase first identified by its overexpression in the LSTRA thymoma cell line(20) . After its initial identification, pp56 was found to be present in all T cells and some B cells but rarely in cells of non-lymphoid origin(20) . Regarding its function, pp56 seems to be crucial for T cell maturation (3, 4, 5, 21) and is also involved in the activation of mature T cells (22) . In particular, pp56 seems to play a central role in the induction of IL-2 production required for T cell proliferation (23, 24) and in mediating the intracellular signals delivered by CD4 and CD8 co-receptors(15) .

In the present report we have compared wild type pp56 and a mutant lacking its unique domain. We have examined their substrate specificities, kinetic constants, and functional activities in T cells. We present data indicating that the specific domain of pp56is involved in regulating substrate preference and is relevant for the specificity of pp56 biological function.


EXPERIMENTAL PROCEDURES

Reagents, Cells, and Antibodies

Triton X-100, phenylmethylsulfonyl fluoride, sodium orthovanadate, aprotinin, leupeptin, poly (Glu, Tyr), poly (Lys, Ala, Glu, Tyr), poly (Glu, Ala, Tyr), myelin basic protein, and enolase were purchased from Sigma. Horse peroxidase-conjugated anti-mouse antibodies, chemiluminiscence developing kit, and [-P]ATP (3000 Ci/mmol; 1 Ci = 37 GBq) were from Amersham. The vector Gex-2T, protein A-Sepharose, and glutathione-Sepharose beads were obtained from Pharmacia. The enzymes required for DNA cloning were purchased from New England Biolabs. Oligonucleotides were synthesized by Paul Morrison (D.F.C.I., Harvard Medical School, Boston, MA). JCam1 cells, a mutated Jurkat cell line that has lost the expression of pp56, was described previously and generously donated by Dr. A. Weiss(19) .

Anti-phosphotyrosine antibody (4G10) was produced and generously donated by Dr. B. Druker (D.F.C.I., Harvard Medical School, Boston, MA). UCHT1 anti-CD3 Ab was described previously (25) and generously donated by Dr. D. Cantrell. Anti-GAP Ab, anti-COOH-terminal-pp56 Ab, and anti-NH(2)-terminal-pp56 Ab, were obtained from Upstate Biotechnology Incorporated.

Jurkat cells and JCam1 cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, L-glutamine (2 mM), HEPES pH 8.0 (10 mM), streptomycin (50 µg/ml), and penicillin (50 units/ml) (RPMIc) at 37 °C, 5% CO(2).

Mutant Construction and cDNAs

To prepare the mutants we used syn-K, synthetic lck gene encoding pp56. (^2)Deletion of the specific domain of pp56 (residues 17-57, named specific^d-pp56) was prepared using ClaI and NaeI restriction sites, located at the 5` and 3` ends of the domain. After restriction, the fragment containing the remaining sequence of lck was purified and ligated. Prior to ligation, overhanging ends were converted into blunt ends using either Klenow enzyme (3` end) and mung bean nuclease (5` end) to keep the pp56 open reading frame intact. The resulting mutant has at the junction the following sequence Asn Met Ala^18 (residue 58 in the wild type enzyme), etc. For the substitution mutant Tyr for Phe (active pp56), the fragment KpnI (located at the end of domain VIII) to EcoRI (located after the stop codon) including the mutation was generated by polymerase chain reaction using the oligonucleotides CAGAGGCCATTAACTACGGTACCTTCACCATC (5`-oligo) and GCCAGTTCCAGCCTCAGCCTTGAGAGGGGGAATTCATCGTG (3`-oligo). The substitution mutant at Lys (inactive pp56) has been previously described(26) .

For Escherichia coli expression, mutants were cloned into Gex-2T (G-pp56 constructs) as described previously(26) . For electroporation of JCam1 cells, wild type and mutated pp56 were cloned into pEF BOS vector(27) . The constructs were created by inserting the 1.5-kilobase NcoI to EcoRI fragment from G-pp56 constructs into the XbaI site of pEF BOS. Prior to ligation all the overhanging ends were blunted using Klenow enzyme. Orientation was determined in each case by restriction analysis. NFAT-CAT (28) has been previously described; it contains three copies of the sequence 5`-GGAGGAAAAACTGTTTCATACAGAAGGCGT-3` (corresponding to the sequence from position -284 to -258 relative to the ATG of the IL-2 gene) upstream of the IL-2 minimal promoter driving the reporter gene CAT. This sequence contains the ARRE-2 site of the human IL-2 enhancer, which is the binding site for NFAT(29) .

Substrate Preparation, Bacterial Enzyme Production and Purification, Kinase Assays, and Enzymatic Analysis

Enolase, poly aminoacids, and myelin basic protein were obtained commercially (Sigma). cdcd2 peptide (30) was generously donated by Helen Piwnica-Worms (Department of Medicine, Beth Israel Hospital, Boston, MA), and src autophosphorylating peptide was obtained from David Wrinkler(31) . Gex-2T-intracytoplasmic--TcR (32) was prepared by Frank Howard and purified using glutathione beads as described(26) . Baculoviral p21-GTPase-activating protein (GAP) preparation was generously donated by Robert Weinberg (Whitehead Institute, Massachusetts Institutes of Technology, Cambridge, MA) and was purified by immunoprecipitation. MAPK was also used as substrate. To avoid background kinase activity, we prepared an inactive MAPK mutant. Gex-inactive-human MAPK was prepared as follows: EcoRI (partial digestion) to BamHI fragment encoding wild type MAPK, obtained from Steven Pellech: University of British Columbia, Vancouver, British Columbia, Canada) was ligated with the vector pLV 393 previously digested with EcoRI and BamHI. To generate inactive MAPK, the conserved Lys at subdomain II, was substituted for Met by polymerase chain reaction using appropriate oligonucleotides. pLV 393 inactive MAPK was subsequently restricted using EcoRI (partial digestion) and BamHI, and the fragment encoding inactive MAPK purified and ligated to pGex-2T previously digested with EcoRI and BamHI. Bacterial inactive MAPK and wild type and mutated G-pp56 proteins were purified as previously detailed(26) .

Kinase reactions were performed as follows: 10 µl containing 50 ng ( Fig. 1and Table 1) or 25 ng ( Fig. 2and Fig. 3) of purified kinase were preincubated at 25 °C for 1 min and then mixed with: (a) 20 µl of 2 times kinase reaction mixture and (b) 10 µl containing 10 µg of acid denatured enolase, or the corresponding substrates (indicated in the figures). The mixture contained 50 mM Tris-HCl, pH 7.4, 10 mM MnCl(2), and serial dilutions of the ATP stock (100 µM ATP, 10 µCi/µl of [-P]ATP, 3000 Ci/mmol). For the enzymatic analysis ( Table 1and Fig. 1), reactions were incubated at 25 °C for 2 min (mixed every 30 s) and terminated by addition of 10 µl of 100 mM EDTA pH 8.0. For substrate specificity analysis ( Fig. 2and Fig. 3), reactions were performed similarly but incubated for 15 min. For kinase reactions using immunopurified anti-pp56 (from 2 times 10^6 cells, Fig. 4), immunoprecipitates were prepared as in (7) , and in vitro reactions were performed as above. Substrate and enzyme were resolved by SDS-PAGE. For the determination of kinetic parameters, phosphate incorporated into enolase was quantitated by liquid scintillation counting. V(max) and K(m) were estimated as described previously(26) . Peptides (1.5 mM final concentration) were used as substrates as described(33) .


Figure 1: Purification and kinase activity of the different bacterially expressed G-pp56 mutants. A, the diagram represents pp56 protein. The different relevant domains are indicated. The position of the different mutations prepared and their nomenclature are shown at the bottom. B, wild type and mutated G-pp56 proteins were produced in E. coli X90. The different fusion proteins present in the bacterial lysates were purified using glutathione-Sepharose beads (200 ng obtained from 400 µg of total soluble protein). Purified proteins were analyzed by SDS-PAGE followed by Coomassie Blue-staining. C, 50-ng aliquots of the different samples were tested for their ability to transfer phosphate into enolase in vitro and resolved by SDS-PAGE. The resulting gel was analyzed by autoradiography. D, autophosphorylation of the different mutants in vivo was analyzed by comparison of similar volumes (20 µl) of each of the crude extracts separated by SDS-PAGE and analyzed by Western blot using anti-phosphotyrosine Abs.






Figure 2: Comparison of the in vitro substrate specificity of specific^d-pp56and wild type-pp56. Wild type and mutated pp56 proteins were produced and purified as explained under ``Experimental Procedures.'' 25 ng of each mutant or wild type enzyme were tested in vitro for their ability to phosphorylate each of the indicated substrates (detailed under ``Experimental Procedures''). Kinase reactions were resolved by SDS-PAGE and analyzed by autoradiography. Substrates are indicated on the left and G-pp56 mutants on the top. A control in the absence of substrate (lanes 1) or enzyme (lanes 2) is run in each case.




Figure 3: Analysis of the in vitro substrate specificity of specific^d-pp56and wild type pp56. The analysis of substrate specificity was performed as in Fig. 2. Controls in the absence of enzyme (lanes 1) or substrate (lanes 2) are run in each case.




Figure 4: Expression of wild type and specific^d-pp56 in JCam1 cells. A, the cDNAs encoding wild type and mutated pp56(50 µg) were electroporated into JCam1 cells. 24 h later cells were lysed, and similar volumes of the different lysates were resolved by SDS-PAGE and developed by Western blot using anti-COOH-pp56antibodies. B, JCam1 lysates, prepared as in A, were immunoprecipitated using anti-COOH-pp56 Abs. Kinase reactions were carried out in pp56 immunoprecipitates, the reaction products were resolved by SDS-PAGE, and the gel was visualized by autoradiography.



Electroporation and CAT Assays

JCam1 cells were transfected via electroporation (Gene Pulser, Bio-Rad), according to manufacturer's instructions. Briefly, JCam1 cells were pulsed (10^7/ml) in RPMIc, at 960 microfarads and 330 V. Cultures were transfected, incubated for a period of 24 h, and then lysed and analyzed. In some cases cells were stimulated 6 h after electroporation as indicated in the figures.

CAT assays (34, 35) were performed using 5-µg aliquots of the different cellular lysates (previously optimized to be within the linear range).

JCam1 Cell Lysis, Immunoprecipitation, and Western Blot

Electroporated JCam 1 cells were collected by centrifugation and resuspended at 2 times 10^7 cells/ml in 1% Triton X-100, 50 mM HEPES pH 8.0, 150 mM NaCl, containing protease and phosphatase inhibitors (1 mM phenylmethylsulfonyl fluoride, 2 mM sodium orthovanadate, 10 mM NaF, 10 µg/ml aprotinin, and 10 µg/ml leupeptin). Lysates were incubated with 2-5 µg of each of the Ab and 30 µl of protein A-Sepharose beads for 3 h. The resulting immunoprecipitates were extensively washed: once in lysis buffer, three times in 0.5 M NaCl, 25 mM Tris-HCl, pH 7.5, and two times in 25 mM Tris-HCl, pH 7.5. Western blotting was carried out as described previously(36) .


RESULTS

Expression of pp56 Mutants in Bacteria

The following mutants have been used for this study (illustrated in Fig. 1A): (i) specific domain deletion mutant of pp56 (named specific^d-pp56); (ii) active pp56 (Tyr505 substituted for Phe) and; (iii) inactive pp56 (Lys substituted for Arg, (26) ). Wild type and mutated pp56 genes were cloned in p-Gex-2T (referred to as G-pp56), and the corresponding proteins were purified from similar volumes of E. coli bacterial cultures. These preparations were compared by SDS-PAGE and Coomassie Blue staining. As shown in Fig. 1B, all the mutants were expressed to a similar extent and, upon purification yielded approximately 50 ng of pure G-pp56 protein from 100 µg of total soluble bacterial protein (quantitated by comparison with bovine serum albumin standards).

As a first approximation to test the kinase activity of the different mutants, 50 ng of each of the pure preparations were tested in an in vitro kinase assay (Fig. 1C). The kinase activity was also evaluated by comparison of similar volumes of the lysates by anti-phosphotyrosine Western blotting (Fig. 1D). Both analysis indicated that specific^d-pp56 displayed a kinase activity that was roughly similar to that displayed by wild type pp56. Interestingly, active pp56 and wild type pp56 also displayed similar phosphotransfer activities (Fig. 1, C and D). This is probably due to the fact that bacteria does not express p50, an enzyme that down-regulates pp56 activity(37) . To compare quantitatively the kinase activity of specific^d-pp56 and wild type pp56, enzymatic parameters were estimated. To this end, we first compared the kinase activity of the fusion protein G-pp56 with the activity of bacterial pp56 upon cleavage of the glutathione S-transferase fragment, and the activity of both preparations was similar(26) . Thus, purified wild type and mutated G-pp56 proteins were used for the enzymatic analysis. To determine the enzymatic parameters of G-pp56, we followed our previously described method(26) . Briefly, the concentration of purified enzyme was estimated by SDS-PAGE followed by Coomassie Blue staining. 50 ng of G-pp56 were mixed with varying amounts of ATP and enolase and subjected to kinase reaction. A time course of the reaction revealed that the incorporation of phosphate was linear at least for the first 5 min using between 10-100 ng of enzyme (not shown). Therefore, for all the assays, 50 ng of enzyme and 2-min incubations were used to remain in the linear range. To measure the K(m) of G-pp56 for ATP in autophosphorylation reactions, enzyme amount was fixed (50 ng) and ATP concentration was varied from 0.25 to 10 µM. To calculate the K(m) for enolase, ATP concentration was fixed at 5 µM and enolase varied from 0.34 to 22 µM (corresponding to <0.1 to >3 times K(m) for enolase of G-pp56). To evaluate the phosphotransfer activity, we determined the apparent V(max) for enolase phosphorylation in excess of ATP (5 µM ATP, corresponding to >5 times K(m)). Enzyme and substrate were resolved by SDS-PAGE. Initial velocity was estimated by measuring the amount of phosphate incorporated into G-pp56 or enolase by liquid scintillation counting. The data obtained were evaluated by Eisenthal Cornish-Bowden(38) , and Lineweaver-Burk (39) approximations, which yielded similar values in every case. The values obtained for wild type G-pp56 ( Table 1and (26) ) were found to be similar to those obtained using purified baculoviral pp56(26, 31) . Using these conditions, we have compared specific^d-pp56 and wild type-pp56 and found that they displayed similar phosphotransfer activities (V(max) for enolase phosphorylation, Table 1). Furthermore, the enzymatic parameters indicated that the affinities (estimated by the K(m)) of specific^d-pp56 and wild type pp56 for ATP and enolase were similar. Thus, deletion of the specific domain does not alter the affinities of pp56 for ATP and enolase and the enzymatic activity of pp56 toward enolase.

Comparison of the Substrate Specificity of Wild Type pp56 and the Specific Domain Deletion Mutant of pp56

The substrate specificity of the mutants was characterized using pure preparations of specific^d-pp56 and wild type pp56 and a collection of substrates. Optimized enzymatic reactions using 20 ng of each of the pure enzymes and excess semipurified substrates were performed as described under ``Experimental Procedures.'' The ability of the pp56 variants to phosphorylate the different substrates was then compared by autoradiography ( Fig. 2and Fig. 3) or upon quantitation of the amount of P incorporated into each of the substrates (Table 2). The substrates analyzed included: 1) some previously described physiological substrates, such as MAPK (40) and GAP(41) ; 2) proposed in vivo substrates such as -TcR ( chain of the T cell receptor); 3) peptide substrates of tyrosine kinases such as those based on the src autophosphorylation site and cdc2 peptide-containing Tyr(30, 31) ; and 4) model substrates for Tyr kinases such as enolase, MBP, and heterogeneous polyaminoacids poly-(EAY), poly-(EY), and poly-(KAEY). An inactive mutant of MAPK was used as a substrate to avoid autokinase activity (see ``Experimental Procedures''). Each of the substrates was incubated in vitro with the different pp56 enzymes, and the reactions were subsequently resolved by SDS-PAGE (see ``Experimental Procedures'').



The results obtained (Table 2, Fig. 2and 3) show that both the wild type and active pp56 enzymes were able to phosphorylate all of the tested substrates. In contrast, the specific^d-pp56 phosphorylated only a subset of these substrates. In fact, even after long exposure, Fig. 2and Fig. 3demonstrate that the physiological substrates GAP and MAPK as well as the polyaminoacids poly-(EY) and poly-(KAEY) were very poorly utilized by specific^d-pp56 (see Table 2for quantitatitative results). This altered substrate specificity is unlikely to be due to a structural change that inactivates the kinase, since the resulting mutant still phosphorylates cdc2 peptide, MBP, enolase, and poly-(EAY) ( Fig. 2and Fig. 3, Table 2) and binds ATP with a similar affinity than the wild type protein (Table 1). Thus, specific^d-pp56 displayed a significantly different substrate specificity profile from the wild type or activated pp56 enzymes. Interestingly, no alteration in substrate specificity was obtained with deletion mutants at SH2 or SH3 domains (our data not shown). Overall, these results indicate that the specific domain contributes in directing the ability of the pp56 enzyme to phosphorylate substrate.

Expression of Wild Type and Specific Domain Deletion Mutant of pp56 in Mammalian Cells

We have compared the ability of wild type pp56 and specific^d-pp56 to mediate the induction of IL-2 production. To carry out these experiments, we have optimized the transient expression of these proteins in pp56 minus Jurkat cells (JCam1 cells). In these cells, the lack of pp56 expression correlates with the inability to produce IL-2(19) . The cells were electroporated with the cDNAs encoding the different mutants and lysed 24 h later to carry out further analysis.

To compare the expression of the different mutants, aliquots of the lysates from the different electroporated cell samples, corresponding to similar number of cells, were analyzed by Western blotting. As shown in Fig. 4A, the different mutants were expressed to a similar extent in JCam1 cells. The stability of wild type and mutated pp56 seems to be similar since the expression of wild type and mutated proteins at 24 h, analyzed by Western blotting (Fig. 4), was similar. Furthermore, fractionation experiments performed as in (7) , indicated that wild type pp56 and specific^d-pp56 display a similar intracellular distribution (not shown). The kinetic parameters from bacterial enzymes demonstrated that wild type pp56 and specific^d-pp56 display similar catalytic activities (Table 1). We have also compared the kinase activity of these mutants upon expression in T cells. As judged by the kinase assay performed in anti-pp56 immunoprecipitates (Fig. 4B), specific^d-pp56 from transfected JCam1 cells displayed a kinase activity similar to wild type pp56. Interestingly, active and wild type pp56 also displayed similar kinase activities (discussed below).

Analysis of the Biological Function of pp56-specific Domain Deletion Mutant in Vivo

The induction of the IL-2 gene was subsequently compared in JCam1 cells expressing specific^d-pp56 or wild type pp56. IL-2 gene induction was quantitated using a reporter composed of the NFAT region of the IL-2 promoter (IL-2P) linked to the chloramphenicol acetyl transferase gene(28) . The reporter DNA was electroporated into JCam1 cells, and IL-2 gene induction was analyzed by performing CAT assays in cell extracts (previously optimized to be within the linear range). Parallel quantitation of pp56 mutant expression was carried out in each experiment to ensure that the enzyme has been similarly expressed in each assay. As shown in Fig. 5, JCam1 cells, lacking pp56 expression, were unable to induce IL-2P in response to TcR triggering. However, expression of wild type pp56 resulted in the induction of the IL-2P, and this induction did not require TcR triggering (Fig. 5). Most likely, the expression of high amounts of pp56 kinase activity bypasses the necessity of cellular activation(42) . Thus, co-electroporation of the IL-2P reporter and 10-fold excess of the different plasmids encoding pp56 mutants was used as the assay to test for IL-2 gene induction.


Figure 5: IL-2 promoter induction in resting and activated JCam1 cells upon expression of pp56. JCam cells were transfected with 5 µg of NFAT-CAT (dark bars) or 5 µg of NFAT-CAT and 50 µg of pEF BOS-pp56 expression vectors (dotted bars), as described under ``Experimental Procedures.'' 6 h after electroporation the cells were activated using anti-CD3 Abs (UCHT1) at 1 µg/ml. 18 h later, the cells were lysed and reporter gene activity assessed. Percent conversion represents the ratio of radioactivity extracted in the organic phase versus total radioactivity in organic plus aqueous phases. Standard deviation from three experiments is indicated.



From the analysis of CAT activity present in cells electroporated with the different pp56 mutants (Fig. 6), we have obtained several conclusions: (i) the kinase activity of pp56 is required for IL-2P activation since the mutant lacking kinase activity did not induce the IL-2P. (ii) Overexpression of active pp56 induced the IL-2P similarly to wild type pp56, confirming that, in certain circumstances, overexpression of pp56 substitutes for pp56 activation(42) . (iii) More importantly, the IL-2P induction was significantly attenuated in cells overexpressing specific^d-pp56 compared to cells overexpressing wild type or activated pp56 (Fig. 6). The fact that the biological activity of specific^d-pp56 was only 15% compared with wild type pp56, indicated that the specific domain is required for pp56 to mediate IL-2 production.


Figure 6: IL-2 promoter induction in resting and activated JCam1 cells upon expression of specific^d-pp56. JCam1 cells were transfected with 5 µg of NFAT-CAT alone or 5 µg of NFAT-CAT plus 50 µg of mutated pEF BOS-pp56 expression vectors as indicated. After 24 h at 37 °C and 5% CO(2), the cells were lysed and reporter gene activity assessed. The percentage of conversion of radioactivity extracted in the organic phase versus total radioactivity in organic plus aqueous phases is represented. Standard deviation from four different experiments is indicated.




DISCUSSION

The src-family tyrosine kinase pp56 mediates early events in signal transduction induced via the T cell antigen receptor (19) and via CD4 and CD8 co-receptors(15) . We have studied the involvement of the unique domain (also named specific domain) of pp56 in determining its biological function. A specific domain deletion mutant was constructed and expressed in Jurkat cells lacking pp56 expression (JCam1 cells). These cells require pp56 to be functional. In fact, the lack of pp56 expression correlates with their inability to produce IL-2(19) . Expression of pp56 in this system induced the recovery of IL-2 production (Fig. 5). Transfection of the pp56 mutant lacking the specific subdomain indicated that this domain is required for pp56 to mediate its specific biological function, as measured by its ability to induce the IL-2 promoter (Fig. 6).

Several explanations may account for the loss of function of the mutant of pp56 lacking the specific domain. First, the lack of the specific domain could affect the transmission of the TcR signal to pp56. However, this is unlikely because under the overexpression conditions used in this analysis IL-2 promoter induction is independent of TcR triggering (Fig. 5). Second, an impaired association of pp56 with CD4 or CD8 (13, 14) as the cause for the defective biological function is also unlikely since neither CD4 nor CD8 seem to participate in the induction of IL-2 production of Jurkat cells. Third, the lack of the specific domain could affect the activation of the enzyme. However, under the overexpression conditions used in this analysis pp56 activation is not required to observe IL-2 promoter induction (Fig. 5). Finally the lack of pp56 function could be due to a structural change. However, the enzyme seems to maintain unaffected its ability to bind ATP (Table 1) and is able to phosphorylate enolase, cdcd2, MBP, and poly-(EAY) ( Fig. 2and Fig. 3, Table 2). Thus, we favor the hypothesis that the unique domain is essential for pp56 to trigger IL-2 production due to the involvement of this domain in regulating substrate preference.

The mechanism through which the specific domain contributes to the phosphorylation of certain substrates is not evident. One possibility is that the specific domain contributes to bind to and orientate the substrate in the enzyme so that it becomes accessible to the catalytic domain. This is supported by the observation that the specific domain of p56 binds MAPK and GAP and that this association is not equally observed with p59(17) . In addition, MAPK has been found to directly phosphorylate pp56 at residue 59 located within the specific/unique domain(43) . The crystal structure of SH2 and SH3 domains of pp56 suggests that this enzyme may form dimers(44) . If this is the case, it would be interesting to know how the specific domain interacts the catalytic core in these structures.

The highly efficient expression system used in this study allowed us to analyze the biological function of the mutants in the absence of any activation requirement. In fact, overexpression of wild type-pp56 did not require TcR signaling and was similar to active pp56 in their ability to induce IL-2P ( Fig. 5and 6). These results are in agreement with previous reports showing that overexpression of src-kinases may result in a phenotype similar to the one obtained with the activated enzyme(42) . This could occur because overexpressed wild type pp56 exceeds the amount of enzyme that endogenous p50 is able to down-regulate (39) and thus, behaves as the activated enzyme. Alternatively, the presence in the cells of very high amounts of kinase, under these expression conditions, may result in increased phosphotransfer activity similar to the one observed upon enzyme activation.

Several authors have suggested that pp56 biological function seems to depend upon the ability of the regulatory domain to associate with cellular proteins(45) . Our results along those lines illustrate that the ability of the specific domain to regulate specific substrate phosphorylation contributes to the enzyme function. However, our results also indicate that the kinase activity is required for pp56 to be functional, since no IL-2P induction was observed in cells expressing inactive pp56. The kinase activity of pp56 is also critical for T cell differentiation(3, 4) . Thus, only in particular situations(45) , the ability of pp56 to bind cellular proteins, conferred mainly by the regulatory domain, seems to be sufficient for pp56 signaling. Under physiological conditions, probably both the kinase activity and the ability to bind cellular proteins cooperate to yield pp56 biological function.

In conclusion, the specific domain of this src-family tyrosine kinase is involved in functions specific for pp56, such as CD4/CD8 binding (as we and others have previously described(13, 14) ) or selection of specific substrates ( Fig. 2and 3). The selection of specific substrates by pp56 seems essential for the enzyme to be functional in T cells. In fact, even in the cellular system used in this report (independent of CD4/CD8 co-receptors, and bypassing TcR signaling requirements) the lack of the specific domain results in impaired pp56 biological function. Thus, substrate selection, regulated by the specific domain, seems to be a novel mechanism that contributes to determine the specific biological function of src-kinases.


FOOTNOTES

*
This work was supported by the CICyT, Pharmacia, and by Public Health Service Grant CA 43803 (to T. M. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by the C.S.I.C. and by the Jose Carreras International Foundation. To whom correspondence and reprint requests should be addressed: Centro Nacional de Biotecnología, Universidad Autonoma, Campus de Cantoblanco, 28049 Madrid, Spain. Tel.: 34-1-585-4537; Fax: 34-1-585-4506 or 34-1-372-0493.

Supported by a fellowship from the Medical Research Council of Canada.

(^1)
The abbreviations used are: TcR, T cell receptor; -TcR, chain of the T cell receptor; IL-2, interleukin-2; IL-2P, interleukin-2 promoter; GAP, p21ras GTPase-activating protein; MAPK, microtubule-associated protein kinase; PAGE, polyacrylamide gel electrophoresis; CAT, chloramphenicol acetyltransferase.

(^2)
A. C. Carrera and T. M. Roberts, unpublished work.


ACKNOWLEDGEMENTS

We thank Dr. L. Ling, Dr. V. Calvo, Dr. I. Merida, and Dr. M. Cohn for their comments on the manuscript; Dr. B. Druker for the anti-phosphotyrosine antibody; Dr. D. Oprian for his help in the construction of the gene; Paul Morrison for the synthesis of the oligonucleotides; Dr. R. Perlmutter for the donation of the natural lck gene; Dr. H. Piwnica-Worms for the donation of the cdc2 peptide; Dr. F. Howard, for the donation of Gex- construct; Dr. D. Winkler for the src autophosphorylating peptide; Dr. Robert Weinberg for the baculoviral GAP; Dr. A. Weiss for the JCam1 cell line; Dr. S. Pellech for the human MAP kinase gene; and Dr. D. Cantrell for his help in the transient expression assays and for the pEF-BOS vector, NFAT-CAT construct, and UCHT1 antibody.


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