J Biol Chem, Vol. 274, Issue 29, 20056-20059, July 16, 1999

COMMUNICATION
Activation of the Lck Tyrosine-protein Kinase by the Binding of the Tip Protein of Herpesvirus Saimiri in the Absence of Regulatory Tyrosine Phosphorylation^*

David A. Hartley^§, Tamara R. Hurley, James S. Hardwick^¶, Troy C. Lund, Peter G. Medveczky, and Bartholomew M. Sefton^**

From the  Molecular Biology and Virology Laboratory, The Salk Institute, La Jolla, California 92037 and the  Department of Medical Microbiology, University of South Florida, Tampa, Florida 33612

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The Tip protein of herpesvirus saimiri 484 binds to the Lck tyrosine-protein kinase at two sites and activates it dramatically. Lck has been shown previously to be activated by either phosphorylation of Tyr³⁹⁴ or dephosphorylation of Tyr⁵⁰⁵. We examined here whether a change in the phosphorylation of either site was required for the activation of Lck by Tip. Remarkably, mutation of both regulatory sites of tyrosine phosphorylation did not prevent activation of Lck by Tip either in vivo or in a cell free in vitro system. Tip therefore appears to be able to activate Lck through an induced conformational change that does not necessarily involve altered phosphorylation of the kinase. Tip may represent the prototype of a novel type of regulator of tyrosine-protein kinases.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Herpesvirus saimiri (HVS)¹ strain C induces T cell lymphomas and lymphoid leukemias in New World monkeys (1) and can transform human T cells in vitro (2, 3). A region of the HVS genome that is essential for T cell transformation (3-5) encodes two proteins, Tip and STP (6). STP, a small, collagen-like membrane protein, has been reported to interact with the Ras protein (7). Tip (a tyrosine-protein kinase-interacting protein) binds to and activates the Lck tyrosine-protein kinase dramatically (8-12).

Lck is a lymphoid-specific member of the Src family of membrane-associated cytoplasmic tyrosine-protein kinases (13). It is essential for both the development of T cells in the thymus and for the response of T cells to signals arising from the antigen receptor (14, 15). It is likely that the interaction of Tip with Lck plays a role in the induction of T cell disease by HVS.

Lck contains two protein interaction domains: an SH3 domain that has been shown to bind to poly-proline type II helices (16), and an SH2 domain that binds to sites of tyrosine phosphorylation (17). The activity of Lck is regulated by phosphorylation. Phosphorylation of Tyr⁵⁰⁵ inhibits its activity (18, 19). Biochemical analysis (20) and structural studies of other closely related Src kinases (21-23) suggest that phosphorylated Tyr⁵⁰⁵ sequesters the SH2 domain of Lck through an intramolecular interaction and that the resultant closed conformation limits activity. Additionally, it is likely that the binding of the SH3 domain to the linker between the SH2 domain and the catalytic domain stabilizes this closed conformation (21-23).

Lck is activated by phosphorylation of Tyr³⁹⁴ (24). This site is subject to autophosphorylation that is likely to occur in an intermolecular fashion and to phosphorylation by an as yet unidentified kinase (24).

Tip appears to be anchored to the cytoplasmic face of cellular membranes by a carboxyl-terminal transmembrane domain (25). It contains two Lck-binding domains. One domain, residues 132-141, is a proline-rich SH3 domain ligand (8, 9, 27) also referred to as the SH3B domain. We will refer to this domain as Lck-binding domain 1 (LBD1). The other binding domain (8, 9, 27), residues 104-113, binds to an as yet unidentified site in the carboxyl-terminal half of Lck.² Referred to previously as the CSKH domain, we will refer to this domain as Lck-binding domain 2 (LBD2). Tip has no other recognizable protein interaction domains or catalytic domains. We have examined here the mechanism by which Tip activates Lck.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cells-- 293 (28), a human kidney cell line, or 293T (29), a derivative stably expressing SV-40 large T antigen, was grown in Dulbecco-Vogt modified Eagle's medium (Cellgro, Mediatech) plus 10% iron-supplemented calf serum (Hyclone). SAKRTLS12.1 (30), a CD4⁺ murine T cell line, was grown in Dulbecco-Vogt modified Eagle's medium plus 10% horse serum (Gemini Bioproducts). Sf9 cells (Life Technologies Inc.), and High Five cells (Invitrogen) were grown at 27 °C in Sf-900 II SFM (Life Technologies Inc.).

Expression Plasmids-- Expression of all genes in 293 cells was under control of the cytomegalovirus immediate early promoter. Wild-type (WT) murine Lck was cloned in pcDNAI/Amp (Invitrogen). Tip from HVS strain 484 was cloned in pCEP4 (Invitrogen). Human CD4 was cloned in pCMX (31). The F394Lck and F394/F505Lck mutants of Lck have been described (24); for this study they were cloned in pCEP4.

Transfection-- 293 or 293T cells, seeded on 5-cm gelatin-coated Petri dishes, were transfected with a calcium phosphate-mediated transfection system (Life Technologies Inc.) using 1-5 µg of each plasmid. Cells were lysed approximately 40-48 h following transfection.

Cell Lysis and Immunoprecipitation-- Techniques for cell lysis and immunoprecipitation have been described previously (32). In general, a lysis buffer containing 1% Nonidet P-40 was used for lysis and washing of the immunoprecipitates. Antigen-antibody complexes were isolated using Pansorbin (Calbiochem).

In Vitro Protein Kinase Assays-- In general, immunoprecipitates were resuspended in kinase buffer (40 mM sodium PIPES, pH 7.2, 10 mM MnCl₂) at 4 °C. For quantification of Lck, a fraction of the suspension was used for Western blot analysis. The remainder of the sample was subjected to a protein kinase assay using [Val⁵]-angiotensin II as an exogenous substrate (24).

Western Blotting-- In general, Western blotting was carried out as described previously (33). Filters were blocked by incubation in 3% bovine serum albumin and then stained with rabbit anti-phosphotyrosine antibodies (33), rabbit anti-Lck antibodies (34), or rabbit anti-Tip antibodies.² Bound antibodies were detected with ¹²⁵I-protein A (ICN) and a PhosphorImager.

Production of GST-Tip and Histidine-tagged Soluble Tip Proteins-- A fragment of the Tip gene encoding amino acids 92-153 (the minimal Lck-binding fragment)² was amplified by PCR using Pfu polymerase (Stratagene) and subcloned into pGEX-2T (Pharmacia). The fusion protein was expressed in BL21/DE3 Escherichia coli and isolated using standard procedures. GST-Tip was eluted in 50 mM Tris-HCl, pH 7.2, 10 mM reduced glutathione. To construct histidine-tagged, soluble Tip proteins, we inserted a stop codon at codon 188 of WT Tip, SFL/AAA Tip, which lacks a functional LBD2,² and 4P/4A Tip, which lacks a functional LBD1,² by PCR using Pfu polymerase. The PCR fragment was then subcloned into the plasmid, pFASTBAC HT (Life Technologies Inc.). The BAC-TO-BAC protocol (Life Technologies Inc.) was used to generate the recombinant bacmids and baculoviruses.

Infected High Five cells were incubated at 27 °C for 50 h. All subsequent steps were performed at 4 °C. Cells were lysed at 1 × 10⁷/ml in 1% Nonidet P-40, 50 mM sodium phosphate, pH 8.0, 150 mM NaCl, 10 mM -mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride for 20 min, and the lysates were clarified by centrifugation. Nickel-nitrilotriacetic acid Superflow beads (Qiagen) were incubated with the clarified lysate for 1-2 h. The beads were washed with 0.5 M KCl, 20 mM sodium phosphate, pH 8.0, 20 mM imidazole, 10% glycerol, and 10 mM -mercaptoethanol and then with the same buffer containing 1 M KCl. The His-tagged protein was then eluted into 100 mM EDTA, 25 mM Tris-HCl, pH 8.0, 300 mM NaCl. Purity and concentration of both proteins were estimated by SDS-polyacrylamide gel electrophoresis followed by staining with Coomassie Blue.

In Vitro Activation of Lck with Purified Tip-- We isolated Lck from SAKRTLS12.1 cells by immunoprecipitation with GK1.5 (35) anti-CD4 antibodies as described previously (36). An immunoprecipitate from 5 × 10⁶ cells was incubated in 145 µl of 0.75% Nonidet P-40, 50 mM Tris-HCl, pH ~8.3, 150 mM NaCl, and 5 µl of the elution buffer alone or approximately 50 ng of purified His-tagged Tip protein or 350 ng of GST-Tip protein in the appropriate elution buffer for 30 min at 4 °C. The immunoprecipitates were then washed twice with 1% Nonidet P-40, 50 mM Tris-HCl, pH 8.4, 150 mM NaCl and once with 50 mM Tris-HCl, pH 7.2, 150 mM NaCl. To obtain complexes of CD4 and mutants of Lck, 293T cells were co-transfected with human CD4 in pCMX and F394Lck or F394/F505Lck in pCEP4 as described above. Complexes of CD4 and Lck were immunoprecipitated using OKT4 (37) anti-human CD4 ascites. The activity of Lck was normalized to the amount of Lck present as determined by Western blotting.

Tryptic Peptide Mapping-- Tryptic digestion and peptide mapping were performed essentially as described previously (38, 39). Transfected 293 cells were labeled biosynthetically with 0.5 mCi/ml ³²P_i (carrier-free, ICN) for 5 h. Peptides were detected by autoradiography. For quantification, maps were analyzed using a Molecular Dynamics PhosphorImager and ImageQuant software.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Mutation of Sites of Tyrosine Phosphorylation Does Not Prevent the Activation of Lck by Tip-- To determine whether the phosphorylation of Tyr³⁹⁴, the site of activating tyrosine phosphorylation in Lck, was required for the activation of Lck by Tip, mutant forms of Lck lacking this site (F394Lck) or both this site and the site of inhibitory phosphorylation, Tyr⁵⁰⁵ (F394/F505Lck), were co-expressed with Tip in 293 cells, and the in vitro protein kinase activity of Lck was measured using [Val⁵]-angiotensin II as an exogenous substrate. F394Lck exhibits reduced catalytic activity when expressed alone (Fig. 1A) (24, 40). Mutation of Tyr⁵⁰⁵, which activates otherwise WT Lck (41, 42), further debilitates F394Lck (24, 40). The activities of both mutant proteins were, however, stimulated by co-expression of Tip when assayed in vitro (Fig. 1A). Additionally, activation of F394Lck and F394/F505Lck by co-expression of Tip was apparent in vivo from the stimulation of tyrosine phosphorylation of cellular proteins in the transfected 293 cells (Fig. 1B). Although the absolute activities of the mutant proteins bound to Tip were noticeably less than that of the WT protein, the degree of activation of the mutant proteins, 8-fold, was greater than the 3-fold activation seen with the WT protein.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   The effect of mutation of sites of tyrosine phosphorylation on the activation of Lck by Tip. 293T cells were transfected with plasmids encoding WT Lck, F394Lck, or F394/F505Lck with or without co-transfection with a plasmid encoding Tip. Tyrosine phosphorylation of cellular protein in the lysates was analyzed by Western blotting with antibodies to phosphotyrosine. Additionally, Lck was isolated from the singly transfected cultures by immunoprecipitation with anti-Lck antibodies and from the co-transfected cultures with anti-Tip antibodies and assayed for in vitro protein kinase activity in triplicate as described under "Materials and Methods." Lck in the immunoprecipitates was quantified by Western blotting, and activities were normalized. The results shown here are from a single experiment. A similar effect of Tip on the activity of F394Lck was seen in two other experiments. A, in vitro protein kinase activities of immunoprecipitated Lck. B, anti-phosphotyrosine Western blot of lysates of transfected cells.

Tip Can Activate Lck in Vitro-- To examine further the role of induced or altered phosphorylation of Lck in activation by Tip and to determine whether other cellular factors were required for activation, we studied the interaction of Lck and Tip in vitro. To do this, we isolated Lck bound to CD4 from the murine T cell line SAKRTLS using a monoclonal antibody to murine CD4. We expressed a soluble, His-tagged version of Tip lacking the transmembrane and extracellular domains in insect cells using a baculovirus vector. This protein was purified with a nickel resin, and preparations were more than 70% pure. Additionally, we isolated a GST fusion protein containing residues 92-153 of Tip from E. coli. This minimal binding domain fragment of Tip contains both Lck-binding domains and activates Lck dramatically when the two proteins are co-expressed in vivo (12). This fusion protein could be obtained in essentially pure form.

Both purified preparations of Tip activated Lck in vitro (Fig. 2). Activation of approximately 2-5-fold was observed routinely. Two sites in Tip bind to Lck (8, 9, 27). Mutant forms of Tip that contain only a single functional Lck-binding domain retain considerable ability to bind to Lck in vivo.² We therefore examined whether Tip containing only a functional LBD1 (SFL/AAA Tip) or only a functional LBD2 (4P/4A Tip) could activate Lck in vitro. Neither mutant Tip protein activated Lck in vitro (Fig. 2), even if used at a 9-fold higher concentration than WT Tip. The lack of activation was not due to a failure of these mutants to bind to Lck. Both mutant Tip proteins bound to Lck and became tyrosine phosphorylated during the kinase reaction (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Tip activates Lck in vitro. Complexes of WT Lck and murine CD4 were isolated from SAKRTLS cells, and complexes of F394Lck and F394/F505Lck and human CD4 were isolated from co-transfected 293T cells by immunoprecipitation with antibodies to CD4. Immunoprecipitates were incubated with purified, His-tagged Tips or with GST-Tip. In the experiment shown in panel B, the His-tagged SFL/AAA (LBD1 only) and 4P/4A (LBD2 only) Tip mutants were used at a 9-fold higher concentration than was the WT Tip protein. The complexes were then washed, and the protein kinase activity of Lck was assayed. Lck in each preparation was quantified by Western blotting. Normalized rates of incorporation of 32P into angiotensin from single, representative experiments are presented. For each panel, similar results were obtained in at least two additional experiments. Panel A: , WT Lck alone; , plus His-Tip; , plus Gst-Tip. Panel B: , WT Lck alone, , plus His-WT Tip; , plus His-SFL/AAA (LBD1 only) Tip; , plus His-4P/4A (LBD2 only) Tip. Panel C: , F394Lck Lck alone; , plus GST-Tip. Panel D: , F394/F505Lck Lck alone; , plus GST-Tip.

Activation of Lck was observed when Tip was allowed to bind to Lck in vitro under conditions where no phosphorylation of Lck could occur. This suggested that the activation observed in vitro might not require altered phosphorylation of Lck. It was possible, however, that the activation resulted from increased autophosphorylation of Lck complexed with Tip during the in vitro kinase assay. The ability of Tip to activate two mutants of Lck lacking the site of activating phosphorylation, F394Lck and F394Lck/F505Lck, was therefore examined in vitro. To do this, mutant Lck proteins and human CD4 were co-expressed transiently in 293T cells, and the complex of CD4 and Lck was isolated by immunoprecipitation with anti-CD4 antibodies. Both GST-Tip (Fig. 2) and His-Tip (data not shown) activated F394Lck and F394/F505Lck in vitro. The binding of Tip activated F394Lck approximately 6-fold and activated F394/F505Lck approximately 20-fold. Although the activity of the two mutant proteins did not rise to that of the WT protein bound to Tip, it did reach the level of the unstimulated WT protein (Fig. 2).

The Effect of the Binding of Tip on the Tyrosine Phosphorylation of the Lck Kinase-- To determine whether Tip-induced activation of Lck was accompanied by changes in the phosphorylation of Lck at either of the two sites of regulatory tyrosine phosphorylation, we carried out tryptic peptide analysis. 293 cells expressing either Lck alone or both Tip and Lck were labeled biosynthetically with ³²P_i, and Lck was isolated from cells expressing only Lck by immunoprecipitation with anti-Lck antibodies and from cells expressing both Lck and Tip with anti-Tip antibodies. 2.6-fold more ³²P was found in the tryptic peptide containing Tyr⁵⁰⁵ than in the peptide containing Tyr³⁹⁴ in Lck expressed in the absence of the Tip protein (Fig. 3). In contrast, less radioactivity (75% as much) was observed in the peptide containing Tyr⁵⁰⁵ than in the peptide containing Tyr³⁹⁴ in the population of Lck bound to Tip and isolated with anti-Tip antibodies (Fig. 3).

View larger version (53K): [in this window] [in a new window]   Fig. 3.   The population of Lck bound to Tip is phosphorylated more extensively at Tyr394. Transiently transfected 293 cells expressing either Lck or both Lck and Tip were labeled biosynthetically with 32Pi. Lck was isolated with anti-Lck antibodies from the cells expressing only Lck or with anti-Tip antibodies from the cells expressing both proteins. The proteins were subjected to tryptic digestion and two-dimensional peptide mapping on cellulose thin layer plates as described (38, 39). The origin of each map was in the lower right corner. Electrophoresis toward the positive terminal was from right to left, and chromatography was from bottom to top. The tryptic peptides containing phosphorylated tyrosines 394 and 505 are indicated.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Our results show that Tip can activate Lck under conditions where no activating changes in tyrosine phosphorylation of Lck can occur. This suggests strongly that Tip can activate Lck, at least partially, through an induced conformational change. The population of Lck bound to Tip in vivo is, however, phosphorylated to a greater extent at Tyr³⁹⁴, the site of activating phosphorylation, than at Tyr⁵⁰⁵, the site of inhibitory phosphorylation. There appear to be two steps in the activation of Lck by Tip. First, the binding of Tip induces a conformational change that leads to greater catalytic activity. Second, the Lck that has been activated by the Tip-induced conformational change undergoes increased phosphorylation at Tyr³⁹⁴ and possibly decreased phosphorylation at Tyr⁵⁰⁵, and this leads to a further increase in catalytic activity.

It is likely that Tip exerts some of its effect as a result of displacement of the SH3 domain of Lck. The SH3 domains of two Src kinases, c-Src and Hck, bind intramolecularly to polyproline type II helices in the linker between their SH2 domains and their catalytic domains (21-23). This interaction may make it difficult for the enzyme to assume a fully active conformation (21-23). The human immunodeficiency virus Nef protein, which contains an SH3 domain-binding motif, can both bind to the SH3 domain of Hck and activate Hck (43, 44). It has been proposed that the activation of Hck by Nef results from displacement of the SH3 domain from the SH2 catalytic domain linker, thereby allowing the catalytic domain to achieve a catalytically more favorable conformation.

Tip contains two Lck-binding domains. LBD1 is a proline-rich domain that binds to the SH3 domain of Lck. LBD2 binds to an as yet unidentified site in the carboxyl-terminal half of Lck.² The binding of the proline-rich LBD1 of Tip to the Lck SH3 domain could displace the SH3 domain and activate Lck by removing conformational constraints, as has been inferred to occur when Nef binds to Hck (43, 44). Consistent with this model are earlier observations that mutation of either the SH3 domain (45, 46) or of the SH2 catalytic domain linker (47, 48) activates Lck partially.

The mechanism of activation of Lck by Tip is not identical to that by which Nef activates Hck. Tip containing only a functional LBD1 can bind to Lck in vitro through the Lck SH3 domain, but this does not activate Lck detectably (Fig. 2). This suggests that the binding of LBD2 to Lck also participates in the activation of Lck. In that LBD2 binds to the carboxyl-terminal half of the protein that consists largely of the catalytic domain, it is possible that LBD2 alters the conformation of the catalytic domain directly. How this might occur is not yet known. LBD2 has sequence identity with the I helix in the Lck catalytic domain (8). It is possible that it displaces this helix and that such a displacement is activating. Alternatively, this binding domain could affect the positioning of the C helix in the catalytic domain in a manner similar to that by which cyclin activates cyclin-dependent kinases (49).

Tip differs from other known regulatory proteins of Src kinases in its bimodal binding to Lck. The middle T antigen of polyoma virus binds only to the carboxyl termini of Src kinases (50) and activates them apparently by interfering with inhibitory phosphorylation (26). The HIV Nef protein appears to function only as an SH3 domain ligand (43, 44) and is not known to interact directly with the catalytic domain of Hck. A question of particular interest is whether Tip has a cellular homologue that functions as a natural, endogenous activator of Lck in T cells. If so, this would suggest that tyrosine-protein kinases may normally be subject to an as yet unappreciated form of regulation and that cellular Tip-like activators of other tyrosine-protein kinases may exist.

	ACKNOWLEDGEMENTS

We thank Bob Hyman for the SAKRTLS cells and Wei Jiang for helpful advice about insect cells.

	FOOTNOTES

^* This work was supported in part by Grants CA14195, CA42350, and CA42364 from the National Cancer Institute of the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Supported by Training Grant T32-CA09523 from the National Cancer Institute of the National Institutes of Health and fellowship PF-4483 from the American Cancer Society. Present address: Dept. of Biology, Boston University, Boston, MA 02215.

^¶ Supported by Training Grant 2T32-GM07240 from the General Medical Institute of the National Institutes of Health. Present address: Dept. of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138.

^** To whom correspondence should be addressed: Molecular Biology and Virology Lab., Salk Inst., 10010 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-453-4100 (ext. 1461); Fax: 619-457-4765; E-mail: sefton@salk.edu.

² D. A. Hartley, K. Amdjadi, T. C. Lund, P. G. Medveczky, and B. M. Sefton, submitted for publication.

	ABBREVIATIONS

The abbreviations used are: HVS, herpesvirus saimiri; LBD, Lck-binding domain; PIPES, 1,4-piperazinediethanesulfonic acid; PCR, polymerase chain reaction; GST, glutathione S-transferase; WT, wild type; HIV, human immunodeficiency virus.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES