©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Lck-binding Elements in Tip of Herpesvirus Saimiri (*)

(Received for publication, May 3, 1995; and in revised form, June 27, 1995)

Jae U. Jung (1)(§) Sabine M. Lang (1) Ute Friedrich (2) Toni Jun (3) Thomas M. Roberts (3) Ronald C. Desrosiers (1) Brigitte Biesinger (2)

From the  (1)Department of Microbiology and Molecular Genetics, New England Regional Primate Research Center, Harvard Medical School, Southborough, Massachusetts 01772-9102, the (2)Institut für Klinische und Molekulare Virologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Schlossgarten 4, 91054 Erlangen, Germany, and the (3)Dana-Farber Cancer Institute, Department of Cellular and Molecular Biology, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A protein called Tip (tyrosine kinase interacting protein) of herpesvirus saimiri associates with Lck in virus-transformed human T cells and is an in vitro substrate for Lck kinase. Mutational analyses of a GST-Tip fusion protein revealed that binding to Lck requires putative SH3 binding sequences and a sequence homologous to the carboxyl terminus of Src-related kinases. These sequences are referred to as SH3-Binding (SH3B) and C-terminal Src-related Kinase Homology (CSKH) elements. Peptide fragments as short as 37 amino acids containing both SH3B and CSKH elements were sufficient to form a stable complex with Lck in vitro. Furthermore, these same sequences of Tip were necessary for in vivo association with Lck when Tip and Lck were expressed transiently in COS-1 cells or stably in Rat-1 cell lines. These results demonstrate that the CSKH element of Tip participates in the binding of sequences within Lck. Tip of herpesvirus saimiri has apparently acquired such CSKH and SH3B elements for the purpose of targeting cellular protein kinases. The interaction of Tip with Lck may influence Lck kinase activity or its binding to other cellular proteins and thereby alter Lck function in T cells infected by h. saimiri.


INTRODUCTION

Proliferation of mature T cells is induced by a multistep process following exposure to antigen-presenting cells. Antigen presentation can be mimicked by the cross-linking of the T cell receptor and certain T cell surface molecules with specific antibodies(1, 2) . Both modes of T cell receptor stimulation lead to rapid tyrosine phosphorylation of cellular proteins followed by an increase in intracellular free calcium. Phosphorylation results from the sequential activation of several tyrosine kinases(1, 3, 4) . A central role in T cell activation has been assigned to the tyrosine kinase Lck. A T cell line defective for lck expression fails to induce tyrosine phosphorylation after stimulation(5) . As a member of the Src kinase family, Lck consists of a short unique region, an SH3 (^1)and an SH2 domain followed by the catalytic domain and a regulatory carboxyl terminus. A myristylation site at the amino terminus attaches the protein to the membrane. The amino-terminal unique sequences are responsible for binding to membrane-anchored surface molecules like CD4 or CD8(6) . The SH2 and SH3 domains bind specific substrates and downstream effectors of Lck(7) .

Herpesvirus saimiri (HVS), a member of the 2 group of herpesviruses, naturally infects squirrel monkeys (Saimiri sciureus) of South America. HVS persists in T lymphocytes of the natural host without any apparent disease, but infection of other species of New World primates results in fulminant lymphomas, lymphosarcomas, and leukemias of T cell origin(8) . A pronounced divergence among different strains of HSV has been localized to the left-end of viral genomic DNA, and this has led to classification into three subgroups, A, B, and C (9, 10) . Strains from subgroups A and C are highly oncogenic and are able to immortalize common marmoset T lymphocytes in vitro to interleukin 2-independent growth(11, 12) . Subgroup C strains are further capable of immortalizing human and rhesus monkey lymphocytes into continuously proliferating T cell lines(13) .

Nucleotide sequence analysis of the entire HVS genome revealed a number of genes with homology to cellular proteins, some of which are likely to contribute to T cell transformation(14) . These include the STP oncogene(15, 16, 17) , superantigen homolog(18) , interleukin 8 receptor homolog(19) , and virus-encoded cyclin(19, 20) . Recently, the product of the gene (orf1) adjacent to STP-C488 at the left end of the viral genome was identified in transformed T cells(21) . Orf1 did not show transforming activity in rodent fibroblast cells(17) , but the protein was found to be associated with the major T cell tyrosine kinase Lck and phosphorylated on tyrosine residues by purified Lck in several cell-free assay systems(21) . Thus, it was designated as tyrosine kinase interacting protein (Tip)(21) . However, any role for Tip in viral-induced cell growth transformation is yet to be defined.

As a first step toward analyzing the function of Tip, we have now localized the Lck-binding domains of Tip protein. Our experiments show that two structural motifs as well as the connecting sequences are necessary and sufficient for efficient Lck binding activity. These two structural elements are a proline-rich segment similar to sequences known to bind to SH3 domains and a motif homologous to the carboxyl-terminal regulatory region of Src-related tyrosine kinases. HVS has apparently acquired these structural elements in Tip for binding to cellular protein kinases.


MATERIALS AND METHODS

Cell Culture and Transfection

Rat-1 and COS-1 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Sf9 cells were maintained at 27 °C in Grace's medium containing 10% fetal calf serum, yeastolate, and lactalbumin hydrolysate. A DEAE-dextran transfection procedure was used for transient expression in COS-1 cells, and CaPO(4) transfection was used for the establishment of stably-expressing Rat-1 cell lines. 48 h after CaPO(4) transfection, G418 (500 µg/ml) or puromycin (5 µg/ml) was added to the medium for selection, and cells were split when confluent.

Antibodies

AU-1 monoclonal antibody recognizing the DTYRYI epitope from bovine papillomavirus L1 capsid protein was purchased from Berkeley Antibody (Richmond, CA). Rabbit polyclonal anti-Lck antibody generated against synthetic peptide based upon residue 22-51 of the amino terminus of human Lck was purchased from UBI (Lake Placid, NY).

Plasmid Constructions

DNA containing the tip open reading frame was amplified from HVS strain C488 virion DNA by polymerase chain reaction using primers containing EcoRI and XhoI recognition sequences at the ends. Amplified DNA was ligated into the EcoRI and XhoI cloning sites of the pSP72 vector (Promega Biotech, Madison WI). For AU-1 tagging, 5` primer CGC GGA TCC ATG GAC ACC TAT CGC TAT ATA GCA AAT GAA GGA GAA was used for polymerase chain reaction amplification and AU-1-tagged tip DNA was cloned into pSP72 vector. AU-1-tagged tip DNA was completely sequenced to verify 100% agreement with the original sequence. pFJ vector was derived from pSRalpha-0 (22) by introducing multicloning restriction enzyme sites at the HindIII site, and pBKCMV vector containing CMV early promoter and neomycin resistance gene was purchased from Stratagene (San Diego, CA). For transient or stable gene expression, the 0.7-kilobase pair EcoRI and NdeI or BglII and XhoI DNA fragment from pSP-AU1-tip described above was inserted into the EcoRI and NdeI sites of the pFJ vector or BamHI and XhoI sites of pBK vector, respectively. For GST fusion expression, the EcoRI and XhoI DNA containing tip gene from pSP72-AU1-tip was cloned into the EcoRI and XhoI sites of pGEX-4T (Pharmacia Biotech Inc.). An EcoRI DNA fragment containing the human lck gene was cloned into pFJ and pBabe-puro retroviral vector.

All mutations in the tip gene were generated with polymerase chain reaction using oligonucleotide-directed mutagenesis(23) . The amplified DNA fragments containing mutations in tip were purified and cloned into pBluescript KS+ vector. Each tip mutant was completely sequenced to verify the presence of the mutation and the absence of any other changes. After confirmation of the DNA sequence, DNA containing the desired tip mutation was recloned into pFJ or pBKCMV vector for gene expression or into pGEX4T for production of bacterial GST-Tip fusion protein.

Metabolic Labeling, Immunoprecipitation, and Immunoblot

COS-1 cells and Sf9 insect cells were labeled with [S]methionine and [S]cysteine (DuPont NEN) as described previously(20) . Cells were harvested and lysed with lysis buffer (0.15 M NaCl, 0.5% Nonidet P-40, and 50 mM Hepes buffer (pH 8.0)) containing 0.1 mM Na(2)VO(3), 1 mM NaF, and protease inhibitors (leupeptin, aprotinin, phenylmethylsulfonyl fluoride, pepstatin, and bestatin). Immunoprecipitated proteins from cleared cell lysates were separated by SDS-PAGE and detected by autoradiography of the dried gel slabs. For protein immunoblots, polypeptides in cell lysates corresponding to 10^5 cells were resolved in SDS-PAGE and transferred to nitrocellulose membrane filter. Immunoblot detection was performed with a 1:2000 dilution of primary antibody and developed as described by the manufacturer of the enhanced chemiluminescence system (ECL, Amersham Corp.).

Construction of Recombinant Baculoviruses

EcoRI-PvuII fragments containing AU-1-tagged tip gene from pFJ-AU1-tip were inserted into the EcoRI-SmaI sites of the baculovirus transfer vector pAcSG1 (Pharmingen, San Diego, CA). For the construction of Lck baculoviral vector, the 1.5-kilobase pair NcoI and KpnI fragment containing the human lck gene was inserted into the NcoI and KpnI sites of the baculovirus transfer vector pAcSG1 (Pharmingen, San Diego, CA). Recombinant baculoviruses were generated as described by the manufacturer of BaculoGold transfection kit (Pharmingen, San Diego, CA).

Phosphoamino Acid Assays

P-Labeled proteins from in vitro kinase reactions with [-P]ATP were resolved by SDS-PAGE, transferred to nitrocellulose membranes, localized by direct autoradiography, and cut from the membranes. The membrane fragments containing labeled proteins were directly hydrolyzed for 1 h at 110 °C in 6 N HCl. Phosphoamino acids were resolved by two-dimensional electrophoresis with unlabeled phosphoamino acids (phosphoserine, phosphothreonine, and phosphotyrosine). The positions of unlabeled phosphoamino acid standards were detected by staining with ninhydrin.

Expression and Purification of GST Fusion Proteins

Bacterial GST fusion expression vector containing the tip gene was introduced into Escherichia coli XL-1. GST fusion protein expression and purification were performed essentially as described by Smith and Johnson(24) . Protein concentration was quantitated by BCA protein assay kit with bovine serum albumin as standard (Pierce).

In Vitro Binding of GST Fusion Proteins to Lck

5 µg of purified GST fusion proteins noncovalently coupled to glutathione-Sepharose beads were mixed with precleared S-labeled insect cell lysates for 1 h at 4 °C and washed 4 times in lysis buffer. Bound proteins were resolved by SDS-PAGE and visualized by autoradiography.

In Vitro Kinase Assays

For in vitro protein kinase assays, immune complexes prepared as described above were washed once more with kinase buffer (10 mM MgCl(2), 1 mM dithiothreitol, 10 µM unlabeled ATP, 20 mM Tris (pH 7.0)) and resuspended with 20 µl of the same buffer containing 5 µCi of [-P]ATP (6000 Ci/mmol, DuPont NEN) for 15 min at room temperature.


RESULTS

In Vitro Binding of GST-Tip to Lck

Inspection of the Tip sequence revealed three features of potential significance (Fig. 1). 10 amino acids near the amino terminus (KLSSCSEETT) are repeated from residues 25-34 and from residues 79-88. 10 amino acid residues of Tip from 146-155 have a high homology with the carboxyl-terminal region of Src-related kinases, which is known to be involved in regulation of these kinases(25) . We refer to this motif as C-terminal Src-related Kinase Homology (CSKH). Finally, a proline-rich region from amino acid residue 174 to 183 shows homology with consensus sequences for binding to SH3 domains of signal transducing proteins(26, 27, 28) . This is designated as an SH3-Binding (SH3B) motif. Although a specific homology to consensus sequences of tyrosine-phosphorylated SH2 binding motifs described by Songyang et al.(29) is yet to be found, four tyrosine residues (Tyr, Tyr, Tyr, and Tyr) are present in the central part of Tip.


Figure 1: Schematic diagram of the structural organization of Tip and summary of in vitro binding of wild-type and mutant forms of GST-Tip to Lck. Box R is the repeat of KLSSCSEETT sequence, Y is tyrosine residue, and hydrophobic at the carboxyl terminus is the hydrophobic domain. Amino acid residues in the shadedbox of the CSKH motif represent the sequence highly homologous to the carboxyl termini of Src-related kinases. XPPLPXR is the consensus sequence for SH3 binding motif(26, 27, 28) . In the Tip mutant called mSH3B, proline residues at amino acids 175, 177, 178, 180, 181, and 183 in SH3B element were changed to alanines. Results for in vitro binding assays with various GST-Tip fusion proteins with Lck from insect cells were summarized in the bottom of figure. ++++, strong binding; -, no binding.



To identify structural elements required for complex formation between Lck and Tip, we first established an in vitro binding system. Gene sequences for Tip lacking the carboxyl-terminal hydrophobic region were fused to the GST gene to produce bacterial fusion protein. Purified GST-Tip protein was mixed with precleared S-labeled insect cell lysates containing Lck, washed extensively, and resolved by SDS-PAGE. As shown in Fig. 1and 2A, GST-Tip fusion protein efficiently bound in vitro to Lck, while GST protein alone did not. Additionally, the GST-Tyr mutant, which lacks the amino-terminal repeat sequence, still bound to Lck as well as wild-type GST-Tip. Thus, purified GST-Tip protein bound efficiently to Lck in vitro, and the amino-terminal repeat sequence was not required for in vitro complex formation with Lck.

SH3B, CSKH, and Spacer Are Necessary and Sufficient for Binding to Lck in Vitro

Binding to SH2 domains requires tyrosine phosphorylation prior to complex formation(29, 30) . Since bacterial proteins do not contain such tyrosine phosphorylations, efficient binding of bacterial GST-Tip to Lck was not likely to occur through the SH2 domain of Lck. To investigate whether the SH3B motif was required for in vitro complex formation with Lck, two mutants were generated by site-directed mutagenesis and cloned into GST fusion expression vector. First, proline residues at amino acid numbers 175, 177, 178, 180, 181, and 183 at the SH3B motif of GST-Tyr were changed to alanine residues to generate GST-Tyr/mSH3B. Second, the SH3B motif was deleted to construct GST-Thr, which contains only 37 amino acids from the carboxyl terminus. As shown in Fig. 2A, GST-Tyr/mSH3B and GST-Thr were severely deficient for binding to Lck in vitro, indicating that the SH3B motif is important for in vitro complex formation with Lck. Finally, GST-SH3B containing only the SH3B motif of Tip from amino acids 175-226 was used for in vitro binding. As shown in Fig. 2A, GST-SH3B was not sufficient to form a complex with Lck in vitro. Thus, the SH3B motif appears to be essential but not sufficient for complex formation with Lck in vitro.


Figure 2: SH3B motif of Tip is essential but not sufficient for binding to Lck. A, SH3B of Tip is important for binding to Lck. B, additional motifs between Tyr(Y) and Tyr(Y) are required for binding to Lck. Glutathione-Sepharose beads containing 5 µg of GST or various GST-Tip fusion proteins were mixed with S-labeled cell lysates containing Lck from insect cells followed by three washing steps with lysis buffer. 5 µg of anti-Lck antibody was used for immunoprecipitation of Lck as control (B, lane1). Associated proteins were resolved in SDS-PAGE and autoradiographed.



Since the SH3B element is essential but not sufficient for binding to Lck in vitro, we investigated which additional regions of Tip may participate in binding to Lck. A series of deletion mutants of the tip gene were generated by polymerase chain reaction and fused to the GST fusion expression vector. As described above, purified bacterial GST fusion proteins were mixed with precleared S-labeled lysates from Sf9 insect cells infected with Lck baculovirus, and associated proteins were resolved by SDS-PAGE. GST-Tip, GST-Tyr, GST-Tyr, and GST-Tyr efficiently bound to Lck in vitro (Fig. 2B). However, the GST-Tyr deletion mutant no longer bound to Lck (Fig. 2B). Again, GST-SH3B did not bind to Lck (Fig. 2B). Thus, amino acids between 127 and 155 together with the SH3B element are required for efficient Lck binding in vitro.

The requirement for amino acids 127-155 suggested that CSKH is likely to be important for Lck binding. To test the importance of this CSKH element for Lck binding, additional deletion mutants were generated and fused into GST expression vector. GST-Tyr, GST-Thr and GST-Glu were capable of binding to Lck in vitro, while GST-Thr/mSH3B, GST-Glu/mSH3B, GST-Tyr, and GST-Pro were deficient in binding to Lck (Fig. 3). To investigate this further, point mutations were introduced into the CSKH element of GST-Glu, whose binding activity is similar to that of wild-type GST-Tip (see Fig. 5). GST-Glu/Ser Arg/Phe His/Leu Met contained changes of serine to arginine, phenylalanine to histidine, and leucine to methionine, and GST-Glu/Phe Cys had change of phenylalanine to cysteine. As shown in Fig. 4, both of GST-Glu/Ser Arg/Phe His/Leu Met and GST-Glu/Phe Cys containing point mutations in CSKH region had significant decreases in binding to Lck. Finally, GST-Glu/Arg, which contained only 37 amino acids spanning CSKH and SH3B elements, was capable of binding efficiently to Lck; GST-Glu/Arg exhibited only a slight reduction of Lck-binding activity when compared with GST-Glu (Fig. 4).


Figure 3: CSKH is the additional motif required for Lck binding to Tip. Experimental procedures and exposure time were the same as described in Fig. 2. Box ``mSH3B'' represents the mutations of proline residues to alanines in the SH3B motif as described in Fig. 1. Boldface letters represent the amino acid sequences in the CSKH motif homologous to carboxyl termini of Src-related kinases. ++++, strong binding; -, no binding.




Figure 5: CSKH, spacer, and SH3B motifs are necessary and sufficient for efficient binding to Lck. Experimental procedures were the same as described in Fig. 2. GST, Tip, Tyr (Y), Glu (E), Glu/Arg (E/R), and SH3B are described in the text and in Fig. 2and Fig. 4. CSKH/SH3B construct derived from GST-Glu/Arg contains the deletion of 18 amino acids between CSKH and SH3B motif. Glu/Gly construct contains the CSKH motif and 18 intervening amino acid sequence without SH3B motif. The gel was overexposed to show the lack of Lck-binding of CSKH/SH3B, Glu/Gly, and SH3B fusion proteins.




Figure 4: Mutational analysis of CSKH motif. Experimental procedures were the same as described in Fig. 2. Glu/Ser Arg/Phe His/Leu Met (E/SR/FH/LM) and Glu/Phe Cys (E/FC) contain the mutations as described in the context. Glu/Arg (E/R) contains 37 amino acids spanning CSKH and SH3B elements. Box SH3B represents the SH3B element. ++++, strong binding; +, weak binding.



18 amino acids that are present between the CSKH and SH3B elements in Tip may link these two elements. We thus analyzed the properties of a mutant with these sequences deleted from the GST-Glu/Arg construct. Overlapping oligonucleotides capable of encoding CSKH and SH3B were fused to generate mutant construct, GST-CSKH/SH3B, which is missing the coding sequences for the intervening amino acid residues 156-173. Again, GST-CSKH/SH3B fusion protein was mixed with S-labeled cell lysates containing Lck protein. GST-CSKH/SH3B mutant protein with the 18 amino acids deleted showed dramatically diminished binding to Lck, while GST-Glu and GST-Glu/Arg bound efficiently to Lck under the same conditions (Fig. 5). This demonstrates that the region between CSKH and SH3B is required for efficient Lck binding and is likely to function as a spacer between CSKH and SH3B elements.

Finally, we examined whether the CSKH motif and the intervening 18 amino acid region were capable of binding to Lck in vitro. To study this, GST-Glu/Gly fusion construct containing CSKH and the 18-amino-acid intervening sequence without the SH3B motif was generated, and binding activity to Lck in vitro was analyzed. As shown in lane6 of Fig. 5, GST-Glu/Gly was grossly deficient for binding to Lck.

Thus, the CSKH, spacer and SH3B motifs are necessary and sufficient for Lck-binding in vitro.

Association of Tip with Lck in Insect Cells

To study the association of Tip with Lck, Lck and Tip were produced in Sf9 insect cells using baculovirus vectors. The tip gene was modified to encode an AU-1 epitope tag at the amino terminus. After baculovirus-infected Sf9 cells were labeled with [S]methionine, anti-AU-1 antibody and anti-Lck antibody were used for immunoprecipitation. S-Labeled Tip from insect cells migrated as 40 kDa in SDS-PAGE similar to that described previously (Fig. 6A)(21) . S-Labeled 56-kDa Lck protein was detected by immunoprecipitation with anti-Lck antibody (Fig. 6A). To investigate the complex formation between Tip and Lck, Sf9 cells were coinfected with recombinant Tip and Lck baculoviruses. As shown in Fig. 6A, coinfection of Sf9 cells with Tip and Lck baculoviruses resulted in the association of Tip with Lck.


Figure 6: Association of Tip with Lck in insect cells. Sf9 insect cells were infected with recombinant baculoviruses expressing Tip and Lck as indicated at the bottom of the figure. A, complex formation between Tip and Lck. After 48 h of infection, cells were labeled with [S]methionine. Cell lysates were used for precipitations with anti-AU-1 (lanes1 and 2) and anti-Lck (lanes3 and 4) antibody and immune complexes were separated by SDS-PAGE. Overnight exposure. B, in vitro kinase assays of immune complexes of anti-AU-1 and anti-Lck antibodies. Immunoprecipitations were performed with anti-AU-1 (lanes1 and 2) and anti-Lck antibody (lanes3 and 4). These immune complexes were assayed for kinase activity with [-P]ATP, and labeled proteins were separated by SDS-PAGE, 2-s exposure. C, complex formation of wild-type and mutant forms of Tip with Lck in insect cells. Mutant Tip/Tyr Ser (Y S) and Tip/mSH3B were described in the text. After 48 h of infection, cells were labeled with [S]methionine. Cell lysates were used for precipitations with anti-AU-1 antibody. After immunoprecipitation, proteins were separated by SDS-PAGE. The molecular markers are ovalbumin (45 kDa) and bovine serum albumin (69 kDa).



Immune complexes from insect cells were subjected to in vitro kinase reaction. Anti-Lck immune complexes from Sf9 cells infected with Lck baculovirus alone or coinfected with Lck and Tip baculoviruses were used for the assay of in vitro kinase activity. 56-kDa phosphorylated Lck was detected in both cells, while 42-43-kDa phosphorylated Tip was additionally detected from Sf9 cells coinfected with Lck and Tip baculoviruses (Fig. 6B, lanes3 and 4). The strongly phosphorylated 42-43-kDa protein in coinfected cells was shown to be Tip on the basis of its presence only when Tip-expressing virus was included and by its precipitation with the AU-1-tag antibody (Fig. 6B). AU-1 immune complexes from Sf9 cells infected with Tip baculovirus alone showed weak phosphorylation of Tip protein (Fig. 6B, lane1). Phosphoamino acid assay of the weakly phosphorylated Tip in the absence of Lck revealed phosphorylation mainly at serine and threonine residues, suggesting the possible presence of serine and threonine kinases in the AU-1 complexes (Fig. 7A). Lck kinase activity in AU-1 immune complexes strongly phosphorylated both Lck and Tip (Fig. 6B, lane2). Phosphoamino acid assays of Tip phosphorylation in the presence of Lck showed that Tip contained P-labeled phosphorylation predominantly at tyrosine residues and a minor amount of phosphorylation at serine and threonine (Fig. 7B). Lck demonstrated phosphorylation only at tyrosine residues (Fig. 7C). The strong phosphorylation of Tip and Lck shown in Fig. 6B was detected with an exposure time of only 2 s. Migration of Tip protein by SDS-PAGE was slightly retarded after association with Lck protein (Fig. 6, A and B, lane2). This slower migration was likely due to the phosphorylation of Tip by Lck.


Figure 7: Two-dimensional phosphoamino acid analysis of in vitro phosphorylated Tip and Lck proteins. Sf9 insect cells were infected with recombinant Tip and/or recombinant Lck baculoviruses. After 48 h of infection, cell lysates were used for precipitations with anti-AU-1 (A and B) or anti-Lck (C) antibody. These immune complexes were assayed for kinase activity with [-P]ATP, and phosphorylated proteins were subjected to phosphoamino acid analysis. Phosphoamino acid analyses were performed with labeled Tip from insect cells infected with Tip recombinant baculovirus (A), labeled Tip from insect cells infected with Tip and Lck recombinant baculoviruses (B), and labeled Lck from insect cells infected with Tip and Lck recombinant baculoviruses (C). S, phosphoserine; T, phosphothreonine; Y, phosphotyrosine.



Since the SH3B motif of Tip is important for in vitro binding to Lck, two mutants of tip were generated and expressed in Sf9 insect cells using baculovirus. The Tip/Tyr Ser mutant contains the change of tyrosine at amino acid number 114 to serine, and Tip/mSH3B has the changes of proline residues within the SH3B motif to alanines as described in Fig. 1. Wild-type Tip, Tip/Tyr Ser, and Tip/mSH3B were expressed in insect cells with or without Lck using the baculovirus expression system. Tip/Tyr Ser migrated slightly faster than wild-type Tip in SDS-PAGE (Fig. 6C, lanes2 and 5). This was also detected in other cell types including COS-1 and Rat-1 cells (data not shown). Coinfection of Sf9 cells with Lck and wild-type or mutant Tip baculoviruses showed that wild-type Tip and Tip/Tyr Ser efficiently formed complexes with Lck, while the Tip/mSH3B mutant showed a dramatic decrease of binding activity with Lck (Fig. 6C).

SH3B and CSKH Motifs Are Required for in Vivo Association of Tip with Lck in COS-1 and Rat-1 Cells

Only 37 amino acids containing CSKH, spacer, and SH3B elements were sufficient to form stable complexes with Lck in vitro. In order to demonstrate the importance of SH3B and CSKH motifs for in vivo association of Tip with Lck, lck and tip genes were expressed in COS-1 and Rat-1 cells. Wild-type and mutant forms of the tip gene were cloned into pBKCMV vector, which contained the SV40 origin and neomycin-resistance gene for selection. The tip gene was expressed from the CMV early promoter in this construct. Mutants Tip/mSH3B, Tip/DeltaCSKH and Tip/mSH3B/DeltaCSKH were used to analyze complex formation with Lck in these cells. Tip/mSH3B contains changes of prolines to alanines in the SH3B motif. Tip/DeltaCSKH contains the deletion of amino acid residue 146-155, which spans the CSKH motif. Tip/mSH3B/DeltaCSKH contains both of the point mutations in the SH3B motif and the deletion of the CSKH motif. Finally, 1.5 kilobase pairs of human lck cDNA were cloned into pFJ vector for transient expression in COS-1 cells and into the retroviral vector pBabe-puro for stable expression in Rat-1 cells.

After transfection of wild-type or mutant forms of tip gene with lck, cells were labeled with [S]methionine and [S]cysteine. Half of the cell lysates were used for in vitro kinase assay after precipitation with anti-Lck antibody, and the other half of the cells were used for immunoprecipitation with anti-AU-1 antibody to show the level of Tip expression. Immunocomplexes precipitated by anti-Lck antibody were employed for in vitro kinase assay with [-P]ATP to show association with and phosphorylation of Tip. Mutations in SH3B and/or CSKH motifs greatly diminished complex formation with Lck, while wild-type Tip was efficiently associated with Lck in COS-1 cells (Fig. 8A). Under these conditions, similar amounts of wild-type and mutant Tip were expressed in COS-1 cells (bottom of Fig. 8A). Thus, the SH3B and CSKH motifs are important for association of Tip with Lck in COS-1 cells.


Figure 8: In vivo association of Tip with Lck in COS-1 and Rat-1 cells. A, association of Tip with Lck in COS-1 cells. Expressed wild-type (wt) and mutant forms of Tip were indicated at the bottom of the figure. COS-1 cells were transfected with pFJ-Lck alone (lane2) or together with pBKCMV expressing wild-type or various mutant forms of Tip as indicated at the bottom of figure. After 48 h of transfection, cells were labeled with [S]methionine and [S]cysteine. Half of the cell lysates was used for in vitro kinase assays of Lck immune complexes (top), and the other half of the cells was used for immunoprecipitation with anti-AU-1 antibody to show the level of the expression (bottom). Untransfected COS-1 cells (lane1) were used as a control. Labeled proteins were fractionated by SDS-PAGE and detected by autoradiography. B, complex formation between Tip and Lck in Rat-1 cells. Stably transfected Rat-babe-Lck cells expressing wild-type Tip, Tip/mSH3B, or Tip/DeltaCSKH were established by transfection with pBKCMV constructs. Expression of wild-type and mutant forms of Tip are indicated at the bottom of the figure. Rat-babe (lane1) and Rat-babe-Lck (lane2) were used for controls. Lysates of 1 10^7 cells were used for immunoprecipitation with anti-Lck and anti-AU-1 antibodies. Immune complexes were subjected to in vitro kinase assays with [-P]ATP. Labeled proteins were fractionated by SDS-PAGE and detected by autoradiography. The level of expression of Tip was detected by immunoblot with whole cell lysates corresponding to 1 10^5 cells. Immunoblot detection was performed with a 1:1000 dilution of primary AU-1 antibody and developed with ECL (bottom). Arrows indicate Lck and Tip proteins.



tip and lck genes were also stably expressed in Rat-1 fibroblast cells. After transfection of Rat-1 cells with recombinant retroviral vector pBabe-Lck, Rat-babe-Lck cell line was selected by growth in medium containing 5 µg/ml of puromycin. Expression of Lck in Rat-babe-Lck cells was confirmed by immunoblot with anti-Lck antibody (data not shown). To express the wild-type Tip and mutant forms of Tip, Rat-babe-Lck cells were transfected with pBKCMV constructs containing wild-type Tip, Tip/mSH3B, or Tip/DeltaCSKH and then selected with 500 µg/ml of G418. Similar amounts of tip gene expression were detected in these cells by immunoblot with AU-1 antibody (bottom of Fig. 8B). To investigate stable complex formation between Tip and Lck in Rat-1 cells, anti-Lck, and anti-AU-1, immune complexes were subjected to in vitro kinase assays. As shown in Fig. 8B, mutant Tip/mSH3B and Tip/DeltaCSKH showed a dramatic reduction in complex formation with Lck in Rat-1 cells when compared with wild-type Tip, which was efficiently associated with Lck under the same conditions. In addition to Lck protein, 62- and 110-kDa phosphorylated proteins were detected in anti-AU-1 immune complexes from Rat-1 cells expressing wild-type tip gene (Fig. 8B). Thus, SH3B and CSKH motifs are essential for in vivo complex formation of Tip with Lck in COS-1 and Rat-1 cells consistent with the in vitro binding assays.


DISCUSSION

Tip protein encoded by the oncogenic herpesvirus saimiri strain C488 was previously shown to be expressed in virus-transformed T cells and to be associated with the major T cell tyrosine kinase Lck(21) . We have now identified sequences within Tip that are responsible for the efficient binding to Lck in vitro as well as in vivo. A segment of only 37 amino acids containing a region homologous to the carboxyl-terminal regulatory domain of Src-related kinases and a proline-rich putative SH3 binding site linked by a short spacer region were sufficient for efficient binding to Lck.

SH3 domains are small units of 55-70 amino acids found in nonreceptor tyrosine kinases and other signaling molecules such as phospholipase C, PI 3-kinase, and Grb2. They mediate protein-protein interactions and also link these proteins to the cytoskeletal architecture(3, 31) . The identification of several SH3 binding proteins by expression cloning and affinity chromatography has revealed that SH3 domains bind to short proline-rich peptide motifs of 9 or 10 amino acids(26, 27, 28) . Binding assays with biased recombinatorial peptide libraries confirmed these findings and defined the roles of the individual amino acid residues(26) . The proline-rich region of Tip has high identity with an SH3 binding consensus sequence and has been shown here to be essential for Lck binding in vitro and in vivo. Thus, the presence of a proline-rich region within Tip with high homology to known SH3 binding sequences and its importance for the binding to Lck suggest that Tip is likely to associate with Lck at least in part through its SH3 domain.

The 10-amino-acid CSKH motif in Tip has high homology with the carboxyl terminus of Src-related kinases, which represents a part of the conserved kinase domain XI and the regulatory region(32) . It has 70-80% identity with the corresponding regions of Src, Yes, Fyn, and Fgr; it has lower homology with that of Lck. Computer searches with the CSKH sequence of Tip revealed a number of other proteins with a high degree of homology; a mitochondrial F1 ATP synthase alpha chain and several genes encoding enzymes in carbohydrate metabolism(33, 34, 35) . However, the role of the homologous sequences in these proteins has not been studied.

Evidence is accumulating that the tyrosine protein kinase activity of the Src family is regulated primarily through phosphorylation of the carboxyl-terminal tyrosine residue such as Tyr for Src or Tyr for Lck(7, 25) . In addition, several reports have suggested that the region surrounding the carboxyl-terminal tyrosine residue is important for kinase activity and protein interaction. For example, the addition or deletion of amino acids from this region of the Src activates the kinase activity and thus the transforming potential(36) , and this same region of Src appears to be required for stable association with polyoma virus middle T antigen (25) . These results suggest that the carboxyl-terminal region may be involved in regulation of kinase activity in the Src family by assisting with the interaction of the phosphorylated carboxyl-terminal tyrosine to the binding pocket within the SH2 domain. The presence of a segment within Tip with high homology to the carboxyl terminus of Src-related kinases and its importance for Lck binding suggest that the CSKH domain of Tip participates in the interaction with Lck analogous to that described above, thereby cooperating with the SH3B motif to enhance binding affinity to Lck. Furthermore, it provides indirect experimental support for the potential role of carboxyl-terminal sequences of individual Src kinases in intramolecular interactions.

The 18-amino-acid segment linking the CSKH and SH3B domains does not share significant homology with any protein in the data base. However, it clearly influences binding of Tip to Lck as shown in Fig. 5. This stretch could possibly associate directly with Lck, or it may simply represent a linker sequence facilitating the alignment of CSKH and SH3B to their target sequences.

Quite a number of cellular proteins have been found to associate with Lck. These include cell surface receptors like CD2, CD4, CD5, CD8 and interleukin 2 receptor(6, 37, 38, 39, 40) , downstream effectors like GPI anchored proteins(41, 42, 43) , PI 3- and PI 4-kinases(44) , p95(45, 46) , Ras GAP(47) , and protein kinases like Raf-related protein, (48) , ZAP-70(49, 50) , and Syk(51) . Tip could alter the interaction of Lck with cellular substrates that physiologically bind to the SH3 and/or SH2 domains of Lck. Alterations in complex formation between cellular proteins and Lck may ultimately deregulate signal transduction through Lck in transformed T cells expressing Tip.

Any role for Tip in altering T cell signal transduction in the process of viral-induced cell growth transformation is yet to be defined. The association of Tip with Lck could conceivably activate Lck activity to achieve virus-induced T cell transformation similar to the constitutive activation of Src by polyomavirus middle T antigen(52) . Alternatively, the association of Tip may interfere with normal Lck function by preventing its interaction with substrates that normally bind to Lck. Analogous to Tip, LMP2A is expressed in latently infected B lymphocytes by Epstein-Barr virus, another member of the group of herpesviruses, and it associates with the B cell tyrosine kinase Syk and Lyn(53) . LMP2A is not necessary for B cell transformation by Epstein-Barr virus, but it has been shown to block the effects of sIg cross-linking on calcium mobilization, tyrosine phosphorylation, and reactivation of Epstein-Barr virus from latent infection in the transformed human B lymphocytes(53, 54) . If Tip were to function analogously in T cells, we may expect it to block Lck-mediated signal transduction. There is good evidence to indicate that an HSV-encoded transforming protein, STP, acts downstream of Lck by binding to Ras and activating the Ras pathway. (^2)Direct activation of the Ras pathway by STP may make Lck activation not only unnecessary for growth transformation but also detrimental to the virus.


FOOTNOTES

*
This work was supported by United States Public Health Service Grants CA31363 and RR00168, Deutsche Forschungsgemeinschaft Grants Bi465/1-3 and 1-4, and by a fellowship from the Deutsches Krebsforschungszentrum (to S. L.). 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.

§
To whom correspondence should be addressed. Tel.: 508-624-8083; Fax: 508-624-8190.

(^1)
The abbreviations used are: SH, Src-homology; HVS, herpesvirus saimiri; STP, saimiri transforming protein; Tip, tyrosine kinase interacting protein; CMV, cytomegalovirus; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; CSKH, C-terminal Src-related kinase homology; SH3B, SH3-binding; PI, phosphoinositol; GPI, glycocyl-phosphatidylinositol; LMP, latent membrane protein.

(^2)
J. U. Jung and R. C. Desrosiers, submitted for publication.


ACKNOWLEDGEMENTS

We thank Drs. A. Veillette, J. Reed, and J. Shin for providing DNAs. We especially thank Dr. J. K. Chung for discussions and technical assistance. We also thank J. Newton and T. Connors for manuscript preparation.


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