COMMUNICATION:
SH3-mediated Hck Tyrosine Kinase Activation and Fibroblast Transformation by the Nef Protein of HIV-1*

(Received for publication, April 25, 1997, and in revised form, May 23, 1997)

Scott D. Briggs Dagger , Mark Sharkey §, Mario Stevenson § and Thomas E. Smithgall Dagger

From the Dagger  Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha, Nebraska 68198-6805 and the § Program in Molecular Medicine, University of Massachusetts Medical Center, Worcester, Massachusetts 01605

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Tyrosine kinases of the Src family are regulated via their Src homology 2 (SH2) and SH3 domains. The Nef protein of human immunodeficiency virus-1 (HIV-1) has previously been shown to bind with high affinity and specificity in vitro to the SH3 domain of Hck, a Src family member expressed primarily in myeloid cells. However, the effect of Nef on Hck activity in living cells is unknown. Here we show that Rat-2 fibroblasts co-expressing Hck and Nef rapidly developed transformed foci, whereas control cells expressing either protein alone did not. Nef formed a stable complex with Hck and stimulated its tyrosine kinase activity in vivo. Mutagenesis of the Nef proline-rich motif essential for SH3 binding completely blocked complex formation, kinase activation, and transformation, indicating that the Nef SH3-binding function is required for its effects on Hck. These results provide direct evidence that SH3 engagement is sufficient to activate a Src family kinase in vivo and suggest that Hck may be activated by Nef in HIV-infected macrophages.


INTRODUCTION

The Src family of non-receptor tyrosine kinases consists of nine members (Src, Lck, Hck, Fyn, Fgr, Yes, Blk, Lyn, and Yrk) that share common structural features and regulation (reviewed in Ref. 1). The N-terminal region of each kinase contains a signal for myristoylation and in some cases palmitoylation; both lipid modifications are involved in membrane targeting. Adjacent to the N-terminal region is a Src homology (SH3)1 domain consisting of approximately 60 amino acids. SH3 domains bind to target proteins via specific proline-rich sequences that adopt a polyproline type II helical conformation (2-4). Protein-protein interactions mediated by Src kinase SH3 domains contribute to substrate recruitment, subcellular localization, and negative regulation of kinase activity (see below). C-terminal to the SH3 domain is the SH2 domain. Comprised of approximately 100 amino acids, SH2 domains bind tightly to specific tyrosine-phosphorylated sequences. As for SH3 domains, SH2 domains also contribute to negative regulation of kinase activity, subcellular localization, and interaction with signaling partners. The SH2 domain is followed by the kinase domain and a C-terminal tail with a highly conserved tyrosine residue essential to kinase regulation.

SH2 and SH3 domains cooperate in the negative regulation of Src family kinase activity (1). Phosphorylation of the conserved tyrosine residue in the C-terminal tail (Tyr-527 in Src) by the regulatory kinase Csk (5) is postulated to promote intramolecular binding to the SH2 domain, contributing to a closed and inactive kinase conformation. A large body of genetic and biochemical evidence supports this model. For example, conversion of the conserved Tyr residue in the tail to Phe or mutation of the SH2 domain results in a constitutively active kinase capable of producing a transformed phenotype in rodent fibroblasts (6, 7). Such mutations presumably interfere with intramolecular SH2-tail interaction. Many studies have shown an essential role for the SH3 domain in the negative regulation of Src family kinase activity as well. Mutants of Src lacking a functional SH3 domain are also resistant to Csk-mediated negative regulation and are often transforming (8-12). These findings suggest that interaction of Src kinase SH2 or SH3 domains with target proteins may destabilize the closed conformation in vivo, leading to kinase activation. Such a mechanism may be responsible for the activation of Src kinases following recruitment via their SH2 domains to autophosphorylated growth factor receptors (13, 14). Whether or not SH3-mediated interaction of Src kinases with target proteins is sufficient to activate the kinase domain in vivo is less clear.

In this study, we investigated the effects of SH3 domain engagement on the activity of a Src-related kinase in living cells. Our model system consisted of Hck, a Src family member expressed primarily in macrophages and neutrophils (15-17), and the Nef protein of the human immunodeficiency virus-1 (HIV-1). Nef is essential for the high titer replication of HIV and SIV and for the induction of AIDS-like disease in monkeys (reviewed in Refs. 18 and 19). Previous reports have shown that Nef specifically binds to the SH3 domain of Hck in vitro with the highest affinity known for an SH3-mediated interaction (20, 21). These findings suggested that Nef may represent an excellent probe of Hck SH3 function in vivo. As shown below, co-expression of Nef with Hck in Rat-2 fibroblasts resulted in the appearance of a transformed phenotype that correlated with the SH3-dependent activation of Hck in vivo. These results suggest that interaction of Src family kinases with SH3 ligands may be sufficient to activate Src family kinases in vivo.


EXPERIMENTAL PROCEDURES

Retrovirus-mediated Expression of Nef and Hck in Rat-2 Cells

The Nef gene from HIV isolate SF2 was amplified by polymerase chain reaction and subcloned into the retroviral vector pSRalpha MSVtkneo (22). Wild-type (WT), kinase-defective (K269E), and activated (Y501F) forms of human p59 Hck were also subcloned into the same vector. The mutant forms of Hck were generated by a standard polymerase chain reaction-based strategy as described elsewhere (23). The resulting constructs were used to generate high titer retroviral stocks by co-transfection of 293T cells (24) with an ecotropic packaging vector. Control retroviruses were prepared using the parent vector. For each infection, 2.5 × 104 Rat-2 cells were plated per well in 6-well tissue culture plates and incubated with viral stocks in a final volume of 6 ml. To enhance the efficiency of infection, cultures were centrifuged at 1,000 × g for 4 h at 18 °C (25) in the presence of 4 µg/ml Polybrene. Following infection, the virus was replaced with fresh medium, and G418 (400 µg/ml) was added 48 h later. Transformed foci were counted after 2 weeks.

Co-immunoprecipitation Assay and Immunoblot Analysis

To detect Hck-Nef protein complexes, cells were sonicated briefly in 1.0 ml of detergent buffer (50 mM Tris-HCl, pH 7.4, 50 mM NaCl, 1 mM EDTA, 10 mM MgCl2, and 1% Triton X-100). Clarified supernatants were incubated with 2 µg of anti-Hck antibody (Santa Cruz Biotechnology) and 15 µl of protein G-Sepharose (50% slurry; Pharmacia Biotech Inc.) for 2.5 h at 4 °C. Immunoprecipitates were washed three times with 1.0 ml of RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, and 1% sodium deoxycholate). Immunoprecipitates were heated in SDS-PAGE sample buffer, resolved by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed for associated Nef by immunoblotting with anti-Nef monoclonal antibodies (kindly supplied by Dr. James A. Hoxie, University of Pennsylvania).

For analysis of protein-tyrosine phosphorylation or protein expression, whole cell lysates were prepared by heating cells directly in SDS-PAGE sample buffer. Lysate proteins were separated by SDS-PAGE, transferred to polyvinylidene difluoride, and immunoblotted with anti-Hck monoclonal antibodies (Transduction Laboratories), anti-phosphotyrosine antibodies (PY20; Transduction Laboratories), or anti-Nef monoclonal antibodies. Immunoreactive bands were visualized colorimetrically using alkaline phosphatase-conjugated secondary antibodies and nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate as substrate.


RESULTS

Co-expression of Nef and Hck Transforms Rat-2 Cells

To determine whether interaction with Nef influences Hck activity under physiological conditions, Rat-2 fibroblasts were co-infected with recombinant retroviruses containing wild-type, activated, or kinase-defective forms of Hck and either a Nef retrovirus or a control virus containing only the bacterial neo resistance marker. After 2 weeks, transformed foci were readily observed in cells expressing wild-type Hck and Nef (Fig. 1 and Table I). In contrast, no transformed foci were observed in cells expressing wild-type Hck or Nef alone. The foci resulting from the co-expression of Nef and Hck were morphologically indistinguishable from those produced by an activated allele of Hck in which the negative regulatory tyrosine residue in the C-terminal tail was mutated to phenylalanine (Hck-YF mutant) (Fig. 1). Fewer foci were observed with the co-infected cells relative to the Hck-YF cells (Table I), probably because transformation is dependent upon successful co-infection and expression of both Hck and Nef within the same cell. No transformed foci were observed following co-expression of Nef with a kinase-defective Hck mutant (Hck-KE; Ref. 23), demonstrating the requirement for Hck tyrosine kinase activity in the initiation of transformation by Nef. We also observed that the transforming activity of Hck-YF was not detectably affected by co-expression with Nef (Table I), despite the ability of these two proteins to associate in vivo (see below).


Fig. 1. Co-infection of Rat-2 fibroblasts with Hck tyrosine kinase and HIV Nef retroviruses induces transformation. Rat-2 cells were co-infected with recombinant retroviruses carrying wild-type (WT), kinase-defective (KE), or kinase-activated (YF) forms of Hck and a control virus carrying the neo selection marker (top three panels) or Nef (middle three panels). Individual foci or infected, untransformed cells were photographed under phase-contrast microscopy after 2 weeks. Additional negative controls include cells singly infected with the control virus carrying the neo selection marker (vector; bottom left) and cells co-infected with Nef and the control virus (bottom center). Also shown is the result of co-expression of wild-type Hck with a Nef mutant bearing a mutation in the SH3-binding domain (WT + PA; bottom right). Magnification, × 100.
[View Larger Version of this Image (114K GIF file)]

Table I. Co-expression of Nef and Hck results in transformation of Rat-2 fibroblasts

Rat-2 cells were plated in six-well tissue culture plates and infected with recombinant retroviruses in the combinations shown. Two days after infection, cells were trypsinized and replated in duplicate 60-mm tissue culture dishes in the presence of G418. Transformed foci were counted after 14 days.

Retroviral combination Number of foci/plate n

Hck-WT + vector 0 6
Hck-KE + vector 0 3
Hck YF + vector >50 3
Nef + vector 0 3
Hck-WT + Nef 17 ± 3.7 (S.E.) 6
Hck-KE + Nef 0 3
Hck YF + Nef >50 3
Hck WT + Nef-PA 0 3
Vector alone 0 3

To verify that the Nef and Hck proteins were expressed in the transformants as well as the non-transformed control cells, immunoblots were performed on whole cell lysates. As shown in Fig. 2, Hck and Nef proteins were readily detected in cells infected with the corresponding retroviruses. Taken together, these results demonstrate that Nef and Hck cooperate to induce fibroblast transformation in a Hck kinase-dependent manner.


Fig. 2. Co-expression of Hck and Nef in Rat-2 fibroblasts. To verify the expression of Nef and Hck proteins in the cultures shown in Fig. 1, immunoblots were performed on whole cell lysates as described under "Experimental Procedures." Results shown are from a control culture infected with the parent retrovirus (vector), cultures expressing wild-type (WT), kinase-defective (KE), or kinase-activated (YF) forms of Hck, Nef alone, or a combination of each Hck protein with Nef (+Nef; right three lanes). Expression controls for the Hck-WT + Nef-PA culture are shown in Fig. 5.
[View Larger Version of this Image (37K GIF file)]

Hck Forms a Stable Complex with Nef Resulting in Constitutive Activation of the Hck Tyrosine Kinase in Vivo

The biological data described above support a model in which Nef binds to the Hck SH3 domain in vivo, leading to activation of the kinase domain and cellular transformation. To test this model, we assessed Hck autophosphorylation and tyrosine phosphorylation of endogenous proteins in lysates from control and transformed cell lines. Cells were lysed directly in SDS-PAGE sample buffer, and tyrosine phosphoproteins were visualized by immunoblotting with anti-phosphotyrosine antibodies. As shown in Fig. 3, transformed cells expressing both Hck and Nef showed several prominent tyrosine-phosphorylated proteins. These include an ~60-kDa phosphoprotein with electrophoretic mobility identical to Hck as well as a second strong band of ~40 kDa. A nearly identical pattern of protein-tyrosine phosphorylation was observed in cells transformed with the activated form of Hck (Hck-YF), including the 40-kDa transformation-related protein. Although we have not identified the endogenous 40-kDa transformation-associated tyrosyl phosphoprotein, immunoblotting experiments indicate that it is not the Src substrates c-Crk, annexin I, or annexin II which are in this molecular mass range (data not shown). None of the untransformed control cell lines showed evidence of protein-tyrosine phosphorylation, including cells expressing Nef alone, Hck-WT alone, or cells co-expressing Nef and the kinase-defective Hck mutant. These results show that co-expression with Nef leads to constitutive activation of the Hck tyrosine kinase in living cells.


Fig. 3. Co-expression with Nef stimulates Hck autophosphorylation and tyrosine phosphorylation of endogenous proteins in vivo. A, cell lines expressing wild-type (WT), kinase-defective (KE), and kinase-activated (YF) forms of Hck in the presence and absence of Nef were lysed directly in SDS-PAGE sample buffer, separated by SDS-PAGE, and immunoblotted with the antiphosphotyrosine antibody, PY20. Additional controls include cell lines infected with a retrovirus carrying the neo selection marker (vector) and cell lines expressing the Nef protein alone. The position of autophosphorylated Hck and the major transformation-related tyrosyl phosphoprotein, p40, are indicated by the arrows. The positions of the molecular mass standards are indicated on the left (from top to bottom: 200, 97, 68, 43, and 29 kDa). B, control immunoblots demonstrate the expression of the Hck and Nef proteins.
[View Larger Version of this Image (57K GIF file)]

We next investigated whether Nef directly interacts with Hck in vivo. Hck was immunoprecipitated from transformed cells and probed for associated Nef by immunoblotting. As shown in Fig. 4, Hck-Nef complexes were readily immunoprecipitated from the Rat-2 cells transformed by co-expression of these proteins. Nef also formed stable complexes with the kinase-defective and activated forms of Hck, indicating that the SH3 domains of all three Hck proteins are accessible to Nef. Control experiments show that Nef does not cross-react with the anti-Hck antibody. These experiments provide the first evidence for stable Hck-Nef complexes in intact cells. However, the possibility that the observed complexes form in solution after lysis of the cells cannot be formally ruled out.


Fig. 4. Hck forms stable complexes with Nef in vivo. Cell lines stably expressing Nef either alone or in the presence of wild-type (WT), kinase-defective (KE), or kinase-activated (YF) forms of Hck were lysed in detergent buffer and incubated with an anti-Hck antibody and protein G-Sepharose. The immune complexes were washed, and associated Nef was visualized by immunoblotting (top panel). Control blots show the tyrosine phosphorylation of the endogenous protein, p40, and verify the expression of Hck and Nef proteins (lower three panels).
[View Larger Version of this Image (35K GIF file)]

Activation of Hck by Nef and Cellular Transformation Requires the Proline-rich Region of Nef

A final series of experiments investigated the dependence of Hck kinase activation and cellular transformation on the SH3-binding activity of Nef. Hck was co-expressed with a mutant of Nef in which the proline residues at positions 72 and 75 were converted to alanine (Nef-PA mutant) (20). Mutagenesis of these prolines has been shown previously to completely block Nef-SH3 binding in vitro (20). Recent structural studies of Nef-SH3 complexes have confirmed that these Nef residues form part of the polyproline type II helix essential for interaction with the SH3 domain (26, 27). Co-expression of Hck with the Nef-PA mutant failed to induce transformation (Fig. 1, Table I), despite strong expression of both proteins in the co-infected cultures (Fig. 5). Furthermore, Nef-PA was unable to stimulate Hck kinase activity in vivo as assessed by tyrosine phosphorylation of the endogenous protein, p40 (Fig. 5). In addition, Hck autophosphorylation was not stimulated by this Nef mutant (data not shown). Consistent with previous in vitro work, the Nef-PA mutant was unable to bind to Hck in the co-immunoprecipitation assay (Fig. 5). Taken together, these findings strongly support the hypothesis that Nef binds directly to the SH3 domain of Hck in vivo, leading to Hck tyrosine kinase activation and cellular transformation.


Fig. 5. Hck-Nef complex formation correlates with transforming activity and requires the proline-rich SH3 domain-binding function of Nef. Detergent extracts from cell lines co-expressing wild-type Nef (WT) or a Nef mutant with alanine substitutions of prolines 72 and 75 (PA) and wild-type Hck were incubated with anti-Hck antibodies. Immune complexes were precipitated with protein G-Sepharose, washed, and probed for associated Nef by immunoblotting (top left). Whole cell lysates were examined for tyrosine phosphorylation of the endogenous Hck substrate, p40, by anti-phosphotyrosine immunoblotting (PY20; top right). Control blots verify the expression of Hck and Nef proteins (lower two panels).
[View Larger Version of this Image (31K GIF file)]


DISCUSSION

Data presented in this report show that engagement of a Src family kinase SH3 domain by a target protein leads to kinase activation, tyrosine phosphorylation of substrate proteins, and cellular transformation. These data provide direct new evidence that SH3 domains contribute to the negative regulation of Src family kinases in vivo. While this manuscript was being prepared for submission, the three-dimensional crystal structures of the inactive, Csk-phosphorylated forms of both c-Src and Hck were reported (28, 29). Both structures revealed the anticipated intramolecular interaction between the SH2 domain and the tyrosine-phosphorylated tail. Unexpectedly, both structures also showed an intramolecular interaction of the SH3 domain with a helical region formed by the linker connecting the SH2 and kinase domains. In light of this new structural information, the most likely mechanistic explanation for our data is that engagement of the SH3 domain by Nef disturbs this intramolecular interaction, releasing the kinase from the inactive, closed state. Such a mechanism implies that SH3-mediated activation can occur in the absence of dephosphorylation of the tail and supports a central role for the SH3 domain in negative regulation. Also supporting this view is the recent observation that Nef can stimulate Hck tyrosine kinase activity in vitro despite the presence of a phosphotyrosine residue in the tail region (30). SH3 domains of Src family kinases have been reported to interact with a wide variety of substrate proteins via their SH3 domains (23, 31-33). Our data suggest that binding of a substrate protein to the SH3 domain may be sufficient to stimulate Src tyrosine kinase activity in vivo, possibly inducing substrate phosphorylation.

Work presented here is consistent with the notion that Nef, which lacks any known catalytic function, acts by influencing kinases or other signaling pathways within the host cell (reviewed in Ref. 18). Also supporting this hypothesis are previous studies of a variant of SIV that may signal by interacting with Src. However, in contrast to our data, the unusual acutely lethal phenotype of this variant (SIVpbj14; Ref. 34) is dependent upon the presence of an SH2 recognition sequence encoded by its Nef gene that is phosphorylated by Src (35). The SIVpbj14 Nef allele was able to transform NIH3T3 cells, although the role of Src activation in the transformation event was not investigated directly (35). SH2-dependent activation of Src may not be required for HIV Nef function in general, since the SIVpbj14 Nef SH2 recognition motifs that govern its acutely pathogenic properties are not found in the Nef genes of known HIV-1 strains (36). We were unable to detect tyrosine phosphorylation of Nef in the fibroblasts transformed by co-expression of Hck and Nef, suggesting that SH2 domain interaction is not involved in the activation mechanism (data not shown).

Our data show that stimulation of Hck protein-tyrosine kinase activity by HIV-1 Nef is sufficient to produce a transformed phenotype in fibroblasts. Given the strong conservation of proline-rich motifs among various Nef alleles, these data argue that SH3-dependent activation of Src family tyrosine kinases may be a general property of primate lentiviruses. Our results, together with previous reports of the remarkable affinity and specificity of HIV-1 Nef for the Hck SH3 domain (20, 21), strongly suggest that Hck may be a relevant target for Nef in macrophages, a major site of Hck expression (15-17). While the role of macrophages in lentivirus replication and pathogenicity is unclear, recent evidence suggests that macrophage infection is necessary for the pathogenic manifestations of both HIV and SIV infection (reviewed in Ref. 37). Hck is rapidly induced following macrophage activation and has been implicated in multiple signaling events including phagocytosis, Fc receptor signal transduction, integrin signaling, and tumor necrosis factor release (38-44). Further investigation should determine which, if any, of these signaling pathways are modulated by HIV infection and whether these effects are mediated through Nef-Hck interaction.


FOOTNOTES

*   This work was supported by National Institutes of Health Grant CA58667 and American Cancer Society Research Grant BE-245 (to T. E. S.), by National Institutes of Health Grants AI 39812 and AI 32890 (to M. S.), and by NCI, National Institutes of Health Cancer Center Support Grant P30 CA36727 to the Eppley Institute.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.
   To whom correspondence should be addressed: Eppley Institute for Research in Cancer, University of Nebraska Medical Center, 600 S. 42nd St., Omaha, NE 68198-6805. Tel.: 402-559-8270; Fax 402-559-4651; E-mail: tsmithga{at}unmc.edu.
1   The abbreviations used are: SH, Src homology; HIV, human immunodeficiency virus; SIV, simian immunodeficiency virus; WT, wild-type; PAGE, polyacrylamide gel electrophoresis.

ACKNOWLEDGEMENTS

We thank Dr. Owen Witte, Howard Hughes Medical Institute, UCLA, for the retroviral vectors and Dr. James A. Hoxie, University of Pennsylvania, for the anti-Nef antibody.


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