©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of a Novel Src-like Adapter Protein That Associates with the Eck Receptor Tyrosine Kinase (*)

(Received for publication, May 24, 1995; and in revised form, June 15, 1995)

Akhilesh Pandey Hangjun Duan Vishva M. Dixit (§)

From the Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0602

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The Eph family of receptor protein tyrosine kinases (RPTKs) is the largest family of RPTKs. The signal transduction pathways initiated by this family have only recently begun to be explored. Using a yeast two-hybrid screen to identify molecules that interact with the cytoplasmic domain of Eck, it was previously shown that activated Eck RPTK bound to and stimulated phosphatidylinositol 3-kinase (Pandey, A., Lazar, D. F., Saltiel, A. R., and Dixit, V. M. (1994) J. Biol. Chem. 269, 30154-30157). Also isolated [Abstract] from this same screen was a novel protein containing SH3 and SH2 adapter modules that had striking homology to those found in the Src family of non-receptor tyrosine kinases. However, unlike other Src family members, it lacked a catalytic tyrosine kinase domain. Hence, this protein was designated SLAP for Src-like adapter protein. Using glutathione S-transferase fusion proteins, it was demonstrated that SLAP bound to activated Eck receptor tyrosine kinase. Therefore, SLAP is a novel candidate downstream signaling intermediate and the first member of the Src family that resembles an adapter molecule.


INTRODUCTION

Receptor protein tyrosine kinases (RPTKs) (^1)are important in mediating cellular responses to growth factors. Several RPTKs and their ligands have been demonstrated to play important roles during development (1, 2, 3) . For example, the Trk subfamily of receptors has neurotrophic functions in the developing nervous system, and the fibroblast growth factor receptor is critical to mesodermal induction (1, 2) . The Eph subfamily is currently the largest subfamily of RPTKs with 13 members having been described to date(1, 4) . Several members of this subfamily show a very restricted pattern of expression limited to the adult brain, while others such as Eck, Eph, and Htk are widely expressed(1) . Eck and its cognate ligand, B61, are highly expressed in a spatially restricted manner during segmental pattern formation in the developing hindbrain and branchial regions(5, 6, 7) . Therefore, it is quite likely that this subfamily of RPTKs also plays an important role in embryonic development, especially that of the nervous system.

The yeast two-hybrid approach was used to identify putative signaling molecules that might interact with the cytoplasmic domain of Eck. Using the cytoplasmic domain as bait in a yeast two-hybrid screen, it had previously been shown that the p85 subunit of phosphatidylinositol 3-kinase bound activated Eck receptor through its C-terminal SH2 domain (8) . During that screen, an additional cDNA was isolated that interacted specifically with the cytoplasmic domain of Eck. Sequence analysis of this clone revealed it to be a novel gene product containing an SH3 and an SH2 domain and a unique C-terminal tail. The SH3 and SH2 domains were highly homologous to those found in the Src family of non-receptor tyrosine kinases. Surprisingly, however, it lacked the catalytic tyrosine kinase domain, which is an invariant feature of the Src family(3, 9) . Therefore, this gene product was named Src-like adapter protein (SLAP).

Src-like kinases relay signals arising from activated RPTKs including receptors for epidermal growth factor, platelet-derived growth factor, colony-stimulating factor-1, nerve growth factor, and fibroblast growth factor(2, 3, 10, 11, 12, 13, 14) . These receptors, upon activation, become autophosphorylated at certain critical tyrosine residues, and a number of Src-like kinases have been shown to bind to the tyrosine-phosphorylated cytoplasmic domains. This binding is mediated via the SH2 domain (composed of approximately 100 amino acids) that binds to tyrosine-phosphorylated proteins(2, 10) . The specificity of the interaction is determined by the amino acids in the immediate vicinity of the phosphorylated tyrosine(11, 12, 15) . Since SLAP contained an SH2 motif, it seemed likely that it bound the activated Eck RPTK. This was demonstrated using GST fusion proteins and lysates prepared from Eck ligand (B61) activated cells.


MATERIALS AND METHODS

Yeast Two-hybrid Screen and cDNA Isolation

The yeast two-hybrid screen using the Eck cytoplasmic domain as bait has been described previously(8) . Briefly, the Eck bait plasmid was cotransformed with a mouse T-cell expression library fused to the GAL4 activation domain in the pACT plasmid (prey). Several of the 10^6 transformants screened were positive as detected by assaying reporter gene (beta-galactosidase) activity. Library plasmid recovered from the positive clones was used in a cotransformation assay with either the Eck cytoplasmic bait or other control heterologous baits. Two of the plasmids encoded the p85 subunit of phosphatidylinositol 3-kinase and have been reported previously(8) . However, a distinct cDNA was also found to interact specifically with the Eck cytoplasmic domain and not with heterologous baits containing the cytoplasmic domains of the p55 tumor necrosis factor receptor, Fas and CD40. The construction of these heterologous baits has been described earlier(27) .

Additional cDNAs were obtained by screening a mouse embryonic brain library constructed in the pcDNA1 vector (Invitrogen) from mouse embryonic brain poly(A) RNA. 1 10^5 transformants were screened with a P-labeled probe corresponding to the first 72 base pairs of the murine SLAP open reading frame using published procedures(16) . DNA sequence analysis was carried out on both strands using the Sequenase kit (U. S. Biochemical Corp.) and custom synthetic oligonucleotide primers. Sequences were aligned by the Clustal method using MegAlign version 1.02 (DNASTAR Inc., Madison, WI). Homology searching against GenPept, PIR, and SwissProt data bases was performed using the on-line BLAST network service.

Production of GST Fusion Proteins

The SLAP cDNA having in-frame clonable ends was obtained by polymerase chain reaction amplification using an upstream primer containing a custom BamHI site (underlined), AAGGGATCCATGGGGAATAGCATGAAATCCAC, and a downstream primer containing a custom SalI site, ACGCGTCGACTTAATCTTCAAAGTACTGGG.

The fragments were subcloned into the GST fusion protein vector pGSTag and transformed into the Escherichia coli strain BL21(DE3) pLysS. GST and GST fusion proteins were prepared using published procedures(17) , and the recombinant proteins were immobilized onto glutathione-agarose beads (Sigma). GST fusion of a control SH2 containing protein Shc was prepared as described previously (18) .

GST Binding Assays

HA-Eck expression plasmid was constructed by subcloning a fragment from the yeast bait vector that encoded HA epitope-tagged Eck cytoplasmic domain into the mammalian expression vector, pcDNA3. 293 T-cells were transfected with the HA-Eck construct, metabolically labeled with 100 µCi/ml [S]methionine and cysteine (Translabel, ICN) for 8 h, and then lysed in lysis buffer containing 50 mM Tris, pH 7.6, 150 mM NaCl, 1% Nonidet P-40, 1 mM sodium orthovanadate in the presence of protease inhibitors (5 µg/ml leupeptin, 5 µg/ml aprotinin, 50 µg/ml soybean trypsin inhibitor, and 5 µg/ml pepstatin). Lysates containing an equivalent number of trichloroacetic acid-precipitable counts were incubated with approximately 2 µg of the indicated GST fusion proteins in 1 ml of lysis buffer for 2 h at 4 °C and washed, and bound material was eluted by boiling in 1% SDS. The eluates were then diluted 10-fold with lysis buffer and immunoprecipitated with anti-HA monoclonal antibody.

Rat vascular smooth muscle cells were metabolically labeled as above, treated with 1 µg/ml B61, the Eck ligand, expressed as an immunoglobulin chimera (B61-Ig) or control-Ig (19) for 5 min, and then lysed in lysis buffer. Cleared cell lysates were incubated with approximately 2 µg of the indicated GST fusion proteins and washed, and bound material was eluted by boiling in 1% SDS. The eluates were then diluted 10-fold with lysis buffer and reimmunoprecipitated with anti-Eck antibody(28) .

Northern Blot Analysis

A mouse multiple tissue Northern blot containing 2 µg of poly(A) RNA per lane was obtained from Clontech (Palo Alto, CA) and hybridized according to the manufacturer's instructions to a P random-prime labeled C-terminal fragment of SLAP amplified using the primers CGCGGATCCCTGGCACAGAACATACCTGC and ACGCGTCGACTTAATCTTCAAAGTACTGGG.


RESULTS AND DISCUSSION

The cytoplasmic domain of the Eck RPTK was fused in-frame to the GAL4 DNA binding domain in the yeast bait vector pAS1CYH2(20) . The cytoplasmic domain expressed in such a fashion had previously been shown to possess constitutive tyrosine kinase activity and was used as bait to detect interacting proteins encoded by a mouse T-cell cDNA library fused to the GAL4 activation domain(8) . A total of 10^6 transformants was screened by expression in a yeast strain harboring lacZ and HIS3 reporter genes under control of the GAL4 upstream activating sequence. Library plasmids that were able to grow in the presence of histidine were then tested for lacZ expression. Library plasmid from one such positive clone was recovered and found to interact specifically with the Eck bait and not with other heterologous baits tested (Table 1).



Sequence analysis revealed that this clone encoded an open reading frame containing SH3 and SH2 domains that bore striking homology to those present in the Src family of kinases. Remarkably, however, there was no catalytic tyrosine kinase domain downstream of the SH2 domain, being replaced instead by a short stretch of unique sequence. To rule out the possibility that this cDNA arose as a cloning artifact, a mouse brain cDNA library was screened to obtain additional full-length cDNAs. A mouse brain library was chosen as Eck and several other members of the Eph family are highly expressed in the brain(1) . Thus, it appeared reasonable that associated downstream signaling molecules would similarly be highly expressed. This screen resulted in several positive clones, all of which proved to be identical to the original cDNA obtained in the yeast two-hybrid screen.

The full-length clone contained an 846-base pair open reading frame that encoded a predicted protein of molecular mass 32 kDa (Fig. 1). The putative initiation codon (AAAGAGATGG) was in agreement with the consensus Kozak's sequence for translation initiation(21) . Comparison of the translated sequence with the protein data base using the BLAST algorithm revealed strong homology to the Src family of tyrosine kinases, particularly in the SH3 and SH2 domains. An alignment of these regions is shown in Fig. 2. Subsequently, the protein encoded by this open reading frame was designated SLAP for Src-like adapter protein. The SH2 domain of SLAP possessed 51% identity to the SH2 domain of the Src family compared with 25 and 27% identity with the SH2 domains of Shc and Grb2, respectively. Similarly, the SH3 domain of SLAP had a 50% identity with the SH3 domain of the Src family compared with 20 and 24% identity with the SH3 domains of phospholipase C1 and p85alpha subunit of phosphatidylinositol 3-kinase, respectively. Additionally, SLAP had a unique N terminus that, unlike the rest of the Src family, lacked any obvious myristoylation or other membrane localization motif(22, 23) . The sequence of SLAP also diverged from Src family members at the C terminus, where it had a short unique sequence instead of a catalytic tyrosine kinase domain. The SH2 domain of SLAP contained the conserved FXXR sequence that is thought to be critical in binding to phosphotyrosines(10, 11, 24) . Likewise, the SH3 domain contained a proline at position 73 that is conserved in all functional SH3 domains (25, 26) . To rule out the possibility that alternatively spliced versions of SLAP containing a catalytic domain might exist, a mouse multiple tissue Northern blot was probed with a fragment from the unique C terminus of SLAP. As shown in Fig. 3A, SLAP is expressed in all tissues examined but only as a single transcript, ruling out the possibility that a larger, alternatively spliced kinase-encoding version exists as a prominent species.


Figure 1: Nucleotide and deduced amino acid sequence of murine SLAP. The open reading frame encoding 281 amino acids of murine SLAP is shown. The SH3 (yellow) and SH2 (orange) domains of SLAP are boxed. The termination codon is indicated by an asterisk. The untranslated regions are shown in lowercase.




Figure 2: Alignment of SH3 and SH2 domains of the Src family and SLAP. The SH3 and SH2 domains of murine SLAP were aligned with murine sequences of members of the Src family using the MegAlign program. Regions of similarity are shaded in yellow (SH3 domain) or orange (SH2 domain). Consensus sequences are indicated at the top of the alignments.




Figure 3: SLAP is expressed ubiquitously and interacts with the Eck cytoplasmic domain. A, Northern blot analysis. A P-labeled DNA fragment from the unique C terminus of SLAP was used to probe a mouse multiple tissue Northern blot (Clontech) containing 2 µg of poly(A) RNA per lane from the tissues indicated. The sizes of the transcripts in kilobases are shown. B, 293 T-cells were either not transfected or transfected with an expression vector containing the cytoplasmic domain of Eck that was HA epitope-tagged. Cell lysates were then incubated with either GST alone, SLAP-GST, or Shc SH2-GST. Bound material was dissociated by boiling in 1% SDS, diluted, and reimmunoprecipitated with anti-HA antibody. IP, immunoprecipitate.



To confirm that SLAP could bind to the cytoplasmic domain of Eck, 293 T-cells were transfected with a HA epitope-tagged cytoplasmic domain of Eck that had previously been shown to possess constitutive kinase activity(8) . Transfected cells were metabolically labeled, lysed, and incubated with GST alone, GST-SLAP, or GST-Shc immobilized onto glutathione-Sepharose. Bound material was eluted and reimmunoprecipitated with an anti-HA antibody. Fig. 3B shows that only GST-SLAP bound to the Eck cytoplasmic domain. This was not due to nonspecific interaction with any SH2 domain containing protein as Shc-GST (which contains an unrelated SH2 domain) did not bind Eck.

Finally, to address the question of whether SLAP bound Eck in a ligand-dependent manner, primary vascular smooth muscle cells that express endogenous Eck were metabolically labeled and treated with the Eck ligand, B61, expressed as an immunoglobulin chimera (B61-Ig) or control chimera (control-Ig)(19) . We had previously shown that B61-Ig is a potent activator of the Eck RPTK on smooth muscle cells(8) . Cell lysates were then incubated with various GST fusion proteins as shown in Fig. 4. It was found that SLAP GST specifically bound Eck in an activation-dependent manner. The SH2 domain of Shc or GST alone did not bind Eck.


Figure 4: SLAP associates with the Eck RPTK. Metabolically labeled smooth muscle cells treated with control-Ig (lanes labeled -) or with B61-Ig (lanes labeled +) were incubated with GST alone, SLAP-GST, or Shc SH2-GST. Bound material was dissociated by boiling in 1% SDS, diluted, and reimmunoprecipitated with anti-Eck antibody. IP, immunoprecipitate.



This report establishes SLAP as a novel adapter protein that binds to the cytoplasmic domain of Eck in a ligand-dependent manner. It is tempting to speculate that it may also bind other receptor protein tyrosine kinases in a similar manner through its SH2 domain. Finally, SLAP is functionally similar to an emerging class of adapter proteins including Grb2, Crk, and Nck (1) in that it possesses SH3 and SH2 domains but no catalytic tyrosine kinase domain. Importantly, however, none of the other adapter proteins share such a striking resemblance to the corresponding SH3 and SH2 domains in the Src family.

The SH2 domains of Grb2, Crk, and Nck adapter proteins share a 27, 25, and 27% identity to the SH2 domains of the Src family whereas SLAP is 51% identical in this region. Similarly, the most N-terminal SH3 domains of Grb2, Crk, and Nck share a 33% identity to the SH3 domain of the Src family compared with 50% identity in the case of SLAP. This suggests that SLAP may have an adapter function that is unique to the function of Src family members.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant DK 39255. 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank®/EMBL Data Bank with accession number(s) U29056[GenBank].

§
Established Investigator of the American Heart Association. To whom correspondence should be addressed: Dept. of Pathology, University of Michigan Medical School, 1301 Catherine St., Ann Arbor, MI 48109-0602. Tel.: 313-747-0264; Fax: 313-764-4308; vishva.dixit{at}med.umich.edu.

(^1)
The abbreviations used are: RPTK, receptor protein tyrosine kinase; SLAP, Src-like adapter protein; GST, glutathione S-transferase; SH, Src homology; HA, hemagglutinin.


ACKNOWLEDGEMENTS

We thank AMGEN for providing anti-Eck antibody and Dr. Alan Saltiel for providing GST fusion of SH2-Shc. We also thank Constance Esposito and Suzanne Genik for expert assistance with sequence analysis. The assistance of Ian Jones and Karen O'Rourke in the preparation of this manuscript is gratefully acknowledged.


REFERENCES

  1. van der Geer, P., Hunter, T., and Lindberg, R. A. (1994) Annu. Rev. Cell Biol. 10,251-337 [CrossRef]
  2. Fantl, W. J., Johnson, D. E., and Williams, L. T. (1993) Annu. Rev. Biochem. 62,453-481 [CrossRef][Medline] [Order article via Infotrieve]
  3. Fry, M. J., Panayotou, G., Booker, G. W., and Waterfield, M. D. (1993) Protein Sci. 2,1785-1797 [Free Full Text]
  4. Bennett, B. D., Wang, Z., Kuang, W. J., Wang, A., Groopman, J. E., Goeddel, D. V., and Scadden, D. T. (1994) J. Biol. Chem. 269,14211-14218 [Abstract/Free Full Text]
  5. Ganju, P., Shigemoto, K., Brennan, J., Entwistle, A., and Reith, A. D. (1994) Oncogene 9,1613-1624 [Medline] [Order article via Infotrieve]
  6. Ruiz, J. C., and Robertson, E. J. (1994) Mech. Dev. 46,87-100 [CrossRef][Medline] [Order article via Infotrieve]
  7. Shao, H., Pandey, A., O'Shea, K. S., Seldin, M., and Dixit, V. M. (1995) J. Biol. Chem. 270,5636-5641 [Abstract/Free Full Text]
  8. Pandey, A., Lazar, D. F., Saltiel, A. R., and Dixit, V. M. (1994) J. Biol. Chem. 269,30154-30157 [Abstract/Free Full Text]
  9. Hanks, S. K., Quinn, A. M., and Hunter, T. (1988) Science 241,42-52 [Medline] [Order article via Infotrieve]
  10. Schlessinger, J. (1994) Curr. Opin. Genet. & Dev. 4,25-30 [Medline] [Order article via Infotrieve]
  11. Songyang, Z., Shoelson, S. E., Chaudhuri, M., Gish, G., Pawson, T., Haser, W. G., King, F., Roberts, T., Ralnofsky, S., Lechleider, R. J., Neel, B. G., Birge, R. B., Fajardo, J. E., Chou, M. M., Hanafusa, H., Schaffhausen, B., and Cantley, L. C. (1993) Cell 72,767-778 [Medline] [Order article via Infotrieve]
  12. Rodrigues, G. A., and Park, M. (1994) Curr. Opin. Genet. & Dev. 4,15-24 [Medline] [Order article via Infotrieve]
  13. Ullrich, A., and Schlessinger, J. (1990) Cell 61,203-212 [Medline] [Order article via Infotrieve]
  14. Yarden, Y., and Ullrich, A. (1988) Annu. Rev. Biochem. 57,443-478 [CrossRef][Medline] [Order article via Infotrieve]
  15. Schlessinger, J., and Ullrich, A. (1992) Neuron 9,383-391 [Medline] [Order article via Infotrieve]
  16. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  17. Studier, F. W. (1991) J. Mol. Biol. 219,37-44 [Medline] [Order article via Infotrieve]
  18. Sasaoka, T., Rose, D. W., Jhun, B. H., Saltiel, A. R., Draznin, B., and Olefsky, J. M. (1994) J. Biol. Chem. 269,13689-13694 [Abstract/Free Full Text]
  19. Pandey, A., Shao, H., Marks, R. M., Polverini, P. J., and Dixit, V. M. (1995) Science 268,567-569 [Medline] [Order article via Infotrieve]
  20. Durfee, T., Becherer, K., Chen, P. L., Yeh, S. H., Yang, Y., Kilburn, A. E., Lee, W. H., and Elledge, S. J. (1993) Genes & Dev. 7,555-569
  21. Kozak, M. (1989) J. Cell Biol. 108,229-241 [Abstract]
  22. Resh, M. D. (1994) Cell 76,411-413 [Medline] [Order article via Infotrieve]
  23. Silverman, L., Sudol, M., and Resh, M. D. (1993) Cell Growth & Differ. 4,475-482
  24. Songyang, Z., Shoelson, S. E., McGlade, J., Olivier, P., Pawson, T., Bustelo, X. R., Barbacid, M., Sabe, H., Hanafusa, H., Yi, T., Ren, R., Baltimore, D., Ratnofsky, S., Feldman, R. A., and Cantley, L. C. (1994) Mol. Cell. Biol. 14,2777-2785 [Abstract]
  25. Clark, S. G., Stern, M. J., and Horvitz, H. R. (1992) Nature 356,340-344 [CrossRef][Medline] [Order article via Infotrieve]
  26. Eck, M. J., Atwell, S. K., Shoelson, S. E., and Harrison, S. C. (1994) Nature 368,764-769 [CrossRef][Medline] [Order article via Infotrieve]
  27. Hu, H. M., O'Rourke, K., Boguski, M. S., and Dixit, V. M. (1994) J. Biol. Chem. 269,30069-30072 [Abstract/Free Full Text]
  28. Lindberg, R. A., and Hunter, T. (1990) Mol. Cell. Biol. 10,6314-6324

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.