(Received for publication, May 24, 1995; and in revised form, June 15, 1995)
From the
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.
Receptor protein tyrosine kinases (RPTKs) ()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.
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
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.
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) .
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) .
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 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 p85
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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank®/EMBL Data Bank with accession number(s) U29056[GenBank].