(Received for publication, October 28, 1996, and in revised form, December 3, 1996)
From the Divisions of Experimental Medicine and Hematology/Oncology, Beth Israel Deaconess Medical Center, Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215
The Csk homologous kinase (CHK), formerly MATK, has previously been shown to bind to activated c-KIT. In this report, we characterize the binding of SH2CHK to specific phosphotyrosine sites on the c-KIT protein sequence. Phosphopeptide inhibition of the in vitro interaction of SH2CHK-glutathione S-transferase fusion protein/c-KIT from SCF/KL-treated Mo7e megakaryocytic cells indicated that two sites on c-KIT were able to bind SH2CHK. These sites were the Tyr568/570 diphosphorylated sequence and the monophosphorylated Tyr721 sequence.
To confirm this, we precipitated native CHK from cellular extracts using phosphorylated peptides linked to Affi-Gel 15. In addition, purified SH2CHK-glutathione S-transferase fusion protein was precipitated with the same peptide beads. All of the peptide bead-binding studies were consistent with the direct binding of SH2CHK to phosphorylated Tyr568/570 and Tyr721 sites. Binding of FYN and SHC to the diphosphorylated Tyr568/570 site was observed, while binding of Csk to this site was not observed. The SH2CHK binding to the two sites is direct and not through phosphorylated intermediates such as FYN or SHC. Site-directed mutagenesis of the full-length c-KIT cDNA followed by transient transfection indicated that only the Tyr568/570, and not the Tyr721, is able to bind SH2CHK. This indicates that CHK binds to the same site on c-KIT to which FYN binds, possibly bringing the two into proximity on associated c-KIT subunits and leading to the down-regulation of FYN by CHK.
The Csk homologous kinase (CHK),1 previously referred to as megakaryocyte-associated tyrosine kinase (MATK), is a recently identified protein tyrosine kinase found predominantly in human brain and hematopoietic cells (1, 2). The kinase is composed of 527 amino acids and has highest homology (~50% identity overall) with the human Csk, a kinase which is known to phosphorylate the C-terminal tyrosine of SRC family kinases. Such phosphorylation results in the inactivation of these SRC kinases (3-6). It has been shown that CHK is also able to phosphorylate the C-terminal tyrosine of pp60SRC (2, 7). Thus, in certain situations, CHK might be a physiological regulator of SRC kinase activity. Like Csk, CHK contains SH3, SH2, and tyrosine kinase domains positioned from the N terminus to the C terminus. Both kinases lack N-terminal myristoylation sites and autophosphorylation sites.
Murine counterparts to CHK have been found by a number of investigators. These have been designated as Ntk, which is cloned from mouse fetal thymus (8), and Ctk, which is cloned from mouse brain (9). The Ntk protein is reported to be of two molecular weights, 52 and 56 kilodaltons, and results from alternate transcriptional splicing, while the Ctk was reported to be of only 52 kilodaltons. It is the 56-kilodalton murine form which most closely resembles human CHK. Human CHK has also been cloned by Sakano et al. (10) from megakaryoblastic leukemia cells and designated HYL. Another human CHK-like form possessing a molecular weight of 57 kilodaltons (designated LSK), which is nearly identical to CHK, was identified in natural killer cells and in activated T cells (11).
We recently found that in CMK cells stimulated by stem cell factor/kit ligand (SCF/KL), CHK associates with tyrosine phosphorylated c-KIT through its SH2 domain (12). c-KIT functions as a growth factor receptor for myeloid and erythroid lineages and promotes the survival of primitive stem cells (13). Mice with defective c-KIT (W) are anemic and deficient in hematopoietic progenitor cells (14).
Since the SRC family kinase FYN is also known to bind to the related PDGF receptor (15), we hypothesized that the binding of CHK by c-KIT would bring CHK into proximity to the bound SRC kinases. In this report, we have characterized the binding of the SH2CHK domain to specific tyrosine phosphorylated sites on c-KIT by phosphopeptide inhibition of c-KIT/CHKSH2, by binding of SH2CHK or native CHK to phosphopeptide beads, and by site-specific mutagenesis of c-KIT cDNA. Interestingly, we also found that the SRC family kinase FYN binds to one of these sites on c-KIT through its SH2 domain. The adapter protein SHC was similarly shown to bind to this site. These findings are in agreement with previous studies on the PDGF receptor (15-17). Our results indicate that SH2CHK binds directly to c-KIT at the Tyr568/570 site and does not bind through intermediates such as FYN or SHC. Thus, it is likely that the biological effect of CHK in hematopoietic cells is through direct, site-specific binding to c-KIT.
Tyrosine-phosphorylated and nonphosphorylated
synthetic peptides were obtained from the Dana Farber Cancer Institute
Molecular Biology Core Facility (Boston, MA). Peptides were analyzed
for purity by high pressure liquid chromatography, mass spectroscopy, and amino acid analysis. SH2CHK-GST fusion protein was
prepared as described previously (12). PI3-kinase p85 SH2-GST fusion
protein was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Anti-GST monoclonal antibodies were produced against Schistosoma
japonicum glutathione S-transferase according to the
procedure of Harlow and Lane (18). Anti-CHK polyclonal antibody
(anti-LSK), anti-SHC polyclonal antibody (C-20), and anti-FYN
polyclonal antibody were purchased from Santa Cruz Biotechnology.
Anti-SHC monoclonal antibody S14620 and anti-p85 polyclonal antibody
were purchased from Transduction Laboratories (Lexington, KY).
Anti-c-KIT polyclonal and recombinant SCF/KL were generously provided
by Dr. B. Bennett, Amgen Inc. (Thousand Oaks, CA). Interleukin-3 and
GM-CSF were purchased from R & D Systems (Minneapolis, MN). All other
reagents were from Sigma unless otherwise specified.
Factor-dependent megakaryocytic Mo7e cells were grown in
RPMI 1640 medium (Mediatech, Washington, D. C.) including 20% fetal
bovine serum, 10 ng/ml interleukin-3, 10 ng/ml GM-CSF, 100 IU/ml
penicillin, and 10 µg/ml streptomycin. Prior to stimulation with
SCF/KL, cells were starved for 15 h in RPMI 1640 medium with 1%
fetal bovine serum, 100 IU/ml penicillin, and 10 µg/ml
streptomycin.
Serum-starved Mo7e cells (2 × 106/ml) were stimulated with 500 ng/ml SCF/KL at 37 °C for 2 min and centrifuged for 2 min at 2200 rpm. Cell pellets were placed on ice and solubilized in 20 mM Tris-HCl, pH 7.4, 137 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 10% glycerol, 1.0% Nonidet P-40, 1 mM Na3VO4, 2 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin (lysis buffer). Cell lysates (40 × 106 cells/ml) were mixed briefly and centrifuged for 15 min at 4800 rpm. During centrifugation, SH2-GST fusion proteins (10 µg/incubation) were preincubated with 0.5 ml of lysis buffer along with synthetic peptides at twice the specified concentration. Cell supernatants (0.5 ml, equivalent to 20 × 106 cells) were added to SH2CHK-GST fusion protein plus peptide. Lysates were rotated during incubation for 1 h at 5 °C and then precipitated by the addition of Glutathione-Sepharose 4B (Pharmacia Biotech Inc.) for 0.5 h at 5 °C. Precipitates were washed by centrifugation and resuspension in 1 ml of lysis buffer (repeated three times). SDS sample buffer was added to washed precipitates, and the samples were run on 7.5% polyacrylamide SDS-PAGE. Proteins were transferred onto nitrocellulose filters (Bio-Rad). Transfers were blocked by 4% bovine serum albumin in 0.1% Tween 20, phosphate-buffered saline (PBST). Primary antibody incubation for 1.5 h was followed by washing in PBST. Horseradish peroxidase-linked secondary antibody incubation was in PBST for 45 min. After washing in PBST, transfers were treated with ECL chemiluminescent reagent (Amersham Corp.) and exposed to x-ray film.
ImmunoprecipitationMo7e cell extract supernatants prepared as described above were also immunoprecipitated by specified antibodies. Incubations of 20 × 106 cell equivalents with 1 µg of affinity-purified antibody were for 1.5 h at 5 °C with rotation. Protein G-Sepharose (Pierce) was added, and incubation was continued for 0.5 h. Precipitates were centrifuged and resuspended in 1 ml of lysis buffer (repeated three times). SDS sample buffer was added to precipitates, and samples were run on 10% or 7.5% polyacrylamide SDS-PAGE. Gels were transferred onto nitrocellulose and immunoblotting was conducted as in the previous section.
Association of SH2CHK-GST or Native CHK with Peptide BeadsPhosphorylated and nonphosphorylated peptides were linked to Affi-Gel 15 by adding 1 µmol of peptide in 40 mM MOPS buffer, pH 7.4, to a 0.5-ml bed volume of washed beads. After reaction for 1 h at room temperature, Tris-HCl, pH 8.0, was added to a final 50 mM concentration to block unreacted sites. After incubation overnight at 5 °C, the resin was washed with 0.5 M NaCl, 50 mM MOPS, pH 7.4, followed by 50 mM MOPS, pH 7.4, and stored at 5 °C with 1 mM sodium azide as a preservative. In order to test for peptide linkage, we measured the A280 of the initial supernatant of the peptide-bead reaction and compared this to the A280 prior to linkage, correcting for dilution. In all cases, 80-90% of the peptide was linked to the beads. Peptide beads (15-µl bead volume) were incubated with 10 µg of SH2CHK in 20 mM Tris-HCl, pH 7.4, 137 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 10% glycerol, 1.0% Nonidet P-40, 1 mM Na3VO4, and 2 mM phenylmethylsulfonyl fluoride (association buffer). Incubation was for 1.5 h at 5 °C. The beads were centrifuged and resuspended in 1 ml of association buffer (repeated three times). SDS sample buffer was added to the washed precipitates, and the samples were subjected to SDS-PAGE followed by transfer to nitrocellulose and immunoblotting as described above with anti-GST as the primary antibody. In order to test binding of native CHK to the peptide beads, 15 µl of beads were incubated with 1 ml of Mo7e extract (20 × 106 cells equivalent) prepared as described in the previous section. Extracts were incubated for 1.5 h at 5 °C, with gentle mixing. Precipitates were treated with SDS sample buffer and subjected to SDS-PAGE/Western blotting with anti-CHK or other antibodies, followed by the appropriate secondary antibody and ECL development. Nitrocellulose filters were stripped for reblotting by incubating in 0.2 M glycine, 0.5 M NaCl, pH 2.8. After neutralization with Tris base, the blots were extensively washed with deionized water.
Construction of Wild Type and Mutant c-KIT Mammalian Expression VectorsSite-directed mutagenesis of full-length c-KIT
cDNA in the pCVN vector (19) (a gift of Dr. Yosef Yarden, Weizmann
Institute, Rehovot, Israel) was performed using the Quickchange
site-directed mutagenesis kit (Stratagene, La Jolla, CA). In order to
change selected tyrosines to phenylalanines, we used the following
synthetic oligonucleotides: GAG GAG ATA AAT GGA AAC AAT
GTT TAC ATA GAC CCA ACA C (Tyr568
Phe) and GC AGC GAT
AGT ACT AAT GAG
ATG GAC ATG AAA CCT GGA G
(Tyr721
Phe). The mutant and wild type c-KIT
inserts were excised from the pCVN vector with
BamHI/XbaI exonucleases and inserted into pcDNA3 vectors for use in the transient transfection
experiments.
Wild type and mutant c-KIT pcDNA3 vectors
were co-transfected into COS 7 cells along with a -galactosidase pSV
reporter plasmid (Promega, Madison, WI). For each 10-cm plate of COS 7 cells, 10 µg of c-KIT pcDNA3 plus 5 µg of
-galactosidase pSV DNA were co-transfected using LipofectAMINE
reagent (Life Technologies, Inc.). After 5 h of transfection, full
medium was added, and cells were continued in culture for another
48-72 h. At this time, cells were serum-starved, stimulated with
SCF/KL (10 min, 350 ng/ml), and harvested into lysis buffer (as
described in the previous section). Supernatants were then subjected to
precipitation by SH2CHK-GST fusion protein as described in
the previous section. Glutathione-Sepharose precipitates were resolved
by SDS-PAGE and transferred to nitrocellulose followed by Western
blotting with c-KIT polyclonal antibody. In each case, amounts of
extract used in precipitation were normalized for the expression of
-galactosidase as measured by an O-nitrophenyl
-D-galactopyranoside reporter assay (Promega).
In order to identify the
tyrosine phosphorylation on c-KIT which is critical for
SH2CHK association, we synthesized a series of
tyrosine-phosphorylated heptapeptides derived from the cytoplasmic
portion of human c-KIT. Since SH2 domains usually associate with
residues on the C-terminal side of phosphotyrosine, we chose peptide
sequences with tyrosine as the N-terminal residue. We did not include
phosphotyrosine sites within the two catalytic domains of c-KIT. These
peptides were used to inhibit the interaction of c-KIT from
SCF/KL-activated Mo7e cells and the SH2CHK-GST fusion
protein as described under "Experimental Procedures." Complexes of
c-KIT and SH2CHK-GST were shown by the presence of c-KIT in
washed SH2CHK-GST precipitates (Fig. 1,
A and B). Of the peptides that inhibited complex
formation, the most significant was the Y*MDMKPG peptide (Fig.
1A, lane 10) with approximately 80% inhibition
of association at 100 µM peptide. The nonphosphorylated
YMDMKPG peptide failed to inhibit complex formation in this assay (Fig.
2, lane 5). We also found a slight inhibition
by the peptide Y*VYIDPT (Fig. 1, lane 9). Recently, it has
been reported that the analogous peptide sequence in the PDGF receptor
is a site for binding of FYN (15) and of SHC (16, 17). In the case of
FYN, binding was significantly greater when both tyrosines of the
sequence were phosphorylated. We tested whether this would be the case
with SH2CHK binding to c-KIT. As shown in Fig. 2, the
diphosphorylated peptide Y*VY*IDPT (lane 1) gave
significantly greater inhibition than did the monophosphorylated
Y*VYIDPT (lane 2). Furthermore, the monophosphorylated
Y*IDPTQL failed to give significant inhibition (Fig. 1, lane
3). Thus, both phosphates are required for inhibition at this
site. In order to test the relative abilities of Y*MDMKPG and Y*VY*IDPT
to inhibit SH2CHK/c-KIT interaction, various concentrations
of peptide from 5 µM to 100 µM were tested
(Fig. 3). Densitometry of c-KIT bands (not shown)
indicated an IC50 of ~50 µM for both
peptides. In a similar experiment substituting SH3-SH2CHK
for SH2CHK, both peptides inhibited to the same extent but
with a slightly lower IC50 of 20 µM. These
results indicated that binding of CHK is primarily at the
Tyr721 and Tyr568/570 sites on c-KIT.
Binding of SH2CHK and Native CHK to Immobilized Peptides
In order to further test the binding of CHK to the
phosphorylated Tyr721 and Tyr768/770 sites, we
linked these peptides to Affi-Gel 15 beads, and the association of
either SH2CHK-GST fusion protein or native CHK from crude
extracts of Mo7e cells was analyzed. As is shown in Fig.
4A, SH2CHK-GST associated in a
phosphotyrosine-dependent manner to Y*MDMKPG and Y*VY*IDPT
linked to beads as shown by an anti-GST immunoblot of the precipitates.
In a control precipitation, GST alone did not associate with the beads
(Fig. 4B). A non-receptor peptide derived from the
Tyr416 phosphorylation site of FYN (IEDNEY*TARQGA) showed
very little phosphate-dependent association to
SH2CHK. We also observed association of SH2CHK
to the phosphorylated C-terminal tyrosine peptide of SRC kinases (EPQY*QPGENL) (Fig. 4A, lane 8). When we looked
at the association of native CHK to peptide beads as shown by anti-CHK
immunoblotting, we observed a similar specificity (Fig.
5A). This specificity was in agreement with
the peptide inhibition experiments and also indicated that the
association was by direct binding of the SH2CHK domain to
the tyrosine-phosphorylated site(s) on c-KIT.
Possible Association of SH2CHK to c-KIT through Other Components
We have shown by peptide inhibition that the
PI3-kinase SH2p85 binds specifically to the Y*MDMKPG
peptide as does SH2CHK (data not shown). To confirm that
CHK binding to c-KIT is not indirectly via PI3-kinase p85, we stripped
and reblotted the transfers from Fig. 1 with anti-p85 subunit antibody
(Fig. 6). The distribution of p85 in this blot was
similar to the distribution of c-KIT seen in Fig. 1. Thus the lack of
p85 in the Y*MDMKPG-inhibited precipitate indicates that both p85 and
CHK are competing for the same site. If CHK was binding through p85,
inhibition with Y*MDMKPG would dissociate only c-KIT from the complex
and would retain p85 in the SH2CHK-GST precipitate. In the
case of the phosphorylated Tyr568/570 site, we have
demonstrated by immunoblotting that association of crude extracts to
peptide beads resulted in not only CHK, but also FYN and SHC binding at
this di-phosphorylated site (Fig. 5, B and C).
Csk did not associate with the diphosphorylated site and only slightly
associated with the monophosphorylated site (Fig. 5D,
left panel). This result was observed in spite of having a
large amount of Csk in the crude extract (Fig. 5D,
right panel). In order to confirm that the CHK association
at this site was direct, and not through either FYN or SHC, we studied
the association of Mo7e extracts to peptide beads with SCF/KL-activated
and nonactivated extracts. As can be seen in Fig. 7,
there was no observable difference in the association of CHK to the
beads when comparing SCF/KL-activated and nonactivated extracts. Thus,
it is unlikely that CHK associated with c-KIT through another component
which itself was tyrosine-phosphorylated in the course of activation.
Experiments attempting to co-precipitate SHC and CHK were negative
(data not shown). Experiments attempting to inhibit the
SH2CHK/c-KIT receptor complex with phosphopeptides present
in SRC kinases were also negative (data not shown), again emphasizing
that FYN and SHC were not intermediaries in CHK/c-KIT binding.
Effect of Site-directed Mutagenesis of c-KIT on Association with SH2CHK
In order to confirm the significance of the
binding of SH2CHK to the activated c-KIT at the
Tyr721 and Tyr568/570 sites, we constructed
mutant c-KIT cDNAs for either Tyr721 Phe
(designated 2162 c-KIT) or Tyr568
Phe
(designated 1702 c-KIT) and attempted to demonstrate whether these mutant proteins were still able to associate with the
SH2CHK. As can be seen in Fig.
8B, the wild type, Tyr568
Phe
(1702), and Tyr721
Phe (2162) c-KIT
constructs expressed comparable amounts of c-KIT protein as observed in
c-KIT Western blots of crude extracts from the transfectants, when
corrected for the extent of transfection as measured by the
-galactosidase reporter assay. When such extracts were precipitated
by anti-c-KIT antibody and blotted with anti-phosphotyrosine antibody,
comparable SCF/KL-dependent phosphorylation was observed for wild type and both mutant c-KIT proteins (data not shown). This
indicated that the expressed protein is capable of ligand stimulated
tyrosine autophosphorylation even with the replacement of the specific
tyrosines by phenylalanines. When wild type c-KIT-expressing cell
extracts were incubated with SH2CHK-GST, c-KIT associated
in an SCF/KL-dependent manner as was observed with
endogenous c-KIT (Fig. 8A, lanes 1 an
2). As expected, no c-KIT association was observed in the
GST control precipitation (Fig. 8A, lane 8). In
these experiments, when SH2CHK was incubated with either of
the two mutant c-KIT-expressing extracts, the SCF/KL-activated
Tyr568
Phe c-KIT was completely unable to associate
(lanes 3 and 4), while the Tyr721
Phe c-KIT still retained the ability to associate with
SH2CHK-GST in an SCF/KL-dependent manner
(lanes 5 and 6).
In this report, we have identified the sites of binding of the SH2CHK domain in the activated human c-KIT. Initial studies involving phosphopeptide inhibition indicated that it was the monophosphorylated Tyr721 and the diphosphorylated Tyr568/570 sites on c-KIT which were important in binding SH2CHK. To test the relative affinities of the two sites, the concentration dependence of the two phosphopeptides for inhibition of the c-KIT/SH2CHK interaction was assessed. Both peptides appeared to inhibit c-KIT/SH2CHK equally well. As has been shown with the PDGF receptor in the binding of SH2FYN to the diphosphorylated site, the second phosphotyrosine of Y*VY*IDPT resembles the glutamic acid which is present in the high affinity Y*EEI peptide as predicted (15, 19). The PI3-kinase site, on the other hand, has a glutamate residue at the +2 position. However, like the diphosphorylated site, the PI3-kinase site has hydrophobic amino acids at the +1 and +3 positions. Thus, it is not surprising that the SH2CHK was capable of being competed by the Y*VY*IDPT and the Y*MDMKPG peptides. The SH2FYN and SH2SHC both favored the binding to the Tyr568/570 site over the Tyr721 site. Studies of SH2SHC binding to activated PDGF receptor indicated binding to multiple sites (17). However, in these studies, binding of the diphosphorylated Tyr579/581 peptide was not analyzed in the peptide association experiments. Thus, it is likely in the case of the PDGF receptor that binding of SHC is favored at the Tyr579/581 site in light of our results. Other studies of FYN binding to the PDGF receptor (15) showed a clear preference for the diphosphorylated Tyr579/581 site over other sites. The binding affinity of FYN to the PDGF receptor (IC50 = 2 µM) was higher than was the binding of c-KIT to CHK (IC50 = 50 µM for SH2CHK/c-KIT and 20 µM for SH3-SH2CHK/c-KIT). This difference may be due to a less native structure in the isolated SH2 compared to the intact protein. The lowering of the IC50 in the case of SH3-SH2CHK/c-KIT is consistent with this. Thus, had we utilized the native CHK, we might have seen an even lower IC50. However, both Tyr568/570 and Tyr721 peptides showed a reduction in IC50 when SH3-SH2CHK was bound to c-KIT, indicating that the relative bindings of native CHK to the two sites are likely to be similar. To demonstrate that the interaction of SH2CHK at the two c-KIT sites is direct and not mediated by other proteins in the system, we showed that native CHK from cell extracts as well as a bacterial SH2CHK-GST fusion protein can bind to phosphorylated peptides linked to beads. This was an unequivocal indication that the CHK/c-KIT interaction was direct and not a result of binding via intermediates in the crude extracts. The possibility of intermediates in the binding was a concern because the sites identified are known to bind other SH2-containing proteins. The Tyr721 site is known to bind the p85 regulatory subunit of the PI3-kinase (20). In the PDGF receptor, the analogous Tyr579/581 juxtamembrane site is known to bind SH2 domains of SHC and SRC family kinases (15-17). The demonstration of direct binding could not completely rule out an additional association via FYN or SHC. Our results of blotting the peptide bead/lysate precipitates with anti-SHC and anti-FYN confirmed that indeed, SHC and FYN were binding to the juxtamembrane phosphorylated Tyr568/570 sequence of c-KIT. If these or other components were to serve as intermediates or adapters in the binding of SH2CHK to c-KIT, they themselves would have to be phosphorylated. However, nearly all tyrosine phosphorylation occurs after growth factor stimulation. Thus, the fact that there was no difference in association of peptide beads to CHK from SCF/KL-activated or nonactivated cells indicated that, again, an intermediate was unlikely. Therefore, it was reasonable to expect that we would not be able to co-precipitate SHC and SH2CHK, or that SRC phosphorylation site peptides would not interfere with SH2CHK/c-KIT interaction.
It is also notable that blotting the precipitates of extracts and peptide beads with anti-Csk did not show significant binding to the Tyr568/570 site, but did show some weak binding to the Tyr721 site. This observation is in spite of the fact that a significant amount of Csk was present in the crude lysates of these cells. Specificity of SH2Csk as determined by peptide libraries (19) shows a preference for Y*TKM. The similarity to the site Y*MDMKPG particularly at the +1 and +3 positions may explain the slight binding to this peptide. Csk has been shown to bind to tyrosine-phosphorylated paxillin, tensin, and focal adhesion kinase (FAK) (21, 22). This emphasizes the role of Csk in the regulation of SRC kinases located in focal adhesions. There have been no reports in the literature of Csk having the ability to bind to growth factor activated receptor tyrosine kinases. Thus, it is more likely that SRC family kinases bound to growth factor receptors such as c-KIT are phosphorylated and down-regulated by CHK and not Csk.
The implication of the c-KIT site directed mutagenesis is that in the association of SH2CHK to intact c-KIT, only the Tyr568/570 site is effectively able to bind while the Tyr721 site is not able to bind this SH2. This is in apparent contradiction to the data involving phosphopeptide inhibition and direct binding of CHK to phosphopeptide beads. A possible explanation for this is that the heptapeptides used in the experiments do not accurately reflect the 3-dimensional structure of the native c-KIT. Thus, the Y*MDMKPG peptide may resemble the Y*VY*IDPT peptide sufficiently such that it could compete for the Tyr568/570 site. It may also be that this Y*MDMKPG peptide linked to beads, is able to bind to the SH2CHK even though the native c-KIT is unable to bind to SH2CHK at the Tyr721 site. The mutagenesis experiment on the other hand does not rely on peptides to distinguish the binding and more accurately reflects the specificities of the protein-protein interactions. Furthermore, the mutation experiment indicates that there are no other sites on c-KIT that are able to bind the SH2CHK since binding was completely abolished by the mutation at the Tyr568 site. Thus, the data taken together indicate that it is only the Tyr568/570 site that is able to bind CHK through its SH2 domain.
A potential model for the regulation of SRC kinase by CHK involves the SCF/KL stimulated dimerization of c-KIT, one bearing an activated SRC kinase and the other bearing the CHK kinase. This dimerization would bring activated SRC kinase into proximity to CHK, leading to the down-regulation of SRC kinase activity. Further studies will be needed to establish whether SRC kinase bound to c-KIT is the primary target for CHK, and to elucidate the downstream effects resulting from these interactions.
This paper is dedicated in memory of our friend and colleague, Dr. Dananagoud Hiregowdara.
We thank Dr. Jerome Groopman for his constructive criticism and advice on this project, Dr. Yosef Yarden (Weizmann Institute, Rehovot, Israel) for providing the wild type c-KIT pCVN vector, and Dr. Brian Bennett for providing polyclonal anti-c-KIT antibody and SCF/KL. We also thank Janet Delahanty for her help in preparation of this manuscript.