From the Department of Physiology and Biophysics, School of Medicine, State University of New York at Stony Brook, Stony Brook, New York 11794-8661
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ABSTRACT |
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Src family protein-tyrosine kinases possess several modular domains important for regulation of catalytic activity and interaction with potential substrates. Here, we explore interactions between the SH2 domain of Hck, a Src family kinase, and substrates containing SH2 domain-binding sites. We have synthesized a series of peptide substrates containing a high affinity SH2 domain binding site, (phospho)Tyr-Glu-Glu-Ile. We show that the presence of this sequence in a peptide results in a dramatic increase in the phosphorylation rate of a second tyrosine located at the N terminus. Enhanced phosphorylation is not a consequence of stimulation of enzymatic activity by C-terminal tail displacement but is imparted instead by a 10-fold reduction in the Km of the phosphotyrosine-containing peptide when compared with a control. The isolated catalytic domain of the non-receptor tyrosine kinase Abl does not show a preference for the pYEEI motif-containing peptide; however, the preference is restored when the SH2 domain of Src is introduced into Abl. Furthermore, enhanced phosphorylation is dependent on the distance between SH2 domain-binding site and phosphorylatable tyrosine, with the minimum distance requirement being seven amino acids. Reversing the orientation of the pYEEI motif with respect to the substrate sequence decreases phosphorylation by down-regulated Hck, but both orientations are utilized equally well by activated Hck. We discuss the possible implications of these results for processive phosphorylation of substrates in vivo by Src family kinases.
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INTRODUCTION |
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There are presently nine identified members of the Src family of non-receptor tyrosine kinases. These enzymes are widely expressed in many tissues and are involved in the conversion of extracellular signals into cellular responses (see Ref. 1 for review). The primary structure of Src family kinases reveals a highly modular organization shared among all members. In addition to the kinase catalytic domain that is responsible for enzymatic activity, Src family kinases possess noncatalytic modules termed Src homology domains (SH2 and SH3). SH2 and SH3 domains mediate specific intramolecular and intermolecular interactions that are important for signal transduction; SH2 domains recognize short peptide motifs containing phosphotyrosine, and SH3 domains bind to proline-rich sequences (2, 3).
For Src family kinases, SH2 and SH3 domains have both a negative and a positive regulatory role in tyrosine kinase activity. The negative regulatory role arises from intramolecular contacts between the SH2 and SH3 domains and the tyrosine kinase catalytic domain. The structural basis for repression of catalytic activity has recently been elucidated by crystal structures of the down-regulated forms of Src and Hck (4, 5). Phosphorylation of Src family kinases at a C-terminal regulatory site by another protein kinase (designated Csk) leads to inhibition. This inhibition is caused by an intramolecular interaction between the phosphorylated tail and the SH2 domain (6-9). A polyproline type II helix is formed by the linker connecting the SH2 domain to the catalytic domain, and this polyproline type II helix serves as a docking site for the SH3 domain. This SH3 interaction appears to regulate kinase activity directly; addition of a high affinity SH3 ligand to down-regulated Hck causes maximal activation (10).
SH2 and SH3 domains also have a positive influence on non-receptor tyrosine kinase signaling. SH2- and SH3-mediated interactions with other proteins are important for at least two reasons: first, they target the tyrosine kinases to specific subcellular locations, and second, they assist the kinases in recognizing certain cellular substrates (11-13).
Processive phosphorylation by Src family kinases is when phosphorylation of a substrate by the catalytic domain creates a site that is recognized by the associated SH2 domain; binding to the SH2 domain increases the local concentration of substrate, allowing the catalytic domain to phosphorylate additional sites. Several proteins have been proposed to be phosphorylated processively by non-receptor tyrosine kinases. One such protein is the focal adhesion protein p130CAS. This protein possesses a Src SH2 domain-binding site near its C terminus (14, 15). Binding of Src to this region is thought to promote phosphorylation of p130CAS at multiple sites closer to the N terminus (15). Hyperphosphorylation of p130CAS has been demonstrated in vitro using a mutant form of Abl containing the Crk SH2 domain (16).
Similarly, the lymphocyte-specific Src family kinase Lck phosphorylates
several tyrosine residues in the chain of the T-cell receptor. Each
chain contains three immunoreceptor tyrosine-based activation
motifs with the consensus
YXX(I/L)X6-8YXX(I/L) (where X is any amino acid) arrayed in tandem (17). Mutation of amino acids involved in phosphotyrosine recognition in the SH2
domain of Lck reduces receptor hyperphosphorylation and signal transduction, consistent with a processive phosphorylation model (18).
Despite these observations for the Src family kinases, the mechanistic details of processive phosphorylation have not been examined closely. Sequence requirements for the interaction between the SH2 domains of Src family kinases and potential substrates are not well understood. In this report, we have tested a basic principle of the processive phosphorylation model by asking whether a high affinity SH2 binding sequence on a substrate enhances phosphorylation of other sites on the same substrate. Using a series of tyrosine-phosphorylated peptides, we show that the SH2 binding sequence indeed enhances phosphorylation of the substrate. We also report the results of experiments aimed at determining the optimal spacing and orientation of the SH2 domain-binding site relative to the potential phosphorylation site.
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EXPERIMENTAL PROCEDURES |
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Peptides--
Synthetic peptides and phosphopeptides were
prepared by solid phase synthesis using standard
Fmoc1 chemistry (19) using an
Applied Biosystems Inc. automated 431A peptide synthesizer.
Phosphotyrosine was incorporated into the peptides using
N-Fmoc-O-phospho-L-tyrosine
(Novabiochem) (20). Peptides were purified by preparative
reversed-phase high performance liquid chromatography. Matrix-assisted
laser desorption ionization time-of-flight mass spectrometry and amino
acid analysis were used to confirm the identity of the final products.
The concentrations of peptide stock solutions were determined by
quantitative amino acid analysis (Commonwealth Biotechnologies,
Richmond, VA).
Proteins-- Expression and purification of C-terminally phosphorylated Hck is described elsewhere (4). To obtain C-terminally dephosphorylated Hck, the enzyme was treated with Yersinia protein-tyrosine phosphatase, as described previously (10). Autophosphorylation of Tyr411 (Tyr416 of c-Src) was performed by incubation of the enzyme (5 mg/ml) on ice for 1 h in kinase assay buffer (20 mM Tris, pH 7.5, 20 mM MgCl2, 1 mg/ml bovine serum albumin) in the presence of 2 mM ATP (10). Full-length v-Src was produced using a baculovirus vector in Sf9 cells and purified by immunoaffinity chromatography as described (21). The Abl catalytic domain was produced in Escherichia coli as a fusion protein with glutathione S-transferase and purified as described (21, 22). The baculovirus vector encoding a chimera with the Src SH2 domain and the Abl catalytic domain was the gift of Dr. Bruce Mayer (13). Baculovirus was generated by established procedures (22), and the chimera was purified on glutathione agarose (22).
Kinase Assays-- Kinase activity measurements were carried out at 30 °C using the phosphocellulose filter binding assay (24, 25). Graphs shown are representative of three or more experiments, each carried out in duplicate. For kinetic measurements, each reaction contained 26 nM Hck that had been previously dephosphorylated at the C terminus and allowed to autophosphorylate at Tyr411 (10). Initial rates of reaction were measured in duplicate, and kinetic constants were determined by fitting to the hyperbolic velocity versus [substrate] plots using the program MacCurve Fit.
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RESULTS AND DISCUSSION |
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Peptides-- The sequences of the synthetic peptides used in these experiments are presented in Table I. The phosphopeptides contain an optimal Src SH2 domain-binding sequence: (phospho)Tyr-Glu-Glu-Ile. This sequence is derived from the polyoma virus middle T antigen, which forms a 1:1 complex with c-Src in cells (26). The pYEEI sequence has been demonstrated to bind with high affinity to the Src SH2 domain (27). Control peptides have a phenylalanine in place of phosphotyrosine. The phosphorylation site sequences on these peptides correspond to a phosphorylation sequence (Glu-Asp-Ala-Ile-Tyr) that we obtained using synthetic peptide libraries (25).
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Enhanced Phosphorylation of Y(11)pY-- To determine whether introduction of an SH2 domain binding site would lead to increased phosphorylation of additional tyrosines within the same peptide, we compared Y(11)pY with its control Y(11)F using C-terminally phosphorylated Hck. We observed a strong preference for Y(11)pY, the peptide containing the SH2 domain binding motif (Fig. 1). In principle, the presence of a high affinity SH2 domain binding site in multiply phosphorylated substrates could facilitate phosphorylation in two ways. First, the pYEEI motif could increase the enzymatic activity of Src family kinases through the displacement of the C-terminal negative regulatory site from the SH2 domain. Second, SH2 domain-pYEEI interactions could promote the further phosphorylation of other tyrosines within the same substrate by raising the effective local concentration of potential phosphorylation sites near the catalytic domain. To discriminate between these possibilities, we performed experiments similar to those shown in Fig. 1 using two enzymes where activation by C-terminal tail displacement is not possible: (i) v-Src, which lacks the C-terminal tail, and (ii) Hck that had been dephosphorylated at Tyr522 (Tyr527 of c-Src) and pre-activated by incubation with ATP. SH2 domain activators, such as the pYEEI peptide, do not activate these enzymes. Both v-Src and dephosphorylated Hck showed increased phosphorylation of Y(11)pY over Y(11)F (data not shown).
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Kinetics-- To clarify further the mechanism of the enhanced phosphorylation of Y(11)pY, we performed initial rate kinetic studies using Hck that had been dephosphorylated at Tyr522 in the C-terminal tail and allowed to autophosphorylate at Tyr411. The results are shown in Table II. Similar values of Vmax were observed for Y(11)pY and Y(11)F. However, introduction of an SH2 domain-binding site in Y(11)pY reduces the Km of the peptide substrate 10-fold, thus increasing the catalytic efficiency (kcat/Km) of the enzyme 10-fold as well. The high affinity interaction between the SH2 domain of Hck and the pYEEI motif of Y(11)pY presumably gives the substrate an advantage because of its high local concentration in proximity to the catalytic domain.
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Role of SH2 Domains-- We carried out experiments to confirm the importance of the SH2 domain in the observed enhanced phosphorylation of Y(11)pY. We first tested for phosphorylation of Y(11)pY versus Y(11)F using the catalytic domain of the non-receptor tyrosine kinase Abl (AblCAT). Because of the absence of an SH2 domain in this construct, we predicted that AblCAT would not discriminate between Y(11)pY and Y(11)F. Fig. 2A shows that, as expected, the isolated catalytic domain of Abl did not preferentially phosphorylate Y(11)pY. In fact, AblCAT favored the phenylalanine control peptide, Y(11)F, over Y(11)pY.
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Length Dependence--
We varied the spacing between the
phosphotyrosine and the phosphorylatable tyrosine to assess the minimum
distance required for enhanced phosphorylation to occur. We tested
peptides of various lengths (Table I) using C-terminally
dephosphorylated (pre-activated) Hck. Fig.
3 shows that there is a length dependence
to the observed enhanced phosphorylation of pYEEI-containing peptides.
For example, when the spacer is reduced to 7 amino acids, as in Y(7)pY,
the rate of phosphorylation is reduced when compared with the longer peptide Y(11)pY but is still above Y(11)F or Y(3)F, the phenylalanine controls. However, when the spacer is further reduced to 3 amino acids,
as in Y(3)pY, the enhanced phosphorylation is no longer observed, and
the phosphorylation rates approach those of the control peptides,
Y(11)F and Y(3)F. These results indicate that in order for SH2
domain-binding motifs to confer an advantage to substrates, there must
be a minimum distance between the phosphotyrosine and the tyrosine.
These spacer lengths are consistent with the 9-11-residue spacers
observed between phosphorylated tyrosines in immunoreceptor
tyrosine-based activation motifs of TCR chains (17).
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Reversed Orientation-- To examine the effect of reversing the orientation between the substrate tyrosine and the SH2 domain-binding site, we produced peptide substrates containing the pYEEI motif (or FEEI control) N-terminal to the tyrosine (Table I). Fig. 4 shows a comparison between Y(11)pY and pY(12)Y using down-regulated Hck. There is a preference for Y(11)pY, the peptide containing the phosphotyrosine at the C- terminus. Both peptides were phosphorylated more efficiently than F(12)Y, the control lacking an SH2 domain-binding site at the N terminus (data not shown). The preference for Y(11)pY over pY(12)Y is consistent with the structure of down-regulated Hck (4), where the phosphotyrosine-containing tail of Hck binds to the SH2 domain in an orientation that places the N terminus of the tail closer to the catalytic domain than the C terminus.
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Concluding Remarks-- Using a synthetic peptide model system, we report here that substrates containing high affinity binding sites for Src family kinase SH2 domains display enhanced phosphorylation at additional sites on the substrate. These experiments on processive phosphorylation suggest that the SH2 domain plays an important role in substrate recognition for non-receptor tyrosine kinases. Based on studies with peptide libraries, it has been proposed that phosphorylation of different peptide sequences by the Src kinase domain correlates with the binding of the sequences to the Src SH2 domain (30). This raises the interesting possibility that the two domains co-evolved to allow the catalytic domain to create sites that will be recognized by the associated SH2 domain (3).
The in vivo substrate specificity of Src family tyrosine kinases reflects both the intrinsic specificity of the kinase catalytic domains and the effective local concentrations of protein substrates. In many cases, the distribution of potential substrates is influenced by interactions with noncatalytic regions of the enzymes such as SH2 and SH3 domains. Previous studies have demonstrated that molecules that bind with high affinity to the SH2 or SH3 domains of Src family kinases can disrupt the relatively low affinity intramolecular interactions, leading to enzyme activation (10, 31). Thus, a variety of protein-protein interactions in the cell can lead to signaling through active Src kinases. Our data imply that the most productive interactions may occur when these high affinity ligands are present together with potential phosphorylation sites on the same molecule, i.e. in the case of processive phosphorylation. This would impart an additional level of specificity in substrate recognition. ![]() |
ACKNOWLEDGEMENTS |
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We thank Drs. John Kuriyan and Frank Sicheri (Rockefeller) for samples of purified Hck and Dr. Bruce Mayer (Harvard Medical School) for the SrcSH2-AblCAT baculovirus expression construct and for a critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant CA58530 (to W. T. M.).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. Tel.: 516-444-3533;
Fax: 516-444-3432; E-mail: miller{at}physiology.pnb.sunysb.edu.
1 The abbreviation used is: Fmoc, N-(9-fluorenyl)methoxycarbonyl; TCR, T-cell receptor.
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REFERENCES |
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