From the Department of Biochemistry and Molecular
Biology, University of Melbourne, Parkville, Victoria 3052, Australia,
the § Ludwig Institute for Cancer Research, Melbourne Tumour
Biology Branch, P. O. Royal Melbourne Hospital, Victoria 3050, Australia, and the ** Department of Medical Biochemistry, University of
Calgary, Calgary, Alberta T2N 4N1, Canada
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ABSTRACT |
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Hck and Src are members of the Src family of protein- tyrosine kinases that carry out distinct and overlapping functions in vivo (Lowell, C. A., Niwa, M., Soriano, P., and Varmus, H. E. (1996) Blood 87, 1780-1792). In an attempt to understand how Hck and Src can function both independently and in concert, we have compared 1) their in vitro substrate specificity and 2) the accessibility of their Src homology 2 (SH2) domain. Using several synthetic peptides, we have demonstrated that Hck and Src recognize similar structural features in the substrate peptides, suggesting that both kinases have the intrinsic ability to carry out overlapping cellular functions by phosphorylating similar cellular proteins in vivo. Using a phosphotyrosine-containing peptide that has previously been shown to bind the SH2 domain of Src family kinases with high affinity, we found that although Src could bind to the phosphopeptide, Hck showed no interaction. The inability of Hck to bind the phosphopeptide was not a result of a stable intramolecular interaction between its SH2 domain and C-terminal regulatory phosphotyrosine residue (Tyr-520), as most Hck molecules in the purified Hck preparation were not tyrosine-phosphorylated. In contrast to intact Hck, a recombinant truncation analog of Hck was able to bind the phosphopeptide with an affinity similar to that of the Src SH2 domain, suggesting that conformational constraints are imposed on intact Hck that limit accessibility of its SH2 domain to the phosphopeptide. Furthermore, the difference in SH2 domain accessibility is a potential mechanism that enables Src and Hck to perform their respective unique functions by 1) targeting them to different subcellular compartments, whereupon they phosphorylate different cellular proteins, and/or 2) facilitating direct binding to their cellular substrates.
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INTRODUCTION |
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The Src family of protein-tyrosine kinases, Src, Yes, Lck, Fyn, Lyn, Fgr, Hck, Blk, and Yrk, are nonreceptor tyrosine kinases, a number of which have been shown to be involved in a wide variety of cellular processes, ranging from cell growth and proliferation to neural functions (see Ref. 1 for review). With the exception of a 40-70 residue unique domain, all Src family kinases are highly homologous in structure. The homologous domains include (i) the fatty acid acylation domain, which targets the kinases to the plasma membrane, (ii) the Src homology 2 (SH2)1 and Src homology 3 (SH3) domains, which facilitate protein-protein interactions, (iii) the kinase domain, and (iv) the C-terminal regulatory domain. A consensus tyrosine residue in the kinase domain can readily undergo autophosphorylation; autophosphorylation of this tyrosine results in autoactivation of the kinase (see ref. 1 for review). In contrast, phosphorylation of a tyrosine in the C-terminal regulatory domain renders the kinase inactive. C-terminal Src kinase (CSK) and/or CSK-related kinases are responsible for phosphorylating the C-terminal regulatory tyrosine. The crystal structures of inactivated Hck and Src reveal that intramolecular interaction between the phosphorylated C-terminal regulatory tyrosine and the SH2 domain contributes to inactivation of the kinases (2, 3).
In addition to stabilizing the inactive conformation of the kinases, the SH2 domain mediates interactions of the Src family kinases with other cellular proteins. The SH2 domain binds to a phosphotyrosine-containing motif in cellular proteins, whereas the SH3 domain binds to proline-rich sequences. Through interactions with other cellular proteins, the kinases can be targeted to specific subcellular compartments, where they have immediate access to their substrate proteins (see Ref. 4 for review). Furthermore, the SH2 and SH3 domains have been shown to mediate binding of Src to some of its substrates, e.g. p130cas, Sin, and the focal adhesion kinase (5, 6). For this reason, the accessibility or specificity of the SH2 and SH3 domains of a Src family kinase can influence the subcellular localization of the kinase and hence the subset of cellular proteins available for phosphorylation by the kinase, as well as the direct recognition of substrate proteins.
Sequences of phosphopeptides capable of high affinity binding to the SH2 domain of Src family kinases have been identified using a degenerate combinatorial phosphopeptide library (7). The local structure around Tyr-315 of the hamster polyoma virus middle T antigen was found to contain the pYEEI motif, which was shown by combinatorial library studies to be preferred by the SH2 domain of Src family kinases (7). Further studies showed that the synthetic middle T antigen phosphopeptide containing the pYEEI motif (pYEEI peptide) can bind the Src family kinase SH2 domain with high affinity (7, 8).
Previous studies by us (9) and other investigators (10) have shown that the local structures flanking the target tyrosine in substrate proteins and peptides contain structural features recognized by the kinase domain of several Src family members. Thus, short synthetic peptides are useful tools for analyzing the in vitro substrate specificities of Src family kinases.
Targeted disruption of the src and/or hck gene in mice indicates that the two kinases carry out specific and overlapping functions in several cell types, including osteoclasts and macrophages (11-13). In an attempt to elucidate the structural basis accounting for the functional specificity and redundancy of Src and Hck, we have used biochemical approaches to compare their in vitro substrate specificity and SH2 domain accessibility. Our results reveal that the kinase domains of both Src and Hck recognize similar structural features in substrate peptides, indicating that both kinases have the potential to carry out overlapping functions by phosphorylating similar cellular proteins. We have also shown that the SH2 domain in intact Src is readily accessible to the pYEEI phosphopeptide, whereas it is inaccessible in intact Hck, supporting the notion that SH2 domain accessibility of the two kinases is regulated by different mechanisms. Because SH2 domain accessibility is one of the factors determining subcellular localizations of the kinases, the different accessibility of the Src and Hck SH2 domains may target them to different subcellular localizations, whereupon they phosphorylate different subsets of cellular proteins, which may contribute in part to the differences in their different cellular functions.
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EXPERIMENTAL PROCEDURES |
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Materials--
The Sephadex G25 Fine gel filtration matrix, the
FPLC Mono-Q (HR5/5) column, and the Mono-S (HR5/5) column were
purchased from Amersham Pharmacia Biotech. Hydroxylapatite and
Affi-Gel-15 were from Bio-Rad. The preparative reverse-phase Econosil
C18 high performance liquid chromatography column was from
Alltech (Deerfield, IL), and the analytical Vydac reverse-phase
C18 high performance liquid chromatography column was from
Separation group Inc. Immobilon polyvinylidene fluoride membranes were
from Millipore (Bedford, MA). The ECL reagents were from Amersham
Pharmacia Biotech. The horseradish peroxidase-linked sheep anti-rabbit
IgG and the alkaline phosphatase-linked anti-rabbit IgG were from
Silenus Laboratory (Hawthorn, Victoria, Australia). The polyclonal
-hck antibody H1077 was a kind gift of Dr. C. Lowell
(University of California, San Francisco, CA) (11). Hybridoma cells
producing the monoclonal Src antibody mAb327 were kindly provided by
Dr. J. Brugge (20). The monoclonal antiphosphotyrosine antibody (pY69)
was purchased from ICN Pharmaceuticals Inc. (Costa Mesa, CA). The
polyclonal anti-phosphotyrosine antibody was from Upstate Biotechnology
Inc. Recombinant Src was expressed and purified as described by Bjorge
et al. (20). Less than 5% of purified Src was
tyrosine-phosphorylated (20). The recombinant CSK was purified from
Spodoptera frugiperda 9 (Sf9) cells infected with the
recombinant baculovirus containing the csk gene, kindly provided by Dr.
D. O. Morgan (20).
Construction of the hck Baculovirus Vector and Generation of the Recombinant hck Baculovirus-- The full-length hck cDNA (21) was subcloned into the NheI site in pBlueBac II (Invitrogen Corporation, San Diego, CA). Sf9 insect cells (Invitrogen) were co-transfected with wild-type baculoviral DNA and pBB2hck by standard calcium phosphate transfection procedures (22). Recombinant hck baculovirus was purified following three rounds of plaque purification by visual screening. The titer of the recombinant hck baculovirus was determined, and Western blotting using an anti-hck antiserum (12) was used to optimize protein production following infection.
Purification of Hck from Crude Cell Lysates of Sf9 Cells
Infected with Recombinant Hck Baculovirus--
A large scale (2-liter)
culture of Sf9 cells was infected with recombinant Hck
baculovirus at a multiplicity of infection of 1.0, and cells were
harvested 3 days after infection for protein purification. All of the
extraction and purification procedures were carried out at 4 °C.
Cells were pelleted at 1000 × g for 5 min, washed once
with Grace's serum-free medium, and homogenized in a buffer consisting
of 25 mM Hepes, pH 7.0, 5% Nonidet P-40, 1 mM
EDTA, 0.1 mg/ml soybean trypsin inhibitor, 0.2 mg/ml benzamidine, 0.1 mg/ml phenymethylsulfonyl fluoride, and 1 mM
dithiothreitol. The homogenate was clarified by centrifugation at
100,000 × g for 40 min. Recombinant Hck was purified
by sequential column chromatography on a Q-Sepharose (Amersham
Pharmacia Biotech) anion exchange column and a hydroxylapatite column,
followed by a Sephacryl-200 gel filtration column. At pH 7.0, the
majority (>95%) of the recombinant Hck was eluted from the
Q-Sepharose column when the column was washed with the column buffer.
For hydroxylapatite column chromatography, the wash fraction from the
Q-Sepharose column was loaded onto the column (length × internal
diameter = 80 × 22 mm) pre-equilibrated with column buffer
consisting of 25 mM Hepes, pH 7.0, 0.1% Nonidet P-40, 10%
glycerol, 0.2 mg/ml benzamidine, 0.1 mg/ml phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol. After the column was
washed, bound proteins were eluted with a 200-ml linear gradient of
0-0.3 M potassium phosphate in column buffer (pH 7.0). Hck
was monitored by both the protein-tyrosine kinase activity assay using
[Lys-19]cdc2(6-20) peptide as the substrate and -hck
immunoreactivity. The peak activity fractions from the hydroxylapatite
column were pooled, concentrated to 10 ml with an Amicon concentration
cell, and then loaded onto a Sephacryl 200 gel filtration column
(length × internal diameter = 760 × 22 mm)
pre-equilibrated with 0.2 M NaCl in column buffer. The peak
Hck activity and immunoreactivity in the eluted column fractions were
determined. SDS-PAGE revealed that the 56- and 59-kDa isoforms of Hck
are the major proteins in the final preparation after S-200 column
chromatography (Fig. 1).
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Construction of the Vectors for the Expression of GST-Hck(1-247) and GST-Hck(1-129)-- Two polymerase chain reaction products were generated: (i) a 751-base pair DNA fragment encoding residues 1-247 of the 59-kDa isoform of murine Hck (21) was amplified using the full-length hck cDNA as a template with primer 1 (5'-GGAATTCCTGGGGGGTCGGTCTAGCTGC) and primer 2 (5'-ATCGAAGCTTTCATTTCTCCCATGGCTTCTG), and (ii) a 405-base pair DNA fragment encoding residues 1-129 was amplified from the same template with primer 1 and primer 3 (5'- ATCGAAGCTTTCAAGCCACATAGTTGCTTGG). In the primer sequences, the restriction sites for EcoRI and HindIII are in boldface italic and the in-frame stop codons are underlined.
The polymerase chain reactions were carried out in a volume of 100 µl containing 50 ng of template DNA, 67 mM Tris-HCl (pH 8.8 at 25 °C), 16.6 mM (NH4)2SO4, 0.45% Triton X-100, 0.2 mg/ml gelatin, 2.5 units of a 16:1 (v/v) mixture of Taq polymerase (Biotech) and rPfu polymerase (Stratagene), 2 mM MgCl2, 100 µM dNTPs, and 0.2 µM of each primer. The amplification was performed in a Perkin-Elmer 9600 cycler and consisted of one cycle at 95 °C for 2 min; 25 cycles of 1 min at 94 °C, 1 min at 60 °C, and 1 min at 72 °C; and a final cycle of 7 min at 72 °C. Site-directed mutagenesis was used to modify the Escherichia coli expression vector pGEX2T (AMRAD, Melbourne, Australia). The SmaI site in pGEX2T was mutated to a HindIII site to allow subcloning of the polymerase chain reaction products. The polymerase chain reaction products from steps (i) and (ii) were digested with EcoRI and HindIII, purified on a 1% low melting temperature agarose gel (SeaPlaque, FMC BioProducts, Rockland, ME) and then directionally subcloned into the modified expression vector pGEX2TH to give (i) pGXHck(1-247) and (ii) pGXHck(1-129). Upon expression, GST-Hck(1-247), consisting of the unique, SH3, and SH2 domains, and GST-Hck(1-129), consisting of the unique and SH3 domains of Hck, were obtained.Purification of the GST Fusion Proteins of Hck
Fragments--
Both GST-Hck(1-247) and GST-Hck(1-129) bound strongly
to glutathione-agarose and could not be released using the high
salt/high glutathione buffer (25 mM Hepes, pH 7.0, 0.1%
Nonidet P-40, 1 mM EDTA, 50 mM glutathione, 2 M NaCl) (data not shown). For this reason, GST-Hck(1-247)
and GST-Hck(1-129) were purified by two sequential steps on a
hydroxylapatite column (length × internal diameter = 90 × 12 mm) and an FPLC Mono-S cation exchange column (HR 5/5). The crude
cell lysate was loaded onto the hydroxylapatite column pre-equilibrated
with Buffer A (25 mM Hepes, pH 7.0, 1 mM EDTA,
1 mM dithiothreitol, 10% glycerol, 0.1% Nonidet P-40, 0.02 mg/ml phenylmethylsulfonyl fluoride, and 0.2 mg/ml benzamidine). After the column was washed with 20 ml of Buffer A, bound proteins were
eluted with a 90-ml linear gradient of 0-0.3 M potassium phosphate in Buffer A at a flow rate of 1 ml/min. Peak protein fractions were identified by SDS-PAGE and immunoblot analysis using
-hck (H1077) antibody. The immunoreactive fractions were pooled, dialyzed overnight in 2 liters of Buffer A, and then loaded onto a Mono-S column pre-equilibrated with Buffer A. After being washed
with 5 ml of Buffer A, bound proteins were eluted using a 15-ml linear
gradient of 0-0.5 M NaCl in Buffer A at a flow rate of 0.5 ml/min. Fractions containing the recombinant proteins were identified
using SDS-PAGE and immunoblot analysis.
Construction of the GST Fusion Vector and Purification of the GST Fusion Protein of Src(1-258)-- Bluescript plasmid (Stratagene) containing the full-length coding region of human Src (20) was digested with NcoI and EcoN1 and then treated with Klenow to create blunt ends. The DNA fragment of 775 nucleotides encoding residues 1-258 of Src was isolated on an agarose gel and ligated into the SmaI site of pGEX-2T vector (Amersham Pharmacia Biotech). The expected orientation and sequence of the resulting expression plasmid was confirmed by restriction enzyme digest and DNA sequencing.
Upon expression, GST-Src(1-258), consisting of the unique, SH3, and SH2 domains of Src, was purified by two sequential steps on a hydroxylapatite column and an FPLC Mono-S cation exchange column under the same conditions as for the purification of GST-Hck(1-247) and GST-Hck(1-129).Preparation of Synthetic Peptides-- Synthetic peptides were synthesized with an Applied Biosystems Model A431 automated peptide synthesizer using N-(9-fluorenyl)methoxycarbonyl-based chemistry. Peptides synthesized included (i) phosphopeptide (pYEEI peptide) derived from the sequence of the hamster polyoma virus middle T antigen THQEEEEPQ(pY)EEIPIYL, and (ii) the substrate peptides. The synthesis and purification of these peptides have been detailed in previous reports (9, 10).
Preparation and Characterization of pYEEI Peptide Immobilized to a Solid Support-- Purified pYEEI peptide was covalently coupled to Affi-Gel-15 agarose following the procedures detailed by the manufacturer (Bio-Rad). The degree of coupling of the peptide to Affi-Gel was determined; a coupling density of 3.04 µmol of pYEEI peptide per ml of packed gel was revealed. The immobilized pYEEI peptide (pYEEI-gel) was diluted with the ethanolamine-treated Affi-Gel-15 agarose (control gel) before use.
SH2 Domain Accessibility Assay--
The ability of Src, Hck, and
the GST fusion proteins GST-Src(1-258), GST-Hck(1-129), and
GST-Hck(1-247) to bind pYEEI peptide was assayed by incubating 280 ng
of protein at 4 °C for 1 h with 20 µl (equivalent to 0.79 nmol of the immobilized pYEEI peptide) of pYEEI-gel equilibrated in the
binding buffer (25 mM Hepes, 1 mM EDTA, 0.2 mg/ml benzamidine, 10% glycerol, 0.1% Nonidet P-40, 0.2 M
NaCl, and 1 mM -mercaptoethanol). The mixture was
centrifuged at 10,000 × g for 5 min, and the
supernatant containing the unbound enzyme was removed and stored for
analysis. The pYEEI-gel was washed with 5 × 1 ml of binding
buffer, and the bound proteins were eluted by boiling in 20 µl of
SDS-PAGE sample buffer. Bound and unbound proteins were quantified
after SDS-PAGE by the procedures described under "Immunoblot
Analysis."
Immunoblot Analysis--
Immunoblot analysis was performed
according to the method of Towbin et al. (23). Briefly,
proteins were separated by SDS-PAGE and then transferred to a
polyvinylidene difluoride membrane filter. The filter was probed with
primary antibody (either -hck polyclonal antibody, the
polyclonal anti-phosphotyrosine antibody,
-Src monoclonal antibody
(M327), or
-phosphotyrosine (PY69) monoclonal antibody) followed by
horseradish peroxidase-conjugated sheep anti-rabbit IgG (for
-hck polyclonal antibody and the polyclonal antiphosphotyrosine antibody) or the horseradish peroxidase-conjugated sheep anti-mouse IgG (for
-Src (M327) and
-phosphotyrosine (PY69) antibodies) and developed using the enhanced chemiluminescence kit
following the protocol detailed by the manufacturer. The relative amounts of Src, Hck, GST-Src(1-258), GST-Hck(1-247), and
GST-Hck(1-129) were determined by densitometry.
Phosphopeptide Mapping of Autophosphorylated and
CSK-phosphorylated Src--
Src (18.8 nM) was
autophosphorylated by incubation for 10 min at 30 °C in kinase
buffer containing 50 µM [-32P]ATP in a
16-µl volume. In another reaction, Src was phosphorylated by CSK upon
incubation of Src with 188 nM CSK under the same conditions for 30 min (Fig. 2A). The
reactions were terminated by addition of 10 µl of 5× SDS sample
buffer (26). Phosphorylated Src was separated from CSK and free
[
-32P]ATP by SDS-PAGE. The proteins on the gel were
then electrotransferred to nitrocellulose, and the phosphorylated Src
was located by autoradiography.
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Characterization of the Immunoreactivity of the Monoclonal (PY69) and the Polyclonal Anti-phosphotyrosine Antibodies against Phosphotyrosines in Different Sequence Contexts-- The PY69 anti-phosphotyrosine antibody may bind to phosphotyrosine sequence contexts with differing affinities; therefore, we examined its immunoreactivity against the autophosphorylation site (Tyr-419) and the phosphorylated C-terminal regulatory tyrosine (Tyr-530) of Src. Src was chosen instead of Hck because Hck is a much poorer in vitro substrate of CSK (data not shown). In addition, we were unable to obtain enough Hck phosphorylated exclusively at the C-terminal regulatory tyrosine for analysis.
Src was first incubated with ATP in assay buffer in both the presence and the absence of CSK. Under the conditions we used, Src was autophosphorylated at Tyr-419 to a stoichiometry of 0.3 mol PO3= per mol of enzyme (Protein Kinase Assay--
The protein-tyrosine kinase activity
of Hck in hydroxylapatite and S-200 gel filtration column fractions was
determined by measuring incorporation of PO3=
from [-32P]ATP into [Lys-19]cdc2(6-20). Routine
enzyme assays were carried out at 30 °C in a 50-µl volume of
kinase buffer (20 mM Tris-HCl, pH 7, 10 mM
MgCl2, 1 mM MnCl2, 83 µM Na3VO4) with 100 µM ATP (specific radioactivity, 300-400 cpm/pmol) and
300 µM [Lys-19]cdc(6-20). The reactions were allowed
to proceed for 6-15 min before they were terminated by the addition of
20 µl of 50% acetic acid. A 30-µl aliquot was spotted onto a
phosphocellulose paper square, which was subsequently washed six times
in 0.3% H3PO4, washed once with acetone, and
then dried. Radioactivity in the dried paper square was monitored by
Cerenkov counting. Under the conditions used, only the initial rates of
phosphorylation of the peptides by the kinases were measured.
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RESULTS |
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Comparison of the in Vitro Peptide Substrate Specificity of Hck and Src-- Because little is known about the identities of the cellular proteins phosphorylated by Src and Hck in vivo, we compared the in vitro substrate specificities of the two kinases using a series of short synthetic substrate peptides. The rationale behind our choice of peptides was as follows. The Src autophosphorylation site peptide is derived from the autophosphorylation site in the Src kinase domain and is known to be an in vitro substrate of the Src family kinases. The cdc2(6-20) peptide has previously been shown to be a specific substrate of several Src family kinases, including Src, Lyn, Lck, and Fyn (9). [Lys-19]cdc2(6-20) and [Val-12,Ser-14,Lys-19]cdc2(6-20), which are substitution analogs of cdc2(6-20), have been used to define the substrate specificity determinants of Src family kinases. We have previously demonstrated that replacement of Glu-12 and Thr-14 in cdc2(6-20) by valine and serine, respectively, adversely affects the ability of Src to phosphorylate the peptide analogs, whereas replacement of Tyr-19 by lysine had no detrimental effect (9). The Src-optimal peptide and abl-optimal peptide were discovered by Songyang et al. (10) and shown to carry all the essential structural determinants for efficient phosphorylation by Src and Abl tyrosine kinase. The peptide library study by Songyang et al. (10) also revealed that the hydrophobic Ile-5 and Phe-9 at the pY-1 and pY+3 positions in the Src-optimal peptide are the two most crucial structural determinants recognized by Src. We therefore prepared the analog [Ala-5,Ala-9]Src-optimal peptide with the two critical hydrophobic residues replaced by the less hydrophobic alanine residue and examined the effect of the substitution on the efficiency of phosphorylation of the peptide by both kinases.
Of the peptides used, [Val-12,Ser-14,Lys-19]cdc2(6-20), [Ala-5,Ala-9]Src-optimal peptide, and Src autophosphorylation site peptides were very poor substrates (Table I). [Lys-19]cdc2(6-20) and cdc2(6-20), however, were phosphorylated by the kinases with catalytic efficiencies (Vmax/Km) 30-50-fold higher than that of Src autophosphorylation site peptide. Similar to Src, Hck phosphorylated the abl-optimal peptide with an efficiency much lower than that of Src-optimal peptide. The Src-optimal peptide was the best substrate for Src and Hck because it was phosphorylated by both kinases at efficiencies that were about 400-fold higher than that of the Src autophosphorylation site peptide. Furthermore, Src and Hck required the presence of Ile-5 and Phe-9 in Src-optimal peptide and Glu-12 and Thr-14 in cdc2(6-20) as crucial substrate specificity determinants, because their substitution dramatically reduced the efficiency of peptide phosphorylation by both kinases (Table I).
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Comparison of the Ability of Hck and Src to Bind the Immobilized pYEEI Peptide-- The ability of a cellular protein to be phosphorylated in vivo by a specific member of the Src family is most likely determined by (i) the presence of specific determinants in the cellular protein that are recognized by the catalytic domain, (ii) the ability of the cellular protein to co-localize with the kinase in the same subcellular compartment, and/or (iii) the ability of the cellular proteins to bind to the SH2, SH3, or unique domain of the kinase. Because of the importance of the SH2 domain in determining the subcellular localization and substrate specificities of Src family kinases, we studied the functional properties of the SH2 domains of Src and Hck.
The pYEEI peptide has previously been demonstrated to bind the SH2 domains of several Src family kinases (Src, Lyn, and Lck) with high affinity (7, 8, 24); we therefore used the pYEEI peptide immobilized to agarose as the ligand to study the binding specificity and accessibility of the SH2 domain of Src and Hck. As shown in Fig. 4, at kinase concentrations ranging from 20 to 80 nM, Src displays significant binding to the immobilized pYEEI peptide. In contrast, no detectable binding of Hck to the phosphopeptide was found under similar conditions. There are three possible reasons for the inability of the recombinant Hck to bind the phosphopeptide: (i) the C-terminal regulatory tyrosine of the recombinant Hck is phosphorylated, and interaction between this phosphorylated tyrosine and the SH2 domain renders the SH2 domain inaccessible to the exogenous pYEEI peptide (2, 3); (ii) the structure of the SH2 domain of Hck differs from that of Src in such a way that they display different binding specificities and different affinity toward the pYEEI peptide; or (iii) the native structure of Hck imposes conformational constraints that limit the accessibility of its SH2 domain. In the following experiments, we showed that the inability of Hck to bind pYEEI peptide is most likely due to the presence of conformational constraints within Hck that limit its SH2 domain accessibility.
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Determination of the Level of Tyrosine Phosphorylation of the Purified Recombinant Hck-- To ascertain whether the inability of Hck to bind the pYEEI peptide is a result of interaction between its SH2 domain and the phosphorylated C-terminal regulatory tyrosine, we determined the level of tyrosine phosphorylation of the purified recombinant Hck. Hck was autophosphorylated in vitro to a stoichiometry of approximately 1 mol PO3= per mol of Hck (data not shown), and the relative levels of its tyrosine phosphorylation before and after autophosphorylation were determined by immunoblot analysis with the monoclonal anti-phosphotyrosine antibody (PY69 mAb) and polyclonal anti-phosphotyrosine antibody. As shown in Fig. 5, weak anti-phosphotyrosine immunoreactivity was detected before autophosphorylation, whereas the autophosphorylated Hck reacted very strongly with both the polyclonal and monoclonal (PY69) anti-phosphotyrosine antibodies. Densitometric analysis of the immunoblot revealed that the anti-phosphotyrosine immunoreactivity of Hck prior to autophosphorylation is less than 5% of that of the autophosphorylated Hck. Because the PY69 antibody displays no difference in its ability to recognize the autophosphorylation site and the C-terminal regulatory phosphotyrosine (Fig. 2) and because the polyclonal antiphosphotyrosine antibody can recognize phosphotyrosines in different sequence contexts (see under "Experimental Procedures"), our results strongly suggest that almost all of the potential tyrosine phosphorylation sites, including the C-terminal regulatory tyrosine and the consensus autophosphorylation site, were not phosphorylated in the purified Hck preparation. Thus, the lack of binding of pYEEI peptide by Hck is not a consequence of occupancy of the SH2 domain by the C-terminal regulatory phosphotyrosine.
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Demonstration of Specific Interaction between the SH2 Domain of Hck and the pYEEI Peptide-- To confirm that the SH2 domain of Hck can indeed specifically bind the pYEEI peptide, we generated two recombinant fragments of Hck as GST fusion proteins and examined their ability to bind the immobilized pYEEI peptide. One of the fragments, GST-Hck(1-247), contains the unique, SH3, and SH2 domains of Hck, whereas the other fragment, GST-Hck(1-129), contains the unique and SH3 domains only. Upon incubation of each sample with the immobilized pYEEI peptide (0.79 nmol), only GST-Hck(1-247) could bind the phosphopeptide (Fig. 6). Because the SH2 domain is present in GST-Hck(1-247) but not in GST-Hck(1-129), our results indicate that the binding of GST-Hck(1-247) to the pYEEI-gel was mediated by the SH2 domain. Upon preincubation of GST-Hck(1-247) with various competitor peptides prior to incubation with the immobilized pYEEI peptide, only the free pYEEI peptide was capable of effectively competing with GST-Hck(1-247) for the immobilized pYEEI peptide, indicating that the binding of GST-Hck(1-247) to pYEEI was specific (data not shown).
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DISCUSSION |
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In the present study, we have shown that Hck and Src display
similar in vitro peptide substrate specificity. This builds
on the previous studies (9, 10) that show that several Src family kinases recognize similar structural features in synthetic peptides and
thus share common substrate specificity determinants. Taken together,
these studies suggest that the catalytic domains of Src family kinases
recognize similar features in the local structures around the
phosphorylation sites in substrate proteins. Because most cellular
functions of a kinase are determined by the cellular proteins that they
phosphorylate in vivo, our results support the notion that
Src and Hck, when co-expressed in the same cells, have the intrinsic
ability to carry out redundant cellular functions by phosphorylating
similar cellular protein substrates. This idea is supported by studies
in which Src family kinases have been disrupted in mice. The milder
than expected phenotype associated with mice deficient in Src family
kinases suggests that many cellular functions normally performed by the
disrupted Src family member(s) are compensated for by other Src family
members co-expressed in the same cells (11-19). Despite an inferred
degree of functional overlap among the Src family kinases in
vivo, mice homozygous for a mutation in one Src family gene
exhibit some unique developmental and functional defects. For examples,
Fc-receptor-independent phagocytosis is perturbed in Hck-deficient
macrophages (16), and bone resorption by osteoclasts is impaired in
src
/
mice (13, 19). This suggests that the
roles of Hck and Src in phagocytosis by macrophages and bone formation,
respectively, cannot be substituted by other co-expressed Src family
members. Thus, in addition to cooperating with other Src family kinases to provide the signals necessary for many cellular functions, each of
the kinases also performs unique functions in vivo.
How might each Src family member carry out its own unique functions if the catalytic domain of all Src family kinases recognizes similar structural features in cellular protein substrates? Because the cellular protein substrates are differentially distributed in various subcellular compartments, src family kinases co-expressed in the same cells can phosphorylate different cellular proteins if they are targeted to different subcellular compartments (see Ref. 4 for review). Furthermore, the SH2, SH3, and perhaps the unique domains can directly interact with the cellular substrates and hence determine the substrate specificity of the kinases. For example, phosphorylation of focal adhesion kinase by Src requires direct interaction of the SH3 domain of Src with the 368RALPSIPKL376 motif of focal adhesion kinase as well as interaction of the SH2 domain of Src and the phosphorylated Tyr-397 of focal adhesion kinase (5); phosphorylation of the cellular protein Sin by Src requires interaction of a Pro-X-X-Pro containing motif of Sin with the SH3 domain of Src (6). For these reasons, the differential SH2 domain accessibility exhibited by Src and Hck is a potential mechanism of targeting the two kinases to different subcellular compartments, thereby allowing them to carry out different functions by phosphorylating the cellular protein substrates in their immediate vicinity. Taken together, our data suggest that the similar in vitro peptide substrate specificity of the catalytic domains of Src family kinases may account for the functional redundancy of the kinases in vivo and the difference in accessibility of their SH2 domains may contribute to their functional specificity. To further substantiate this notion, future studies should focus on identification of common and specific in vivo cellular protein substrates of the kinases and demonstration of targeting of Src and Hck to different subcellular compartments or binding to different cellular proteins as a consequence of the differential SH2 domain accessibility displayed by both kinases.
Our study also suggests the presence of conformational constraints limiting the SH2 domain accessibility of intact Hck even if its C-terminal regulatory tyrosine is not phosphorylated. Elucidation of the structural basis of these conformational constraints is essential for our understanding of the regulatory properties of Hck. The fact that deletion of the kinase domain and the C-terminal regulatory domain renders the SH2 domain in GST-Hck(1-247) accessible to the pYEEI peptide is consistent with the notion that only the intact form of Hck can impose these conformational constraints. In contrast to our observation that intact Src and Hck display different SH2 domain accessibility, Xu et al. (2) and Sicheri et al. (3) reported that the crystal structures of the inactive conformation of Hck and Src, which lack the unique and fatty acid acylation domains, are virtually identical. It is therefore reasonable to propose that the unique and/or fatty acid acylation domains confer differences in SH2 domain accessibility of the two kinases. Future studies should test this hypothesis by comparing the SH2 domain accessibility of intact Hck and the truncated form of Hck without the fatty acid acylation and the unique domains. Alternatively, Src and Hck may exhibit a difference in the ability of the C-terminal tail to move away from the SH2 domain after dephosphorylation. Src may have a rather flexible C terminus that can move away from the SH2 domain after dephosphorylation, thereby allowing binding to the pYEEI peptide resin. In contrast, Hck may have a more rigid C terminus that, even upon dephosphorylation, does not allow it to fully move away from the SH2 domain, thereby limiting the accessibility of the SH2 domain to the pYEEI resin.
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ACKNOWLEDGEMENTS |
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We thank Loretta Gibson for assistance with the generation of the recombinant Hck baculovirus, Nick Sotirellis for assistance with the purification of recombinant mouse Hck, Daisy Lio for assistance with the purification of the GST fusion proteins of Hck and Src fragments, Jing Wang for kindly providing the GST fusion vector encoding GST-Src(1-258), Dr. Joan Brugge for providing the hybridoma cells producing the monoclonal Src antibody mAb327, and Ben Kreunen for preparing the figures. We thank Drs. Serge Roche, Christian Dumas, and Christine Benistant for comments on the manuscript.
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FOOTNOTES |
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* This work was supported in part by grants from the National Health and Medical Research Council of Australia, the Anti-Cancer Council of Victoria, Australia, the Medical Research Council of Canada, and the National Cancer Institute of Canada.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.
¶ Recipient of a Senior Research Fellowship from the Australian Research Council.
Current address: Universite de Montpellier I, Faculte de
Pharmacie, Laboratoire de Biochimie des Membranes, CNRS EP-612,
15 avenue Charles Flahault 34060, Montpellier, Cedex, France.
Scientist of the Alberta Heritage Foundation for Medical
Research.
§§ To whom correspondence should be addressed. Tel.: 61-3-9344-5947; Fax: 61-3-9347-7730; E-mail: Heung-Chin_Cheng.BioChem{at}muwaye.unimelb.edu.au.
1 The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; SH2, Src homology 2; CSK, C-terminal Src kinase; GST, glutathione S-transferase; mAb, monoclonal antibody.
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REFERENCES |
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