From the Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
Received for publication, October 1, 2002, and in revised form, November 10, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The protein-tyrosine phosphatase SHP-1 plays a
variety of roles in the "negative" regulation of cell signaling.
The molecular basis for the regulation of SHP-1 is incompletely
understood. Whereas SHP-1 has previously been shown to be
phosphorylated on two tail tyrosine residues (Tyr536
and Tyr564) by several protein-tyrosine kinases, the
effects of these phosphorylation events have been difficult to address
because of the intrinsic instability of the linkages within a
protein-tyrosine phosphatase. Using expressed protein ligation, we have
generated semisynthetic SHP-1 proteins containing phosphotyrosine
mimetics at the Tyr536 and Tyr564 sites. Two
phosphonate analogues were installed, phosphonomethylenephenylalanine (Pmp) and difluorophosphonomethylenephenylalanine (F2Pmp).
Incorporation of Pmp at the 536 site led to 4-fold stimulation of the
SHP-1 tyrosine phosphatase activity whereas incorporation at the 564 site led to no effect. Incorporation of F2Pmp at the 536 site led to 8-fold stimulation of the SHP-1 tyrosine phosphatase
activity and 1.6-fold at the 564 site. A combination of size exclusion chromatography, phosphotyrosine peptide stimulation studies, and site-directed mutagenesis led to the structural model in which tyrosine
phosphorylation at the 536 site engages the N-Src homology 2 domain in an intramolecular fashion relieving basal inhibition. In
contrast, tyrosine phosphorylation at the 564 site has the potential to
engage the C-Src homology 2 domain intramolecularly, which can modestly
and indirectly influence catalytic activity. The finding that
phosphonate modification at each of the 536 and 564 sites can promote
interaction with the Grb2 adaptor protein indicates that the
intramolecular interactions fostered by post-translational modifications of tyrosine are not energetically strong and susceptible to intermolecular competition.
The SHP-1 protein-tyrosine phosphatase
(PTPase)1 (initially
designated SHPTP-1, SHP, HCP, and PTP1C) is thought to play a role as a
negative regulator of cell signaling in cells of hematopoietic lineage
(1). Its involvement has been implicated in colony stimulating factor 1 receptor signaling pathways (2), B cell receptor-induced apoptosis and
signaling (3, 4), Fc SHP-1 is a 68-kDa protein composed of two SH2 domains, a tyrosine
phosphatase catalytic domain and a flexible C-terminal domain which has
been proposed to play a regulatory role (Fig. 1A). There is
a high resolution crystal structure of the SHP-1 catalytic domain
indicating the classical PTPase fold (10). These enzymes have a highly
conserved cysteine residue that serves as the catalytic nucleophile,
generating a phosphocysteine covalent intermediate (11). The
phosphoenzyme intermediate is then hydrolyzed producing inorganic
phosphate. Whereas the authentic physiologic protein substrates of SHP1
have not been determined with certainty, several proposed
phosphoprotein targets include Lyn, Syk, BLNK/SLP-65, Lck, ZAP-70,
phosphatidylinositol 3-kinase, SLP-76, interleukin 2 receptor,
IRF1, and the interferon consensus sequence-binding protein (1, 12,
13). Typically phosphopeptides or para-nitrophenol phosphate
(pNPP) have been used to study the catalytic mechanism and
regulation of SHP-1 in vitro (14).
The mechanisms of the regulation of SHP-1 are incompletely understood.
No high resolution structure has been reported for the full-length
protein. The two SH2 domains may be important in recruiting
phosphotyrosine-containing proteins, controlling substrate specificity,
and cellular localization of the enzyme. In addition, the N-terminal
SH2 domain appears to play a role as a negative regulator of SHP-1
catalytic activity by directly binding to the SHP-1 catalytic domain
(15). This inhibition can be relieved by phosphotyrosine containing
peptides with appropriate sequences and possibly phospholipids (15,
16). Two C-terminal tyrosine phosphorylation sites at
Tyr536 and Tyr564 have been mapped (Fig.
1A) and possible tyrosine kinases that catalyze these
reactions include Lck, Abl, and the insulin receptor tyrosine kinase
(7, 17-19). The function(s) of these phosphorylations have been
difficult to address in detail because of the instability of these
modifications because of the inherent catalytic nature of the
phosphatase. Possible roles for the phosphotyrosines include the
recruitment of phosphotyrosine-binding modules such as SH2 and PTB
domain-containing proteins as well as activation of the SHP-1 catalytic
activity. One or more of these phosphotyrosines could bind in an
intermolecular or intramolecular fashion to the SH2 domains of
SHP-1.
Recently, expressed protein ligation (20, 21) has been used to probe
the function of phosphotyrosine modifications in the related
protein-tyrosine phosphatase enzyme, SHP-2 (22). In this study, the
phosphotyrosine mimetic phosphonomethylenephenylalanine (Pmp) was
incorporated into the C terminus of SHP-2 at two phosphorylation sites
and the effects on SHP-2 catalytic behavior determined. Because the
SHP-2 and SHP-1 tails show little sequence homology (Fig.
1B), and their SH2 domains appear to show different
phosphotyrosine sequence preferences, it was not known whether the
results with SHP-2 would extend to SHP-1. Here we use expressed protein
ligation to probe the importance of C-terminal tyrosine phosphorylation of SHP-1 and find evidence both for stimulatory and adaptor functions.
Preparation of the Shp-1 Constructs--
The full-length
human shp-1 tyrosine phosphatase encoded in a DNA plasmid
(gift of Dr. Jun Wang) was used to amplify DNA encoding amino acids
1-556 by use of primers containing NdeI and SmaI
restriction digestion sites at the 5' and 3' ends, respectively. The
PCR product was purified and ligated into the pTYB2 vector (New England
Biolabs) in-frame with the intein and chitin binding domain open
reading frames. The extra SmaI site at the 3' end of the
shp-1 gene was deleted using QuikChange site-directed
mutagenesis (Stratagene) to give the plasmid
pTYB2-shp-1-(1-556). This plasmid was used to generate
564-modified proteins (after ligation) and as a template to produce all
other mutants by the QuikChange method including pTYB2-shp-1-(1-531),
which was used to generate 536-modified proteins. All constructs were
confirmed by DNA sequencing the entire open reading frames of the
shp-1 gene.
Preparation of Phosphotyrosine (Phosphonate)
Analogues--
Fmoc-phosphonomethylene-L-phenylalanine
(Fmoc-Pmp) was purchased from Advanced ChemTech and used without
further purification. Fmoc-difluorophosphonomethylene-L-phenylalanine
(Fmoc-F2Pmp) was synthesized and characterized as described
by Lawrence and colleagues (23).
Peptide Synthesis--
Standard Fmoc-protected amino acids,
peptide synthesis reagents, and Wang resins were purchased from
Novabiochem. All peptides were synthesized on Wang resin using the
standard Fmoc strategy on a Rainin PS-3 machine. Pmp,
F2Pmp, or phosphotyrosine (Tyr(P)) containing
peptides were prepared by incorporating the corresponding non-natural
amino acids during the coupling reactions. Crude peptides were purified
by reversed-phase high performance liquid chromatography on a
preparative or semipreparative C18 column. The purity (>95%) of the
peptides was established by reversed-phase analytical high performance
liquid chromatography and the molecular weights were confirmed by both
MALDI and electrospray mass spectrometric analysis.
Expressed Protein Ligation--
Protein ligations were carried
out as described (20, 22). Briefly, the shp-1 DNA constructs
were transformed into Escherichia coli BL21(DE3) cells and
the transformed cells grown at 37 °C until
A595 = 0.6-0.8. The cells were then treated
with isopropyl-1-thio- Gel Filtration--
The molecular weights of the semisynthetic
proteins, SHP-1/Tyr564, SHP-1/Pmp564,
SHP-1/Tyr536, and SHP-1/Pmp536 were determined
by gel filtration chromatography on a Pharmacia FPLC system.
Semisynthetic SHP-1 proteins (100 µl of 2 mg/ml) were loaded onto a
Superdex 200 column (Amersham Biosciences), which was
pre-equilibrated with 50 ml of buffer (25 mM Na-Hepes, pH
7.2, 200 mM NaCl, and 2 mM dithiothreitol). A
molecular weight marker kit containing cytochrome c,
carbonic anhydrase, bovine serum albumin, alcohol dehydrogenase, and
Phosphatase Assays--
pNPP (purchased from
Acros) was used as substrate to determine the phosphatase activities of
the semisynthetic SHP-1 proteins. It has previously been observed that
pNPP shows nonclassical Michaelis-Menten kinetics with SHP-1
(14) and so reaction rates were measured as specific activities using a
fixed and subsaturating concentration of pNPP (2 mM). Reactions were carried out typically with 1 µM SHP-1 protein and incubated in 50 µl of reaction
buffer (0.2 mg/ml bovine serum albumin, 100 mM Na-Hepes, pH
7.4, 150 mM NaCl, 1 mM EDTA, and 10 mM dithiothreitol) at 25 °C for 30 min. It should be
noted that pH 7.4 is above the pH optimum for SHP-1 (14) but was
selected because it is likely to be more physiologic. The reactions
were quenched with 950 µl of 1 N NaOH and the turnover rates of the proteins were calculated from the amount of the released p-nitrophenolate, which was determined from its
spectrophotometric absorbance at 405 nm. Reaction rates were shown to
be linear with enzyme concentration and time in the ranges used (Fig.
4A). To assess the stimulatory effects of synthetic
(phospho)peptides on the phosphatase reactions, 70 µM
peptide was included in the reaction buffer prior to initiation with
SHP-1.
Grb2 Binding Studies--
GST-Grb2 fusion protein was expressed
in E. coli BL21(DE3) cells and purified by the use of
glutathione-agarose (Sigma) as previously described (26, 27). The Grb2
binding studies were carried out as follows: 50 µl of GST-Grb2 bead
slurry containing ~50 µg of fusion protein (estimated by 10%
SDS-PAGE), was centrifuged at 2000 × g for 5 min in a
small plastic (Eppendorf) tube. The supernatant was discarded and the
pellet was washed twice with 0.1 ml of binding buffer (25 mM Na-Hepes, pH 7.5, 10% glycerol, and 100 mM
NaCl), and then 10 µl of binding buffer was added to the pellet to
resuspend the beads. To the resuspended beads was added 10 µg of
SHP-1 protein in 30 µl of binding buffer. After 4 min incubation at
16 °C, the reaction mixture was centrifuged at 2000 × g for 5 min. The pellets were then washed three times with
200 µl of binding buffer. The beads were finally mixed with 30 µl
of 1× SDS loading dye at 95 °C for 5 min and the supernatant was
loaded onto 10% SDS-PAGE. The dried gels were scanned and the amount
of SHP-1 proteins bound to Grb2 was quantified using ImageQuant software.
Generation of Semisynthetic SHP-1--
The N-terminal portion of
SHP-1 including its SH2 domains and catalytic domain but lacking most
of the C-terminal tail was subcloned into the pTYB2 vector in-frame
with the intein-chitin binding domains. Two constructs were developed
covering residues 1-531 and 1-556, which enable the ligation of 15- and 16-residue N-terminal cysteine containing peptides, respectively,
to replace the physiologic sequences in these positions (Fig.
1C). Based on previously
published studies, it was expected that partial deletions in the
C-terminal sequence would resemble full-length protein catalytic
behavior (14, 15), and this proved to be the case in our hands (data
not shown). The requisite synthetic peptides containing either tyrosine
or Pmp at the sites of phosphorylation were prepared by solid phase
peptide synthesis. Cysteines were installed at the N terminus of these
peptides replacing Gln (for 564-modified proteins) or Ser (for
536-modified proteins) in the natural sequences as required for the
expressed protein ligation method. To facilitate ligation, the
C-terminal residues of the recombinant protein were Gly, which was the
natural residue of the 536-modified proteins but replaced a Ser in the
564- modified proteins. These mutations did not significantly affect
the activities of the proteins as revealed by pNPP assay
(data not shown). Expressed protein ligation performed in the presence
of thiophenol2 as previously described (20, 22) led to
robust protein production (>2 mg/liter of E. coli cell
culture), which appeared to be greater than 90% pure eluting from the
chitin resin (Fig. 2A). Mass
spectra supported the efficiency of the ligation process (Fig.
2B). It was also demonstrated that the phosphatase activity
of standard SHP-1 recombinant protein identical in sequence length to
the semisynthetic proteins showed similar catalytic behavior (data not
shown).
Gel Filtration--
There was a theoretical
possibility that tail phosphorylation could promote intermolecular
protein interactions via SH2 domain-phosphotyrosine interactions. This
potential was assessed with the semisynthetic proteins by size
exclusion chromatography (Fig. 3). A
comparison of elution profiles of Pmp-containing versus
tyrosine containing proteins showed no significant differences in the
elution profiles (Fig. 3). Moreover, by comparison to the profiles of
protein standards (25) it was shown that each of the semisynthetic
proteins showed sizes consistent with monomers. Thus it can be
concluded that at the concentration investigated (~1-2 mg/ml), the
Pmp groups do not promote protein dimerization or higher order
oligomers.
Enzyme Activity of Pmp-substituted Semisynthetic SHP-1
Proteins--
To address the possibility that the Pmp substitutions
could modulate the enzyme activity of SHP-1, the relative activities of
the semisynthetic proteins were studied with the well established substrate, pNPP. Because wild-type SHP-1 does not display
Michaelis-Menten kinetic behavior, a fixed concentration of
pNPP was employed. As can be seen, SHP-1/Pmp536
showed a substantial 4-fold increase in phosphatase activity compared
with SHP-1/Tyr536 (Fig. 4).
In contrast, SHP-1/Pmp564 showed very similar catalytic
behavior compared with SHP-1/Tyr564. These results suggest
site-selective activation of SHP-1 by tyrosine phosphorylation at
Tyr536 (Fig. 4). Analogous results were seen using
phosphorylated RCM-lysozyme as substrate (data not shown).
To probe the structural basis of Pmp536 activation, the
phosphatase activity of the semisynthetic proteins was measured in the presence of phosphotyrosine peptide EpoR-pY-429
(AcHN-PHLKYLpYLVVSDK-CO2H). This peptide has been shown to
stimulate the phosphatase activity of SHP-1 by relieving basal
inhibition mediated by the N-terminal SH2 domain (15). As can be seen
(Fig. 4B), 70 µM EpoR-pY-429 caused a similar
4-fold stimulation of SHP-1/Tyr564 and
SHP-1/Pmp564. In contrast, whereas EpoR-pY-429 led to a
4-fold activation of SHP-1/Tyr536, it caused a more modest
1.5-fold activation of SHP-1/Pmp536. This is consistent
with the possibility that Pmp536 can partially relieve the
basal suppression mediated by the N-terminal SH2 domain.
Generation of a Semisynthetic SHP-1 with a Consensus
Sequence--
Because Pmp536 may be interacting with the
N-terminal SH2 domain of SHP-1 to enhance the phosphatase activity of
SHP1, we considered the possibility that an amino acid sequence
surrounding Pmp536, which was designed to interact with the
N-SH2 domain, might show more robust stimulation of SHP-1. Based on the
analysis of the affinity of the SHP-1 N-terminal SH2 domain using
peptide libraries (28), a peptide containing the consensus sequence LHpYMNF (Fig. 5A) was
synthesized for use in expressed protein ligation. This semisynthetic
protein (SHP-1/Pmp536con) was prepared as
efficiently as SHP-1/Pmp536 and subjected to catalytic
activity measurements. As shown in Fig. 5B, the activity of
SHP-1/Pmp536con was greater than SHP-1/Pmp536
and minimally affected by the addition of EpoR-pY-429. This provides further evidence of an interaction between Tyr(P)536 and
the N-terminal SH2 domain.
Effect of F2Pmp Incorporation on SHP-1--
It has
previously been shown by Burke and colleagues (29) that
F2Pmp is a closer mimic of phosphotyrosine compared with Pmp. Because the consensus sequence for N-SH2 binding led to greater SHP-1 activation relative to the natural sequence, we pursued incorporation of F2Pmp at residues 536 and 564 of SHP-1,
respectively. Synthesis of the difluoro analogue was carried out as
described by Guo et al. (23) and incorporated into the same
synthetic peptide sequences used with Pmp (Fig. 1C). These
peptides were used to produce the desired semisynthetic proteins in
similar efficiency compared with the Pmp derivatives. Strikingly,
F2Pmp induced greater phosphatase activity at both
positions 536 and 564 (Fig. 5C). An 8-fold activation in
phosphatase activity was detected for
SHP-1/F2Pmp536 compared with
SHP-1/Tyr536, and SHP-1/F2Pmp536
did not show enhanced activity in the presence of EpoR-pY-429. Moreover, SHP-1/F2Pmp564 actually displayed a
1.6-fold increase in activity compared with SHP-1/Tyr564 or
SHP-1/Pmp564. These results unmask a potential for tyrosine
phosphorylation to provide modest stimulation of SHP-1 at the 564 position. This modest stimulation in the absence of peptide EpoR-pY-429
was less apparent in the presence of stimulatory peptide (Fig.
5C), presumably because the peptide effect was more dominant.
Effect of SH2 Mutations on the Activities of SHP-1--
The
structural basis of Pmp (and F2Pmp) activation of SHP-1
plausibly involves intramolecular interactions between the phosphonates and the SH2 domains. The molecular basis of SH2-phosphotyrosine recognition generally involves a highly conserved SH2 Arg (within the
conserved motif FLVRES) forming an electrostatic interaction with the
phosphate moiety of the ligand (30). It is typically the case that
mutation of this Arg to other residues (including Lys) will greatly
weaken the interaction with ligand but otherwise be structurally
tolerated. We changed this key residue (Arg30) within the
N-SH2 domain of SHP-1 to several other residues including Ala, Met,
Ile, Leu, and Lys in SHP-1 semisynthetic proteins. These semisynthetic
mutant proteins were prepared identically to the "wild-type"
versions described above. In each case, in the absence of
phosphotyrosine ligands significant increases (2-6-fold) in the basal
activity of the SHP-1 PTPase were observed (see Fig. 6A). This unusual finding
confirms and extends an earlier observation of Pei et al.
(15) and suggests that Arg30 is directly or indirectly
contributing to the presumed intramolecular inhibitory interaction
between the N-SH2 domain and the catalytic domain. In contrast,
mutation of the homologous residue in the C-SH2 domain
(Arg136) does not affect basal SHP-1 activity (see Fig.
6A).
Perhaps more surprisingly, the phosphopeptide EpoR-pY-429 was able to
further stimulate each of these Arg30 mutant SHP-1 enzymes
at a concentration identical to that used in the wild-type SHP-1
studies (Fig. 6A). Using the corresponding nonphosphorylated
form of the peptide (EpoR-Y-429) at the same concentration, no
activation of the Arg30 mutants was detected (data not
shown). Thus the putative interaction of phosphopeptide ligand with the
N-SH2 domain of SHP-1 is unconventional compared with typical cases and
the role of the FLVRES Arg is likely substituted by another residue in
this case.
The effects of mutation of Arg30 on several of the
phosphonate containing semisynthetic proteins were also examined (Fig.
6, B and C). It can be seen that
SHP-1/R30A-Pmp536 shows a 2-fold greater PTPase rate
compared with SHP-1/R30A-Tyr536 (Fig. 6B). These
results are consistent with the intermolecular studies with
phosphopeptide EpoR-pY-429 in which it was shown that the presence of
Arg30 is not critical for PTPase stimulation. It was also
shown that SHP-1/R136A-Pmp536 displays a 4-fold greater
rate compared with SHP-1/R136A-Tyr536 arguing against the
role of the C-SH2 domain in mediating PTPase activation by the
Pmp536 modification. Finally, it was found that
SHP-1/R136A-F2Pmp564 shows an identical rate to
SHP-1/R136A-Tyr564 (Fig. 6C) suggesting that the
C-SH2 domain is most likely responsible for the modest activation by
the F2Pmp564 modification (Fig. 6C).
Taken together, the data support a structural model of SHP-1 activation
by phosphorylation shown in Fig. 7.
Grb2 Binding--
The role of the phosphonate modifications of
SHP-1 in mediating interactions with Grb2, an SH2 domain containing
adaptor protein (22, 26), were studied by a variant of the GST
pull-down assays. GST-Grb2 was immobilized on glutathione-agarose and
incubated with SHP-1 semisynthetic proteins for 4 min prior to several
brief buffer washes. It was found that both SHP-1/Pmp536
and SHP-1/Pmp564 proteins showed significantly greater
binding than the unphosphonylated semisynthetic proteins (Fig.
8). Likewise, F2Pmp SHP-1
proteins showed greater binding to Grb2 than unmodified proteins.
Interestingly, Pmp containing SHP-1 proteins showed somewhat more
efficient binding (about 2-fold) to Grb2 compared with
F2Pmp proteins. This could be the case because
F2Pmp have a lower relative affinity for Grb2, a slower
rate of release from their intramolecular interactions with the SHP-1
SH2 domains, or a slower on-rate with respect to the Grb2 domains
(these assays were designed to look at kinetic effects). However, the
importance of slow intramolecular dissociation is argued against
because mutations in the N-SH2 or C-SH2 domains did not have a large
effect on the relative proportion of SHP-1 proteins bound to Grb2.
The value of expressed protein ligation in addressing
structure-function relationships in reversible phosphorylation of
signaling proteins is becoming increasingly well demonstrated (20-22,
31, 32). It is most conveniently applied when the region of the protein
to be modified is in the C terminus of a signaling protein because the
C-terminal thioester generated using intein technology can be fused
with a readily prepared N-terminal cysteine containing peptide.
Recently published studies on transforming growth factor- The poor homology of SHP-2 and SHP-1 C-terminal tail regions (Fig.
1B) made it difficult to predict based on the results of SHP-2 what to expect with SHP-1. It is now clear that phosphorylation at Tyr536 can activate the SHP-1 PTPase activity in analogy
with Tyr542 in SHP-2. However, the degree of activation,
about 8-fold with F2Pmp in SHP-1, is significantly greater
than the 2-3-fold observed with Pmp and SHP-2. It remains to be seen
if this difference is related to the choice of the phosphotyrosine
analogue although given that only a 4-fold effect was seen with
Pmp536 and SHP-1, this has to be considered a possibility.
It should also be pointed out that a study with enzymatically
phosphorylated SHP-1 at the 536 position also reports PTPase activation
by this modification (18), although this was characterized with a less pure protein mixture and uncertain stoichiometry in which the SHP-1
undergoes dephosphorylation during the PTPase measurements.
The model of activation based on the findings with the consensus
sequence engineered SHP-1, stimulation with EpoR-pY-429 peptide, and
mutagenesis support the proposition that phosphoryl modification at 536 activates SHP-1 by interaction with the N-terminal SH2 domain (Fig. 7).
Whereas mutation of the FLVRES Arg of the N-SH2 leads to somewhat
unconventional behavior, it is clear that mutation of the C-SH2 domain
does not play a role in activation by Pmp536 (Fig. 7). The
gel filtration experiments showing that SHP-1/Pmp536 is
monomeric support the likelihood that this interaction is intramolecular (Fig. 7). Furthermore, this activation model would be
similar to the effect of phosphorylation of Tyr542 in SHP-2
(22). Not addressed in these studies or in Fig. 7 is the interesting
finding that deletion of the C-terminal tail 35 amino acids but not the
final 60 amino acids appears to be activating for SHP-1 (15). The basis
of catalytic activation by this truncation is not yet understood and
may add complexity to the simple model in Fig. 7. It should be stated
that deletion of the C-terminal 49 amino acid residues as present in
SHP-1/Tyr536 or 23 amino acids in SHP-1/Tyr564
showed similar catalytic activity to full-length SHP-1 in our hands.
It is worthwhile to consider the reason why mutation of the FLVRES Arg
itself leads to PTPase stimulation and yet these mutant proteins are
still susceptible to phosphotyrosine activation. One possibility is
that the SHP-1 N-SH2 domain is noncanonical and the FLVRES Arg
contributes relatively little to binding in this peculiar case. Another
explanation would be that the SHP-1 catalytic domain is perturbing the
structure of the N-SH2 domain such that it behaves anomalously. Related
to this idea, the catalytic domain could even be contributing residues
that substitute for SH2 domain residues in the function of the domain.
Finally, it should be mentioned that a loss of affinity of the N-SH2
domain for the catalytic domain may occur when Arg30
is mutated, leading to PTPase activation. This weaker interaction may
make the N-SH2 domain, even though damaged by Arg30
mutation, more available for phosphotyrosine binding, which would offset the loss of affinity resulting from the
Arg30-phosphate interaction. Further structural studies
will be needed to distinguish among these possibilities.
In contrast to the relatively large stimulation by phosphonates at the
536 position of SHP-1, the catalytic stimulation from F2Pmp
at the 564 site is rather modest at 1.6-fold. It is noteworthy that the
effect seen with F2Pmp was absent when the initial Pmp analogue was employed. This argues for the utility of the
fluorophosphonate analogues bringing out more subtle structural effects
because they are more faithful mimics. Whereas a small effect, the
F2Pmp effect can be structurally rationalized because it
disappears upon mutation of the C-SH2 Arg136 residue. Thus,
it is likely that this phosphorylation of Tyr564 can
modestly enhance SHP-1 activity by intramolecular C-SH2 domain engagement, presumably by an indirect effect (Fig. 7). This is qualitatively similar to the behavior of Tyr580 in SHP-2
although in that case the activation is more robust and observable with
Pmp (2-3-fold), comparable with that of Tyr542
phosphorylation in SHP-2 (22).
Interestingly, although phosphonylation of SHP-1 at 536 and 564 can
allow for intramolecular interactions with the SHP-1 SH2 domains, they
are still quite efficiently able to facilitate binding to the adaptor
protein Grb2 (Fig. 8). These results should be contrasted with
Tyr542 phosphorylation of SHP-2 where intramolecular
engagement with the N-SH2 domain effectively competes with Grb2 binding
kinetically. Thus, tail phosphorylation of SHP-1 could presumably
impact signal transduction in vivo by either catalytic
activation or by an adaptor function. The particular pathway and
interacting molecules may dictate which effects predominate in the
cellular environment.
Summary--
Expressed protein ligation has been used to generate
site-specific and stoichiometric phosphonate-modified forms of SHP-1 to
simulate the effects of tyrosine phosphorylation of SHP-1. These
studies describe the first comparative analysis of the effects of
difluoromethylenephosphonate and methylenephosphonate as
phosphotyrosine mimetics site-specifically incorporated within the
context of a protein. It was shown that phosphonate at the 536 position
of SHP-1 is capable of up to 8-fold stimulation of the SHP-1 tyrosine phosphatase activity, likely by intramolecular engagement of the N-SH2
domain, relieving basal inhibition. In contrast, phosphonate modification of the 564 position of SHP-1 results in a smaller (1.6-fold) stimulation of the tyrosine phosphatase activity, probably by an indirect effect via interaction with the C-terminal SH2 domain.
The phosphonate-modified SHP-1 proteins are readily able to recruit the
SH2-containing adaptor protein Grb2 suggesting that the intramolecular
interactions promoted by tyrosine phosphorylation are not highly
favorable energetically. These studies suggest that tyrosine
phosphorylation of SHP-1 could play distinct roles in cell signaling
either by direct catalytic activation of the enzyme or by recruitment
of other signaling molecules to a specific cellular location.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
receptor-mediated phagocytosis (5),
HoxA-mediated transcriptional repression (6), Abl-induced DNA damage
response (7), erythropoitein receptor signaling (8), and T cell
receptor signaling (1). Mice lacking wild-type SHP-1 show a
"motheaten" phenotype characterized by patchy alopecia, severe
combined immunodeficiency, and the lethal onset of hemorrhagic
interstitial pneumonitis (1, 9). There has been a continuing interest
in delineating the complete biological functions of SHP-1.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside (final
concentration = 0.2 mM) and harvested after 20 h
culture at 16 °C. The resuspended cells were lysed by French press
and the lysates were eluted over chitin beads and the beads washed as
previously described (20, 22). The immobilized SHP-1 fusion proteins
were ligated to synthetic peptides in the presence of 2%
thiophenol.2 The purity of
the semisynthetic proteins was determined by 10% SDS-PAGE stained with
Coomassie Blue and by MALDI mass spectrometry (>90%). The yield was
2-10 mg of protein/liter in E. coli cell culture as
revealed by Bradford assay (24).
-amylase proteins (Sigma) was used to obtain a standard curve. The
molecular weights of the SHP-1 semisynthetic proteins were calculated
from the retention volumes by the use of the standard curve (25).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (23K):
[in a new window]
Fig. 1.
Scheme of the SHP-1 structure and the
strategies for modification. A, SHP-1 protein consists of
two SH2 domains followed by one catalytic domain (PTPase) and a
C-terminal tail. R30 and R136 are conserved Arg
residues within the FLVRES motifs thought to be important for the N-SH2
and C-SH2 domains, respectively, for interaction with phosphotyrosine
containing peptides. C453 (Cys453) is critical
for the activity of the SHP-1 protein. Two tyrosine residues at the
C-terminal tail, Tyr536 and Tyr564, can be
phosphorylated by tyrosine kinases; B, comparison of the
C-terminal tail sequences of the SHP-1 and SHP-2 proteins. Tyrosine
residues that can be phosphorylated are indicated; C,
sequences of the C-terminal tails of the semisynthetic proteins. The
sequences of the synthetic peptides used in expressed protein ligation
are underlined. To facilitate the protein ligation,
Gln532 (536-modified SHP-1), Ser556
(564-modified SHP-1), and Ser557 (564-modified SHP-1) were
mutated to Cys532, Gly556, and
Cys557, respectively. The mutated residues are depicted in
bigger font. X represents tyrosine or phosphotyrosine
analogues, either Pmp or F2Pmp; D, the
structures of tyrosine, Pmp, and F2Pmp are shown.
View larger version (33K):
[in a new window]
Fig. 2.
SDS-PAGE and MS of several SHP-1
semisynthetic proteins. A, 10% SDS-PAGE stained with
Coomassie Blue of several of the semisynthetic SHP-1 proteins. The
molecular weights of the marker used are indicated at the
left. The contents of the lanes are labeled at the
top of each lane. B, MALDI-MS profiles of the
semisynthetic proteins. The smaller peak in each profile represents the
double-charged protein. The calculated masses
(m/z) of Tyr536, Pmp536,
Tyr564, and Pmp564 are 61,819, 61,897, 64,836, and 64,914 Da, respectively.
View larger version (23K):
[in a new window]
Fig. 3.
Gel filtration profiles of the semisynthetic
SHP-1 proteins. The molecular weight of the SHP-1 proteins were
calculated based on the standards, eluting at 17.6 ml (12,400), 16.1 ml
(29,000), 14.3 ml (66,000), 12.5 ml (150,000), and 12.0 ml (200,000).
Calculated semisynthetic SHP-1 molecular weights were
Tyr536 = 62,000, Pmp536 = 62,000, Tyr564 = 65,000, Pmp564 = 65,000.
View larger version (16K):
[in a new window]
Fig. 4.
Effects of phosphonate modification on PTPase
activities of SHP-1 proteins. A, time course of
Tyr536 and Pmp536. The release of the reaction
product, p-nitrophenolate, is linear for at least 40 min;
B, the phosphatase activities of the SHP-1 semisynthetic
proteins were measured in the presence of 2 mM
pNPP, without (open bar) or with 70 µM EpoR-pY-429 peptide (solid bar).
View larger version (19K):
[in a new window]
Fig. 5.
Phosphatase activities of the semisynthetic
proteins containing consensus sequences or F2Pmp analogue.
A, comparison of wild-type and consensus peptide sequences.
The synthetic peptide containing the consensus sequence of the N-SH2
domain ligand was used to generate Tyr536con and
Pmp536con semisynthetic SHP-1 proteins; B,
relative phosphatase activities of SHP-1/Tyr536con and
SHP-1/Pmp536con; C, relative phosphatase
activities of semisynthetic SHP-1 proteins containing
F2Pmp. The phosphatase activities of the SHP-1
semisynthetic proteins were measured in the presence of 2 mM pNPP, without (open bar) or with
70 µM EpoR-pY-429 peptide (solid bar).
View larger version (23K):
[in a new window]
Fig. 6.
Phosphatase activities of the SHP-1 proteins
containing mutations in the N-SH2 (Arg30) or C-SH2
(Arg136) domains. A, phosphatase activities of
semisynthetic SHP-1 proteins containing various SH2 domain point
mutations; B, relative phosphatase activities of
semisynthetic SHP-1 proteins with Pmp536 substitution and
SH2 mutations; C, relative phosphatase activity of
semisynthetic SHP-1 with 564-F2Pmp substitution and C-SH2
mutation. The phosphatase activities of the SHP-1 semisynthetic
proteins were measured in the presence of 2 mM
pNPP, without (open bar) or with 70 µM EpoR-pY-429 peptide (solid bar).
View larger version (21K):
[in a new window]
Fig. 7.
Regulation of the SHP-1 protein activity by
the phosphorylation of the C-terminal tyrosine residues. When both
Tyr536 and Tyr564 are unphosphorylated, the
N-SH2 domain interacts with the PTPase domain, inhibiting the
phosphatase activity. Phosphorylation at Tyr536 leads to
interaction with the N-SH2 domain, releasing the inhibitory effect of
the N-SH2 domain on the PTPase domain. Phosphorylation of
Tyr564 leads to interaction with the C-SH2 domain,
partially and indirectly releasing the inhibitory effect of the N-SH2
domain, and modestly increasing the PTPase activity.
View larger version (39K):
[in a new window]
Fig. 8.
Grb-2 interactions with semisynthetic SHP-1
proteins. A, relative level of
Pmp536-modified SHP-1 semisynthetic proteins carrying SH2
mutations binding to Grb2; B, comparison of Pmp- and
F2Pmp-modified SHP-1 semisynthetic proteins binding to
Grb2; C, relative levels of
SHP-1/F2Pmp564 carrying SH2 mutations binding
to Grb2. GST-Grb2 was immobilized on glutathione resin and the level of
SHP-1 protein "pulled-down" was measured as described under
"Experimental Procedures." One SDS-PAGE, stained with Coomassie
Blue, of three independent experiments, with good reproducibility, is
shown. The mean relative intensities from the three experiments are
indicated below each lane.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-substrate interactions (31), Csk-Src regulation (20, 32), and SHP-2 tyrosine phosphatase (22) employing expressed protein ligation have
illustrated the power and scope of the method. The recent work on SHP-2
illustrates the strength of the use of nonhydrolyzable phosphotyrosines
to examine the structure and function of a signaling protein (22).
Whereas for many years Glu and Asp have been used as mimics of
phosphoserine and phosphothreonines, often successfully, no such
encoded amino acid comes close to resembling phosphotyrosine. Thiophosphates, resistant to enzymatic hydrolysis, have been introduced into signaling proteins by enzymatic modification of recombinant proteins with ATP
S. These are often sluggish reactions that
generally afford low stoichiometries and often show poor
regioselectivity (33). Moreover, thiophosphates can indeed be
hydrolyzed chemically and enzymatically, by some phosphatases almost as
rapidly as phosphate substrates (34), in contrast to the
nonhydrolyzable analogues used here.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Jun Wang for the gift of the SHP-1 plasmid DNA and members of the Cole lab for helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant CA74305 (to P. A. C.) and a National Institutes of Health ACDD Training Grant (to K. S.).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.: 410-614-0540;
Fax: 410-614-7717; E-mail: pcole@jhmi.edu.
Published, JBC Papers in Press, December 4, 2002, DOI 10.1074/jbc.M210028200
2 Whereas mercaptoethylsulfonate is a useful co-reagent for expressed protein ligation (21, 31), and was initially used for some of the experiments here, the semisynthetic SHP-1 proteins showed aberrant catalytic behavior compared with the fully recombinant proteins. In contrast, thiophenol in place of mercaptoethylsulfonate did not lead to this unnatural catalytic activity and was thus employed for all of the work described here.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
PTPase, protein-tyrosine phosphatase;
SH2, Src homology 2;
N-SH2, N-terminal
SH2 domain;
C-SH2, C-terminal SH2 domain;
pY, phosphotyrosine
residue;
EpoR, erythropoietin receptor;
pNPP, p-nitrophenyl phosphate;
Pmp, phosphonomethylene-L-phenylalanine;
F2Pmp, difluorophosphonomethylene-L-phenylalanine;
Fmoc, N-(9-fluorenyl)methoxycarbonyl;
MALDI, matrix-assisted laser
desorption ionization;
GST, glutathione S-transferase;
ATPS, adenosine 5'-O-(thiotriphosphate).
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Zhang, J., Somani, A.-K., and Siminovich, K. A. (2000) Semin. Immunol. 12, 361-378[CrossRef][Medline] [Order article via Infotrieve] |
2. | Chen, H. E., Chang, S., Trub, T., and Neel, B. G. (1996) Mol. Cell. Biol. 16, 3685-3697[Abstract] |
3. |
Mizuno, K.,
Tagawa, Y.,
Mitomo, K.,
Watanabe, N.,
Katagiri, T.,
Ogimoto, M.,
and Yakura, H.
(2002)
J. Immunol.
169,
778-786 |
4. |
Otipoby, K. L.,
Draves, K. E.,
and Clark, E. A.
(2001)
J. Biol. Chem.
276,
44315-44322 |
5. |
Kant, A. M., De, P.,
Peng, X., Yi, T.,
Rawlings, D. J.,
Kim, J. S.,
and Durden, D. L.
(2002)
Blood
100,
1852-1859 |
6. |
Eklund, E. A.,
Goldenberg, I., Lu, Y.,
Andrejic, J.,
and Kakar, R.
(2002)
J. Biol. Chem
277,
36878-36888 |
7. |
Kharbanda, S.,
Bharti, A.,
Pei, D.,
Wang, J.,
Pandey, P.,
Ren, R.,
Weichselbaum, R.,
Walsh, C. T.,
and Kufe, D.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
6898-6901 |
8. | Klingmuller, U., Lorenz, U., Cantley, L. C., Neel, B. G., and Lodish, H. F. (1995) Cell 80, 729-738[Medline] [Order article via Infotrieve] |
9. | Tsui, H. W., Siminovitch, K. A., de Souza, L., and Tsui, F. W. L. (1993) Nat. Gen. 4, 124-129[Medline] [Order article via Infotrieve] |
10. | Yang, J., Liang, X., Niu, T., Meng, W., Zhao, Z., and Zhou, G. W. (1998) J. Biol. Chem. 43, 28199-28207[CrossRef] |
11. | Burke, T. R., and Zhang, Z.-Y. (1998) Biopolymers 47, 225-241[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Chiang, G. G.,
and Sefton, B. M.
(2001)
J. Biol. Chem.
276,
23173-23178 |
13. |
Kautz, B.,
Kakar, R.,
David, E.,
and Eklund, E. A.
(2001)
J. Biol. Chem.
276,
37868-37878 |
14. | Pei, D., Neel, B. G., and Walsh, C. T. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1092-1096[Abstract] |
15. | Pei, D., Lorenz, U., Klingmuller, U., Neel, B. G., and Walsh, C. T. (1994) Biochemistry 33, 15483-15493[Medline] [Order article via Infotrieve] |
16. | Frank, C., Keilhack, H., Opitz, F., Zschornig, O., and Bohmer, F.-D. (1999) Biochemistry 38, 11993-12002[CrossRef][Medline] [Order article via Infotrieve] |
17. | Lorenz, U., Ravichandran, K. S., Pei, D., Walsh, C. T., Burakoff, S. J., and Neel, B. G. (1994) Mol. Cell. Biol. 14, 1824-1834[Abstract] |
18. |
Uchida, T.,
Matozaki, T.,
Noguchi, T.,
Yamao, T.,
Horita, K.,
Suzuki, T.,
Fujioka, Y.,
Sakamoto, C.,
and Kasuga, M.
(1994)
J. Biol. Chem.
269,
12220-12228 |
19. |
Yeung, Y.-G.,
Berg, K. L.,
Pixley, F. J.,
Angeletti, R. H.,
and Stanley, E. R.
(1992)
J. Biol. Chem.
265,
14777-14783 |
20. |
Muir, T. W.,
Sondhi, D.,
and Cole, P. A.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
6705-6710 |
21. |
Evans, T. C.,
Benner, J.,
and Xu, M.-Q.
(1998)
Protein Sci.
7,
2256-2264 |
22. | Lu, W., Gong, D., Bar-Sagi, D., and Cole, P. A. (2001) Mol. Cell 8, 759-769[Medline] [Order article via Infotrieve] |
23. |
Guo, X. L.,
Shen, K.,
Wang, F.,
Lawrence, D. S.,
and Zhang, Z. Y.
(2002)
J. Biol. Chem.
277,
41014-41022 |
24. | Bradford, M. M. (1976) Anal. Biochem. 72, 248-252[CrossRef][Medline] [Order article via Infotrieve] |
25. | Cantor, C. R., and Schimmel, P. R. (1980) Biophysical Chemistry Part II: Techniques for the Study of Biological Structure and Function , pp. 670-675, W. H. Freeman and Co., New York |
26. | Sondhi, D., and Cole, P. A. (1999) Biochemistry 38, 11147-11155[CrossRef][Medline] [Order article via Infotrieve] |
27. | Li, W., Nishimura, R., Kashishian, A., Batzer, A. G., Kim, W. J. H., Cooper, J. A., and Schlessinger, J. (1994) Mol. Cell. Bol. 14, 509-517[Abstract] |
28. | Beebe, K. D., Wang, P., Arabaci, G., and Pei, D. (2000) Biochemistry 39, 13251-13260[CrossRef][Medline] [Order article via Infotrieve] |
29. | Burke, T. R., Jr., Smyth, M. S., Otaka, A., Nomizu, M., Roller, P. P., Wolf, G., Case, R., and Shoelson, S. E. (1994) Biochemistry 33, 6490-6494[Medline] [Order article via Infotrieve] |
30. | Kuriyan, J., and Cowburn, D. (1997) Annu. Rev. Biophys. Biomol. Struct. 26, 259-288[CrossRef][Medline] [Order article via Infotrieve] |
31. | Wu, J. W., Hu, M., Chai, J., Seoane, J., Huse, M., Li, C., Rigotti, D. J., Kyin, S., Muir, T. W., Fairman, R., Massague, J., and Shi, Y. (2001) Mol. Cell. 8, 1277-1289[Medline] [Order article via Infotrieve] |
32. | Wang, D., and Cole, P. A. (2001) J. Am. Chem. Soc. 123, 8883-8887[CrossRef][Medline] [Order article via Infotrieve] |
33. |
Tailor, P.,
Gilman, J.,
Williams, S.,
Couture, C.,
and Mustelin, T.
(1997)
J. Biol. Chem.
272,
5371-5374 |
34. |
Cho, H.,
Krishnaraj, R.,
Itoh, M.,
Kitas, E.,
Bannwarth, W.,
Saito, H.,
and Walsh, C. T.
(1993)
Protein Sci.
2,
977-984 |