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*
Howard Hughes Medical Institute,
Rheumatology Division, Department of Medicine, and
Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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RIIB, CD22), macrophages (paired Ig-like
receptor-B) (2, 3, 4), and NK cells (Ly-49,
KIR3). Despite their
functional similarity, these receptors have apparently evolved
separately, as suggested by their different structures. For example,
the NK receptors belonging to the Ly-49 and KIR families appear to
subserve similar functions, but are either type II membrane proteins
with homology to C-type lectins, or type I membrane proteins belonging
to the Ig superfamily, respectively (5). Despite the differences in
cell distribution and structure, many inhibitory receptors signal
through a common mechanism: the immunoreceptor tyrosine-based
inhibitory motif (ITIM), which is a 6-amino-acid stretch consisting of
V/L/IxYxxL/V (6, 7, 8, 9, 10). When phosphorylated, ITIMs bind inhibitory
cytoplasmic phosphatases. The role of the ITIM was first elucidated in B cells, where it was shown that the ITIMs of CD22 were responsible for transmitting the CD22 inhibitory signal by engaging and activating SHP-1 (8), a tandem SH2 domain-containing cytoplasmic tyrosine phosphatase (11, 12, 13, 14). The importance of the inhibitory signals delivered by SHP-1 is highlighted by the motheaten (me) mouse (15), which has a natural mutation in the SHP-1 locus (16). Mice with this deficiency are characterized by a widespread autoimmune phenomena, caused by an inability to negatively regulate immune responses. Consistent with the pleiotropic defects observed in the motheaten mouse, SHP-1 has also been implicated in negative signaling through many receptors, including receptors for growth factors, cytokines, and Ag, as well as through integrins (17). More recently, an important role for SHP-1 in negative signaling through NK cell inhibitory receptors has emerged. Experiments utilizing phosphorylated ITIM peptides implicated SHP-1 in signaling through the ITIM-bearing receptors in the KIR and Ly-49 family (18) before these associations were verified by coimmunoprecipitation studies (19, 20, 21, 22). Another NK cell receptor, the CD94/NKG2A complex, also associates with SHP-1 (23, 24). Thus, SHP-1 plays a prominent and still expanding role in controlling immune responses.
The mechanism of binding and activation of SHP-1 by ITIM-bearing receptors is not yet fully understood. It is clear that tandem ITIMs, either on the same polypeptide (KIR) or as a result of dimerization (Ly-49A), optimally stimulate SHP-1 phosphatase activity (9), suggesting that occupation of both SHP-1 SH2 domains is required for maximal enzymatic activity. However, both SH2 domains are not required for binding of SHP-1 to receptor ITIMs based on binding studies using truncated fusion proteins (10, 19, 25, 26). The binding specificities of the SHP-1 SH2 domains in the context of the full-length protein have not been extensively analyzed. Such studies are especially important given that the crystal structure of the related molecule SHP-2 predicts that the phosphatase domain influences the binding of its SH2 domains (27). It is possible that ITIM binding by the SHP-1 SH2 domains is regulated in a similar fashion, although this has not been demonstrated.
The receptor gp49B is expressed on mast cells (28) and NK cells (29, 30, 31) and contains two putative ITIMs in its cytoplasmic tail separated by 16 amino acids. These ITIMs appear to be functional in NK cells, as suggested by Rojo et al., who have demonstrated that the gp49B cytoplasmic tail is able to inhibit killing by murine NK cells (30). In this study, we examine the signaling pathway utilized by gp49B and specifically its ability to associate with SHP-1. Additionally, we define the structural requirements of SHP-1 that mediate its interaction with gp49B.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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A20 (murine B cell) and Jurkat (human T cell) cells were
obtained from American Type Culture Collection (Manassas, VA) and
maintained in complete DMEM or RPMI 1640 media (10% FCS (Harlan,
Indianapolis, IN), L-glutamine, penicillin, streptomycin,
and 0.5 µM 2-ME), respectively. Neither A20 nor Jurkat cells express
gp49B (L.L.W., W.M.Y., unpublished observations). The murine NK cell
clone KY-2 (32) was maintained in complete RPMI 1640 supplemented with
100 U/ml human rIL-2 (Chiron, Emeryville, CA). Immunoblotting reagents
used were rabbit anti-SHP-1 polyclonal antisera (33),
anti-SHP-2 (Transduction Laboratories, Lexington, KY),
anti-phosphotyrosine 4G10-biotin (Upstate Biotechnology, Lake
Placid, NY), anti-gp49B, and horseradish peroxidase-conjugated
avidin or donkey anti-rabbit IgG (Amersham, Arlington Heights, IL).
For generation of anti-gp49B, rabbits were immunized with the
peptide YAQVKPSRLQKDTAC (Quality Controlled Biochemicals, Hopkinton,
MA), corresponding to amino acids 300–314 of gp49B, conjugated to
keyhole limpet hemocyanin. Rabbits were injected at 0, 4, 8, and 12 wk
with 100 µg of peptide in CFA (week 0) or in IFA (weeks 4, 8, and
12). In week 12, rabbits were exsanguinated, and anti-gp49B was
purified by peptide affinity chromatography. Anti-gp49B detects a
55-kDa protein in lysates from KY-2 cells and gp49B-transfected
Chinese hamster ovary cells, which is absent in Jurkat and
untransfected Chinese hamster ovary cells (data not shown).
Immunoprecipitation Abs used were B23.1 mAb (rat IgM) (34) purified by
MAR18.5 (anti-rat IgG 
) affinity chromatography, H3 (hamster
polyclonal antiserum generated against gp49B-Fc), and isotype control
rat IgM (PharMingen, San Diego, CA). For production of H3, Armenian
hamsters were injected with 100 µg of gp49B-Fc in Ribi adjuvant (RIBI
ImmunoChem Research, Hamilton, MT) at 0, 3, and 6 wk. To construct
gp49B-Fc, the extracellular domain of gp49B (amino acids 23–238) was
amplified using the PCR primers: forward,
5'-TATATGCTAGCAGGACACCTCCCAAAGCCCATC-3', and reverse,
5'-ATATAGGATCCGCATGCTTCTGGTATGTTTCCAGTCCATC-3', which introduced
NheI and BamHI restriction sites at the 5' and 3'
ends, respectively. The amplified fragment was cloned into Pcd5lneg1 (a
generous gift from Brian Seed, Harvard Medical School, Boston,
MA) and gp49B-Fc was produced and purified as previously described
(35).
Peptides
All peptides were purchased from Quality Controlled Biochemicals and synthesized conjugated to biotin on the amino terminus. Unphosphorylated or tyrosine-phosphorylated peptides with the following sequences were synthesized: SEQEVTYSMVRF (Ly-49A ITIM), QGIVYAQVKPS (gp49B proximal ITIM), QDVTYAQLCIR (gp49B distal ITIM), VNRHYAKI (ZAP-70 Y474), and GIHYSELIQFG (derivative of CD22-Y5).
Cell stimulation and immunoblot analysis
Cells were washed in PBS and left untreated or stimulated with 10 µM pervanadate for 10 min at room temperature. Pelleted cells were lysed in either 1% Nonidet P-40 lysis buffer (peptide and GST SHP-1 adsorptions) or 1% Triton X-100 lysis buffer (immunoprecipitations) containing 150 mM NaCl, 10 mM Tris, pH 8, PMSF, aprotinin, leupeptin, pepstatin, EDTA, and soybean trypsin inhibitor. For immunoprecipitation and GST SHP-1 studies, sodium fluoride and sodium orthovanadate were also included in the lysis buffer. Insoluble material was removed by centrifugation at 14,000 x g for 20 min at 4°C. Following affinity purification with the indicated reagent, precipitates were washed four times in lysis buffer, separated by SDS-PAGE, and transferred to nitrocellulose, and immunoblot analysis was performed. For membrane stripping, blots were submerged in 100 mM 2-ME, 2% SDS, and 62.5 mM Tris-HCl, pH 6.7, for 30 min, 50°C.
Peptide adsorption and immunoprecipitation
Cell lysates representing 3 x 107 cells/lane (silver stain or immunoprecipitation) or 1 x 107 cells/lane (immunoblot) were mixed with peptide at the indicated concentration, 2 µg of Ab, or 3 µl of hamster serum. Peptides were precipitated with avidin-agarose (Sigma, St. Louis, MO), rat IgM was precipitated by goat anti-rat IgG agarose (Sigma), and hamster IgG was precipitated with protein G-Sepharose (Pharmacia, Piscataway, NJ). Bound proteins were separated under reducing conditions by 7.5% SDS-PAGE and analyzed by silver stain (Daichii, Tokyo, Japan), or transferred to nitrocellulose, followed by immunoblot analysis.
SHP-1 phosphatase assays
Baculoviral-expressed full-length SHP-1 was purified by conventional chromatography and HPLC. The reaction mixture contained 0–200 µM peptide, 250 ng SHP-1, 50 mM HEPES (pH 7), 5 mM EDTA, 1 mM DTT, and 5 mM p-nitrophenyl phosphate (pNPP) as substrate. Reactions were stopped after 30 min at 37°C, and the relative phosphatase activity was measured by absorbance at 405 nm. Results are expressed as fold increase over basal phosphatase activity of SHP-1 in the absence of peptide. A phosphorylated CD22 peptide was used as a positive control for the phosphatase assay.
GST fusion proteins
For generation of the GST fusion proteins containing the SH2 domains of murine SHP-1 (amino acids 1–219), PCR was used to amplify the tandem SH2 domains from full-length SHP-1 cDNA as template: forward primer, 5'-GTGAGAATTCCCAGGATGGTGAGGTG-3', and reverse primer, 5'-CAATGTAAGCTTCGTTTACCCGAGT-3'. EcoRI and HindIII sites were incorporated into the forward and reverse primers, respectively, and used as the cloning sites for ligation into pGEX-KG (Pharmacia). The mutations of the amino and carboxyl SH2 domains were introduced by the PCR site overlap extension method. To introduce the amino SH2 domain mutations (R30, 33KE), the internal primers used were forward primer, 5'-GTCCCCGGGAGCTTCCTGGCTAAGCCCAGCGAGAAGAACCAGGG-3', and reverse primer, 5'-CCCTGGTTCTTCTCGCTGGGCTTAGCCAGGAAGCTCCCGGGGAC-3'. For mutation of the carboxyl SH2 domain (R136K), the PCR primers used were forward primer, 5'-GCGAGCCATGGACATTTCTTGTGAAGGAGAGTC-3', and reverse primer, 5'-GACTCTCCTTCACAAGAAATGTCCATG-3'. Site-directed mutagenesis of the full-length SHP-1 cDNA was used to replace Cys453 with Ser in the phosphatase domain. The mutated SH2 domains were cassetted using internal unique restriction enzyme sites. All mutations were verified by automated nucleotide sequencing (Applied Biosystems, Foster City, CA). GST fusion proteins were transformed into Escherichia coli strain BL21(DE3) for protein production. Adsorption experiments were performed with the indicated quantity of GST fusion protein conjugated to glutathione agarose (Sigma).
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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gp49B contains two putative ITIMs in its cytoplasmic domain. To
identify signaling molecules associated with the ITIMs, we synthesized
peptides representing each of the two ITIMs of gp49B (membrane proximal
and distal) in either the tyrosine-phosphorylated or unphosphorylated
state. The Ly-49A ITIM, which has previously been shown to bind to and
signal through SHP-1 (18, 22), was also synthesized. These peptides
were used to adsorb lysates from A20 cells (Fig. 1
A) or KY-2 cells (data not
shown), and bound proteins were analyzed by SDS-PAGE and silver stain.
The primary product that associated specifically with the
gp49B-phosphorylated ITIMs and not the unphosphorylated peptides
migrated at 66 kDa (p66). To determine the identity of p66, we
performed anti-SHP-1 immunoblot analysis on lysates adsorbed with
the ITIM peptides. Each ITIM peptide was able to interact with SHP-1 in
a phosphorylation-dependent manner (Fig. 1B, top,
lanes 2, 4, and 6). However, a
tyrosine-phosphorylated ZAP-70 peptide did not interact with SHP-1,
demonstrating the importance of not only the phosphorylated tyrosine,
but also the surrounding ITIM sequence for SHP-1 binding. By our silver
stain analysis, only one other protein, migrating at 70 kDa (Fig. 1A), bound specifically to the phosphorylated ITIMs and most
likely represents SHP-2 (Fig. 1B, bottom). From
these data, we conclude that the phosphorylated gp49B ITIMs interact
primarily with SHP-1 from hemopoietic cell lysates.
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The relative binding capacities of the phosphorylated gp49B
ITIMs for SHP-1 were assessed by titrating each
phosphorylated ITIM peptide with equivalent amounts of Jurkat cell
lysate (Fig. 2). Among the gp49B ITIMs,
the distal ITIM appeared to interact more strongly to SHP-1, as it
retained binding at 0.16 µM (Fig. 2, lane 11), a
concentration at which the proximal ITIM had lost SHP-1 binding (Fig. 2, lane 7). Binding of the distal gp49B ITIM was comparable
with that observed for the Ly-49A ITIM, with both peptides losing
binding at 0.04 µM.
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). As previously published, a
phosphorylated CD22 ITIM peptide activates SHP-1 very strongly (8). The
gp49B-proximal ITIM demonstrated intermediate levels of SHP-1
activation, comparable with the levels seen with the Ly-49A peptide. In
contrast, the distal ITIM of gp49B was a very weak stimulator of SHP-1
activity, but still activated at levels >2-fold above baseline
activation with unphosphorylated peptides. Taken together, even though
the distal ITIM demonstrates stronger binding to SHP-1 (Fig. 2
), it is
a far weaker stimulator of the SHP-1 phosphatase than the proximal ITIM
(Fig. 3), suggesting that ITIM affinity to SHP-1 does not directly
correlate with its phosphatase-activating potential.
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To verify the association between gp49B and SHP-1, we
immunoprecipitated gp49B, using either mAb B23.1 or the polyclonal
antiserum H3, from untreated or pervanadate-treated KY-2 cells,
previously shown to express gp49B (29). Antiphosphotyrosine
immunoblotting indicated that gp49B becomes phosphorylated following
pervanadate treatment (Fig. 4A, lane 3).
Coimmunoprecipitated proteins were subjected to anti-SHP-1
immunoblotting (Fig. 4B, top). gp49B associated
with SHP-1 at very low levels in untreated NK cells (Fig. 4B, top, lanes 2 and 5), an
interaction that is greatly augmented following pervanadate treatment
of cells (Fig. 4B, top, lanes 3 and
6). Anti-gp49B immunoblotting confirmed that comparable
levels of gp49B were precipitated in each sample (Fig. 4B,
bottom). SHP-1 did not coprecipitate with rat IgM, an
isotype control Ab for B23.1, or preimmune hamster serum. Additionally,
B23.1 and H3 did not precipitate SHP-1 from pervanadate-treated Jurkat
cells that contain SHP-1, but lack gp49B. This establishes that
tyrosine-phosphorylated gp49B interacts with SHP-1 in NK cells.
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To further define the interaction between gp49B and SHP-1, we
synthesized GST fusion proteins containing only the tandem SH2 domains
of SHP-1 (2SH2). In addition to synthesizing the wild-type SH2 domains
of SHP-1, mutations were introduced in either the amino SH2 domain
(R30, 33KE) or the carboxyl SH2 domain (R136K), which severely impair
the binding of the respective SH2 domain (36). Untreated or
pervanadate-treated KY-2 lysates were precipitated with the wild-type
SH2 domains or the single mutated SH2 domains. Bound proteins were
separated by SDS-PAGE and subjected to gp49B immunoblotting. Wild-type
GST SHP-1 bound to gp49B only from pervanadate-stimulated cells (Fig. 5A, top, lane
3). Furthermore, mutation of either the amino or carboxyl SH2
domain of SHP-1 did not appear to affect the association with
phosphorylated gp49B (Fig. 5A, top, lanes
6 and 9). Consistent with our phosphopeptide-binding
data, binding of each SHP-1 fusion protein to unphosphorylated gp49B
was undetectable. SHP-1 immunoblotting was also performed to confirm
comparable loading of GST SHP-1 (Fig. 5A,
bottom). Thus, the SHP-1 SH2 domains interact with
tyrosine-phosphorylated gp49B, and either SH2 domain of SHP-1 is
sufficient for this interaction.
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We tested the involvement of the phosphatase domain in regulating the
interaction between gp49B and SHP-1 by synthesizing full-length GST
SHP-1 with an inactive phosphatase domain (C453S) (33). GST fusion
proteins were made containing mutations in the amino SH2 domain (R30,
33KE) or the carboxyl SH2 domain (R136K). Full-length GST SHP-1 with a
mutated amino SH2 domain (R30, 33KE) retained binding to phosphorylated
gp49B (Fig. 5B, top, lanes 2 and
3), although the binding appeared to be slightly diminished
as compared with that of the tandem SH2 domains (Fig. 5A).
However, mutation of the carboxyl SH2 domain (R136K) completely
abrogated binding to gp49B (Fig. 5B, top,
lane 5), as did mutation of both SH2 domains (data not
shown). One possible explanation is that the lack of binding by the
R136K mutant is a result of improper folding of this protein. However,
we feel this is highly unlikely because of the following: 1) The R136K
mutation was produced and isolated in equally abundant amounts.
Improperly folded proteins may be produced in far lesser quantities and
are oftentimes insoluble. 2) The R136K mutation was identical to the
mutation introduced in the tandem SH2 domain fusion (Fig. 5A, top, lanes 8–10), which is
functional. If the R136K mutation altered the structure of the protein,
the tandem SH2 domain mutant should also be nonfunctional. Therefore,
the lack of gp49B binding by the full-length SHP-1 R136K is not likely
to be caused by improper folding of the fusion protein.
Taken together, these data indicate that the amino SH2 domain of SHP-1,
in the absence of the phosphatase domain, is sufficient for the
association with phosphorylated gp49B (Fig. 5A,
top, lane 9). However, the presence of the
phosphatase domain prevents this interaction (Fig. 5B,
top, lane 5). Moreover, the carboxyl SH2 domain
of SHP-1, even in the context of full-length SHP-1, is sufficient for
binding to gp49B (Fig. 5B, top, lanes
2 and 3). These results are consistent with predicted
models of SHP-2 regulation (27), and we propose a similar model for
SHP-1 association with receptor ITIMs (Fig. 6). In the basal state, the amino SH2
domain forms an intramolecular association with the phosphatase domain,
which greatly diminishes the affinity of the amino SH2 domain for
phosphorylated ITIM (Fig. 6A). The carboxyl SH2 domain of
SHP-1 initiates an interaction by binding one site in a
biphosphorylated ligand, thus increasing the local concentration of
phosphorylated ITIM (Fig. 6B). This allows the amino SH2
domain to bind phosphorylated tyrosine, leading to release and
activation of the phosphatase (Fig. 6C). The crystal
structure of SHP-1 may ultimately prove this model.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Wayne M. Yokoyama, Rheumatology Division, Box 8045, Department of Medicine, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail address:
3 Abbreviations used in this paper: KIR, killer inhibitory receptor; GST, glutathione S-transferase; ITIM, immunoreceptor tyrosine-based inhibitory motif; pNPP, p-nitrophenyl phosphate; SH2, Src holomology 2.
Received for publication August 12, 1998. Accepted for publication October 14, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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RIIB modulates B-cell receptor signalling. Nature 368:70.[Medline]
RIIB1. Science 268:293.1 upon B cell activation. J. Exp. Med. 183:547.This article has been cited by other articles:
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  K. Oishi, K. Gaengel, S. Krishnamoorthy, K. Kamiya, I.-K. Kim, H. Ying, U. Weber, L. A. Perkins, M. Tartaglia, M. Mlodzik, et al. Transgenic Drosophila models of Noonan syndrome causing PTPN11 gain-of-function mutations Hum. Mol. Genet., February 15, 2006; 15(4): 543 - 553. [Abstract] [Full Text] [PDF]
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J. Yang, L. Liu, D. He, X. Song, X. Liang, Z. J. Zhao, and G. W. Zhou Crystal Structure of Human Protein-tyrosine Phosphatase SHP-1 J. Biol. Chem., February 14, 2003; 278(8): 6516 - 6520. [Abstract] [Full Text] [PDF]
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D. Nandan, T. Yi, M. Lopez, C. Lai, and N. E. Reiner Leishmania EF-1alpha Activates the Src Homology 2 Domain Containing Tyrosine Phosphatase SHP-1 Leading to Macrophage Deactivation J. Biol. Chem., December 13, 2002; 277(51): 50190 - 50197. [Abstract] [Full Text] [PDF]
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 T. Oka, M. Ouchida, M. Koyama, Y. Ogama, S. Takada, Y. Nakatani, T. Tanaka, T. Yoshino, K. Hayashi, N. Ohara, et al. Gene Silencing of the Tyrosine Phosphatase SHP1 Gene by Aberrant Methylation in Leukemias/Lymphomas Cancer Res., November 15, 2002; 62(22): 6390 - 6394. [Abstract] [Full Text] [PDF]
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 Y.-H. Feng, Y. Sun, and J. G. Douglas Gbeta gamma -independent constitutive association of Galpha s with SHP-1 and angiotensin II receptor AT2 is essential in AT2-mediated ITIM-independent activation of SHP-1 PNAS, September 17, 2002; 99(19): 12049 - 12054. [Abstract] [Full Text] [PDF]
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