Molecular Mechanism of the Syk Activation Switch
2008; Elsevier BV; Volume: 283; Issue: 47 Linguagem: Inglês
10.1074/jbc.m806340200
ISSN1083-351X
AutoresEmily K. Tsang, Anthony M. Giannetti, David E. Shaw, Marie Dinh, Joyce Tse, Shaan C. Gandhi, Hoangdung Ho, Sandra Wang, Eva Papp, J. Michael Bradshaw,
Tópico(s)Antimicrobial Peptides and Activities
ResumoMany immune signaling pathways require activation of the Syk tyrosine kinase to link ligation of surface receptors to changes in gene expression. Despite the central role of Syk in these pathways, the Syk activation process remains poorly understood. In this work we quantitatively characterized the molecular mechanism of Syk activation in vitro using a real time fluorescence kinase assay, mutagenesis, and other biochemical techniques. We found that dephosphorylated full-length Syk demonstrates a low initial rate of substrate phosphorylation that increases during the kinase reaction due to autophosphorylation. The initial rate of Syk activity was strongly increased by either pre-autophosphorylation or binding of phosphorylated immune tyrosine activation motif peptides, and each of these factors independently fully activated Syk. Deletion mutagenesis was used to identify regions of Syk important for regulation, and residues 340–356 of the SH2 kinase linker region were identified to be important for suppression of activity before activation. Comparison of the activation processes of Syk and Zap-70 revealed that Syk is more readily activated by autophosphorylation than Zap-70, although both kinases are rapidly activated by Src family kinases. We also studied Syk activity in B cell lysates and found endogenous Syk is also activated by phosphorylation and immune tyrosine activation motif binding. Together these experiments show that Syk functions as an "OR-gate" type of molecular switch. This mechanism of switch-like activation helps explain how Syk is both rapidly activated after receptor binding but also sustains activity over time to facilitate longer term changes in gene expression. Many immune signaling pathways require activation of the Syk tyrosine kinase to link ligation of surface receptors to changes in gene expression. Despite the central role of Syk in these pathways, the Syk activation process remains poorly understood. In this work we quantitatively characterized the molecular mechanism of Syk activation in vitro using a real time fluorescence kinase assay, mutagenesis, and other biochemical techniques. We found that dephosphorylated full-length Syk demonstrates a low initial rate of substrate phosphorylation that increases during the kinase reaction due to autophosphorylation. The initial rate of Syk activity was strongly increased by either pre-autophosphorylation or binding of phosphorylated immune tyrosine activation motif peptides, and each of these factors independently fully activated Syk. Deletion mutagenesis was used to identify regions of Syk important for regulation, and residues 340–356 of the SH2 kinase linker region were identified to be important for suppression of activity before activation. Comparison of the activation processes of Syk and Zap-70 revealed that Syk is more readily activated by autophosphorylation than Zap-70, although both kinases are rapidly activated by Src family kinases. We also studied Syk activity in B cell lysates and found endogenous Syk is also activated by phosphorylation and immune tyrosine activation motif binding. Together these experiments show that Syk functions as an "OR-gate" type of molecular switch. This mechanism of switch-like activation helps explain how Syk is both rapidly activated after receptor binding but also sustains activity over time to facilitate longer term changes in gene expression. Syk is a tyrosine kinase that functions immediately downstream of antigen receptors in immune cells including B lymphocytes, mast cells, and macrophages (1Sada K. Takano T. Yanagi S. Yamamura H. J Biochem. (Tokyo). 2001; 130: 177-186Crossref PubMed Scopus (235) Google Scholar, 2Siraganian R.P. Zhang J. Suzuki K. Sada K. Mol. Immunol. 2002; 38: 1229-1233Crossref PubMed Scopus (122) Google Scholar, 3Berton G. Mocsai A. Lowell C.A. Trends Immunol. 2005; 26: 208-214Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). The central role of Syk in cell types associated with disorders such as rheumatoid arthritis and allergic rhinitis suggests that strategies to block Syk activation may have therapeutic benefit (4Bajpai M. Chopra P. Dastidar S.G. Ray A. Expert Opin. Investig. Drugs. 2008; 17: 641-659Crossref PubMed Scopus (47) Google Scholar, 5Ulanova M. Duta F. Puttagunta L. Schreiber A.D. Befus A.D. Expert Opin. Ther. Targets. 2005; 9: 901-921Crossref PubMed Scopus (58) Google Scholar, 6Wong B.R. Grossbard E.B. Payan D.G. Masuda E.S. Expert Opin. Investig. Drugs. 2004; 13: 743-762Crossref PubMed Scopus (141) Google Scholar). After receptor ligation and phosphorylation, Syk becomes localized to immune receptors and proceeds to phosphorylate downstream targets leading to Ca2+ mobilization, initiation of the extracellular signal-regulated kinase and p38 mitogen-activated protein kinase cascades, and activation of transcription factors such as NF-κB (7Dal Porto J.M. Gauld S.B. Merrell K.T. Mills D. Pugh-Bernard A.E. Cambier J. Mol. Immunol. 2004; 41: 599-613Crossref PubMed Scopus (415) Google Scholar). Although increased Syk activity is known to be indispensable for each of these cellular events, a molecular-level understanding of the steps leading to Syk activation has not been clearly defined. The Syk domain structure consists of an N-terminal pair of Src homology 2 (SH2) 2The abbreviations used are: SH2, Src homology 2; ITAM, immune tyrosine activation motif; PTP1B, protein-tyrosine phosphatase 1B; FcϵRIγ, γ chain of the FcϵRI receptor; DTT, dithiothreitol; BCR, B cell receptor. 2The abbreviations used are: SH2, Src homology 2; ITAM, immune tyrosine activation motif; PTP1B, protein-tyrosine phosphatase 1B; FcϵRIγ, γ chain of the FcϵRI receptor; DTT, dithiothreitol; BCR, B cell receptor. domains separated by an inter-SH2 linker, an SH2-domain-kinase linker, and a C-terminal kinase domain. Recruitment of Syk to immune receptors involves binding of the tandem SH2 domains of Syk to motifs in the receptor known as immune tyrosine activation motifs (ITAMs), which are two YXXL sequences typically separated by 7–12 intervening residues (8Cambier J.C. J. Immunol. 1995; 155: 3281-3285PubMed Google Scholar, 9Flaswinkel H. Barner M. Reth M. Semin. Immunol. 1995; 7: 21-27Crossref PubMed Scopus (59) Google Scholar). Syk is known to have multiple sites of phosphorylation which both regulate activity and serve as docking motifs for other proteins (1Sada K. Takano T. Yanagi S. Yamamura H. J Biochem. (Tokyo). 2001; 130: 177-186Crossref PubMed Scopus (235) Google Scholar). These sites include Tyr-348 and Tyr-352 within the SH2-linker region (10Brdicka T. Kadlecek T.A. Roose J.P. Pastuszak A.W. Weiss A. Mol. Cell. Biol. 2005; 25: 4924-4933Crossref PubMed Scopus (108) Google Scholar), Tyr-525 and Tyr-526 within the activation loop of the kinase domain (11Zhang J. Billingsley M.L. Kincaid R.L. Siraganian R.P. J. Biol. Chem. 2000; 275: 35442-35447Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar), Tyr-630 in the C terminus of Syk (12Kulathu Y. Hobeika E. Turchinovich G. Reth M. EMBO J. 2008; 27: 1333-1344Crossref PubMed Scopus (67) Google Scholar), and other sites. Many of the key structural features and sites of phosphorylation found in Syk are conserved in its homolog Zap-70 (13Chu D.H. Morita C.T. Weiss A. Immunol. Rev. 1998; 165: 167-180Crossref PubMed Scopus (206) Google Scholar). A greater understanding of the activation process for Syk and Zap-70 was recently gleaned from the crystal structure of a full-length version of Zap-70 (14Deindl S. Kadlecek T.A. Brdicka T. Cao X. Weiss A. Kuriyan J. Cell. 2007; 129: 735-746Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). In this structure a unique network of interactions involving the kinase-SH2 linker, the inter-SH2 linker, and the kinase hinge region was observed that appeared to stabilize an inactive Zap-70 conformation. Disruption of this network was proposed to cause kinase activation, and indeed mutations in this region increased Zap-70 activity. The similarity between Syk and Zap-70 and the electron microscopy structure of Syk (15Arias-Palomo E. Recuero-Checa M.A. Bustelo X.R. Llorca O. Biochim. Biophys. Acta. 2007; 1774: 1493-1499Crossref PubMed Scopus (21) Google Scholar) suggest that Syk may adopt a similar inactive conformation, and hence, show a similar activation mechanism, as Zap-70. However, for Syk, functional data directly testing this model is lacking. Previous studies have linked both binding to phosphorylated ITAM sequences and phosphorylation at several sites to increased Syk activity (16Rolli V. Gallwitz M. Wossning T. Flemming A. Schamel W.W. Zurn C. Reth M. Mol. Cell. 2002; 10: 1057-1069Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). However, many questions still remain regarding the molecular mechanism of Syk activation. For instance, how much do phosphorylation and ITAM binding each contribute to Syk activation? What is the magnitude of activation? Does maximal activation require both phosphorylation and ITAM binding, or is each process itself sufficient to fully activate? What are the similarities and differences between activation of Syk and Zap-70? In this study we set out to better understand the molecular basis of Syk activation. Full-length Syk was studied in vitro using both a real time kinase assay and Western blotting with phosphospecific antibodies. It was observed that either Syk phosphorylation or binding of phosphorylated ITAM peptides was sufficient to fully activate Syk. Mutagenesis indicated that residues 340–356 of the SH2 kinase linker are important for phosphorylation-dependent regulation. Furthermore, endogenous Syk from B cell lysates was found to demonstrate similar activation as recombinant Syk in vitro. These experiments suggest a model whereby multiple different stimuli can each fully activate Syk, and hence, Syk can be viewed as an OR-gate type of kinase switch (17Dueber J.E. Yeh B.J. Chak K. Lim W.A. Science. 2003; 301: 1904-1908Crossref PubMed Scopus (243) Google Scholar). Reagents—The following enzymes were obtained from Invitrogen: Syk (catalog # PV4089), Zap-70 (catalog # PR3017A), Lck (catalog # P3043), Lyn (catalog # PR3722A), PTP1B (catalog # PR4454A). Syk from Invitrogen was used for the studies described here. Syk was also obtained from Carna Biosciences (Kobe, Japan) (catalog # 08-761) and SignalChem (Richmond, BC, Canada) (catalog # s52) and confirmed to have the same regulation by ITAM binding and phosphorylation as Syk from Invitrogen (data not shown). All Syk proteins contained an N-terminal glutathione S-transferase tag for purification purposes. ITAM phosphopeptides were custom synthesized by Quality Controlled Biochemicals (Hopkinton, MA) and obtained at 95% purity level. The following antibodies were employed: Syk monoclonal from BD Biosciences, Syk phosphospecific 525/526 polyclonal from Cell Signaling Technologies (Danvers, MA), Syk phosphospecific 348/352 and Zap-70 315/319 phosphospecific polyclonal from Cell Signaling Technologies. Sox-fluorophore-containing peptide substrates for Syk (Y7, KNZ3071) and Zap-70 (Y8, KNZ3081) were obtained from Invitrogen. Dephosphorylation and Autophosphorylation Conditions—To dephosphorylate the basal state of Syk (which was observed to be phosphorylated following purification), 4 μm Syk was incubated together with 0.08 μg/μl phosphatase PTP1B for 60 min at 27 °C in a buffer of 20 mm Hepes, pH 7.15, 0.1 mm EGTA, 0.1 mm DTT, 0.5 mg/ml bovine serum albumin, 10 mm MgCl2. After dephosphorylation with PTP1B, all subsequent evaluations of Syk activity were performed in a buffer containing 10 μm phosphatase inhibitor sodium vanadate. We demonstrated that adding 10 μm sodium vanadate to the buffer blocked the basal phosphatase activity of PTP1B (supplemental Fig. 1) and, hence, prevented PTP1B from affecting evaluations of Syk activity. Furthermore, we demonstrated that 10 μm sodium vanadate had no affect on basal Syk activity in the absence of PTP1B (supplemental Fig. 1). To re-autophosphorylate Syk before analysis of activity, 0.8 μm Syk was incubated with 100 μm ATP for 15 min at 27 °C in a buffer of 20 mm Hepes, pH 7.15, 0.1 mm EGTA, 0.1 mm DTT, 0.5 mg/ml bovine serum albumin, 10 mm MgCl2, and 10 μm sodium vanadate. Enzyme Activity Assay—Syk enzyme activity was monitored as described previously (18Papp E. Tse J.K. Ho H. Wang S. Shaw D. Lee S. Barnett J. Swinney D.C. Bradshaw J.M. Biochemistry. 2007; 46: 15103-15114Crossref PubMed Scopus (23) Google Scholar). Briefly, activity was assessed by monitoring the increase in fluorescence after phosphorylation of a commercially available peptide substrate (pep7) that contains the phosphorylation-sensitive amino acid Sox (19Shults M.D. Carrico-Moniz D. Imperiali B. Anal. Biochem. 2006; 352: 198-207Crossref PubMed Scopus (66) Google Scholar, 20Shults M.D. Janes K.A. Lauffenburger D.A. Imperiali B. Nat. Methods. 2005; 2: 277-283Crossref PubMed Scopus (182) Google Scholar). Assays were performed in 20 mm Hepes, pH 7.15, 0.1 mm EGTA, 0.1 mm DTT, 0.5 mg/ml bovine serum albumin, 10 mm MgCl2, 10 μm sodium vanadate, and typically 25 μm ATP in 96-well black plates (Corning Inc., Corning, NY) using a final assay volume of 50 μl. Data were collected on either a Spectra-Max GeminiXS fluorescence plate reader (Molecular Devices, Sunnyvale, CA) or PheraStar microplate reader (BMG LABTECH, Durham, NC) using excitation and emission wavelengths of 360 and 455 nm, respectively. The concentration of Syk in the assay was typically 5 or 10 nm (except for experiments with Syk356 and Syk360, which employed 1 or 2 nm Syk due to the high activity of these particular Syk variants). Michaelis-Menten Analysis—The initial velocity (vin) of Syk product formation measured in μm/min was assessed by evaluating the rate of product formation from the initial phase of the fluorescence progress curve. This time course was typically linear until ∼50% of substrate was converted to product for autophosphorylated Syk, Syk with ITAM peptides, and truncated Syk variants. For dephosphorylated Syk, vin was estimated from the first few time points (∼2 min) of the reaction time course. The product formation rate of a given experiment (kcat) was calculated by dividing vin by the concentration of enzyme used in the experiment. To determine the kinetic constants of maximum turnover rate ((kcat∗)) and Michaelis constant (Km) of different Syk constructs with respect to ATP, the ATP concentration was varied at quarter log scale from 1 to 300 μm at a constant pep7 concentration of 5 μm. Likewise, to determine the kinetic constants ((kcat∗), Km) with respect to pep7, the pep7 concentration was varied at quarter log scale from 1 to 30 μm at a constant ATP concentration of 25 μm. These data were evaluated using the Michaelis-Menten equation. Data analysis was performed with GraphPad Prism 4.0 (GraphPad Software, Inc., San Diego, CA). Western Blotting—Syk samples containing 0.1 μg of protein were analyzed by electrophoresis using the NuPAGE gel system and transferred to nitrocellulose via Iblot transfer (Invitrogen). Membranes were probed with antibodies against total Syk at a 1:10,000 dilution, phosphorylated Tyr residues 348 and 352 at 1:500 dilution, or phosphorylated Tyr residues 525 and 526 at 1:500 dilution. Blots were probed with horseradish peroxidase-conjugated secondary antibodies at a 1:10,000 dilution and developed with ECL+ solution (GE Healthcare). Other Western blot procedures were performed as described (21Dinh M. Grunberger D. Ho H. Tsing S.Y. Shaw D. Lee S. Barnett J. Hill R.J. Swinney D.C. Bradshaw J.M. J. Biol. Chem. 2007; 282: 8768-8776Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Surface Plasmon Resonance—Surface plasmon resonance experiments were carried out using a Biacore S51 biosensor instrument (GE Healthcare). Interactions between the protein immobilized on a biosensor surface and peptides flowed over the surface were monitored in real time as a change in surface plasmon resonance as measured in resonance units (22Malmqvist M. Nature. 1993; 361: 186-187Crossref PubMed Scopus (523) Google Scholar). An anti-glutathione S-transferase (GST) antibody was captured to the surface of a CM5 sensor chip using a commercially available anti-GST kit and the manufacturer's instructions (GE Healthcare). Full-length glutathione S-transferase-Syk was capture by flowing freshly thawed protein aliquots over the antibody surface until saturation of the antibody surface was achieved. Peptides were diluted in running buffer (50 mm Hepes, pH 8.0, 150 mm NaCl, 10 mm MgCl2, 1 mm Tris(2-carboxyethyl)phosphine, and 0.01% Tween 20) to the top concentrations in the dilution series. From this, a 2-fold dilution series was made, and the peptides were injected over the surface at a flow rate of 100 μl/min. Raw sensogram data were reduced and double referenced using the Scrubber II software package (BioLogic Software, Campbell, Australia). Equilibrium fits to a 1:1 binding model were insufficient to describe the data, but data were well described by a 2-site equilibrium model, reporting two KD values. Data for the γ chain of the FcϵRI receptor (FcϵRIγ) ITAM exhibited measurable kinetics and were fit using the CLAMP99 software package (23Morton T.A. Myszka D.G. Methods Enzymol. 1998; 295: 268-294Crossref PubMed Scopus (267) Google Scholar). KD values derived from the kinetics are very similar to those obtained from the equilibrium fits. Expression and Purification of Truncated Syk Protein—Truncated versions of human Syk were cloned into a PVL 1392 vector and expressed by baculovirus in Sf9 insect cells. Cells were harvested 3 days after infection and resuspended in 250 ml of disruption buffer (50 mm Hepes pH 7.5, 150 mm NaCl, 5% glycerol, 200 mm arginine, 0.1% Ipegal) and disrupted on ice using a microfluidizer with 2 passes at 15,000 p.s.i. Supernatant was then collected after centrifugation at 13,000 × g, and the lysate was filtered using a 0.45-μm SuporCap Capsule. Material was then incubated for 3 h with a nickel-Sepharose FF resin that had been pre-equilibrated in disruption buffer with 20 mm imidazole. Resin was collected at 1000 × g and then washed 4 times using disruption buffer before bound proteins were eluted twice with 20 ml of elution buffer (25 mm Hepes pH 7.5, 5% glycerol, 150 mm NaCl, 10 mm methionine, 200 mm imidazole). The sample was then concentrated to 5 mg/ml and passed through a His-trap column to remove uncleaved protein. Flow-through was collected and concentrated to 2 ml using a Superdex XK16/60 column equilibrated with buffer (50 mm Hepes, pH 7.5, 150 mm NaCl, 5 mm DTT, 10 mm methionine), and samples were run at 1 ml/min. 1-min fractions were then collected before being analyzed on 8–16% Tris-glycine gels. The clean fraction was pooled and concentrated to 1 mg/ml and stored at −80 °C. Zap-70 Activation—The enzyme activity of Zap-70 was assessed using 10 μm Sox-containing substrate pep8 at a final concentration of 50 nm Zap-70. Other buffer conditions included 20 mm Hepes, pH 7.15, 0.1 mm EGTA, 0.1 mm DTT, 0.5 mg/ml bovine serum albumin, 10 mm MgCl2, 10 μm sodium vanadate, and typically 500 μm ATP using a final assay volume of 50 μl. To preactivate Zap-70 before enzymatic analysis, Zap-70 was incubated with 1 mm ATP for varying times at 30 °C in a buffer of 20 mm Hepes, pH 7.15, 0.1 mm EGTA, 0.1 mm DTT, 10 mm MgCl2, and 10 μm sodium vanadate. Note that the requirement for a high ATP concentration in the preactivation conditions prevents determination of Zap-70 activity at very low ATP concentration in the Michaelis-Menten analysis of Zap-70. Zap-70 samples were analyzed by Western blot as described above for Syk using the dual Syk 348/352 and Zap315/319 phosphospecific antibody. Here, total Zap-70 concentration was determined by Coomassie staining. To assess how the activity of Zap-70 and Syk is modulated by Src family kinases, 10 nm concentrations of either Lck or Lyn was added to the standard enzyme activity mixture for either Zap-70 or Syk, respectively, and the time course of product formation was monitored. Monitoring Syk Activity in Ramos B Cell Lysates—Ramos B cells were cultured in media containing RPMI 1640 with l-glutamate, 10% fetal bovine serum, and 1 mm sodium pyruvate and incubated in 5% CO2 at 37 °C. To prepare lysates, cells were harvested at a density of 1 million cells/ml by centrifugation at 100 × g for 5 min. Cells were then resuspended at 100 million cells/ml in RPMI 1640 and 10 mm Hepes, pH 7.4. Cells were then incubated for 10 min at 27 °C and centrifuged at 600 × g for 10 min, and the pellet was snap-frozen at stored at −80 °C. On the day of the experiment, cells were reconstituted and lysed with cold buffer containing 0.5% Triton-X, 50 mm Tris, pH 7.5, 150 mm NaCl, and protease inhibitor tablet. Samples were centrifuged at 3600 × g for 10 min at 4 °C. The supernatant and pellet were both retained. For activity analysis in whole lysates, a dilution of the final prepared Ramos lysate of 1:10 was typically employed into the standard enzyme assay buffer containing sodium vanadate. To isolate endogenous Syk by immunoprecipitation for analysis of activity, lysates were incubated with the total Syk monoclonal antibody at 1:100 for 1 h at 4 °C then with protein A-conjugated agarose beads (Thermo Scientific) for 1 h at 4 °C, and the beads were isolated by centrifugation at 1000 × g. Phosphorylation-dependent Syk Activity—In this study we first set out to explore the relationship between Syk kinase activity and the phosphorylation state of the kinase. To monitor the enzymatic activity of Syk, a recently developed fluorescence kinase assay was employed. This assay monitors phosphorylation of a peptide substrate, pep7, in real time using the change in fluorescence of a non-natural amino acid (the "Sox" amino acid) incorporated directly into the peptide substrate (19Shults M.D. Carrico-Moniz D. Imperiali B. Anal. Biochem. 2006; 352: 198-207Crossref PubMed Scopus (66) Google Scholar, 20Shults M.D. Janes K.A. Lauffenburger D.A. Imperiali B. Nat. Methods. 2005; 2: 277-283Crossref PubMed Scopus (182) Google Scholar). Utilizing Syk that was not treated after purification ("basal Syk"), it was found that product formation was initially linear with time and then plateaued after substrate was completely converted to product (Fig. 1A). In contrast to basal Syk, Syk that was treated with protein-tyrosine phosphatase PTP1B before activity analysis (dephos Syk) demonstrated a clear lag phase in product formation (Fig. 1A). This nonlinear time course is consistent with Syk being in a less active conformation at the outset of the experiment, which then converts to a more active conformation during the reaction due to autophosphorylation. If PTP1B-treated Syk was incubated with Mg2+/ATP before activity analysis (autophos Syk), the lag in the reaction time course was eliminated (Fig. 1A). The product formation rate (calculated from the initial velocity) of the basal Syk, dephos Syk, and autophos Syk conditions were determined, respectively, to be 33 ± 1, 2.8 ± 0.6, and 52 ± 1 min-1 (Table 1), indicating that phosphorylation causes a greater than 10-fold increase in Syk activity.TABLE 1Activity of Syk in different statesEnzyme formITAM peptidekcatmin–1Basal SykNone33 ± 1Dephos SykNone2.8 ± 0.6Autophos SykNone52 ± 1Dephos SykBCR50 ± 1Dephos SykFcϵRIγ56 ± 1Dephos SykFcγRIIa51 ± 1Autophos SykBCR54 ± 1 Open table in a new tab The preceding results suggest that autophosphorylation increases Syk activity. To confirm that Syk undergoes a change in phosphorylation after treatment with PTP1B, we used Western blotting with phosphospecific antibodies. Antibodies against Tyr-352 and Tyr-525/526 were employed. Both the basal and autophosphorylated forms of Syk were observed to be phosphorylated at Tyr-352 and Tyr-525/526, whereas neither site showed significant phosphorylation in the PTP1B-treated sample (Fig. 1B). Western blotting also demonstrated that, during the course of an enzyme assay, PTP1B-treated Syk became autophosphorylated at both Tyr-352 and Tyr-525/526 (Fig. 1C), confirming that the increase in Syk activity over time observed is attributable to Syk autophosphorylation. Together, these findings indicate that 1) dephosphorylated Syk has low activity toward the pep7 substrate, 2) Syk can autophosphorylate and autophosphorylation increases Syk activity, and 3) a lag phase in product formation reflects Syk being in the inactive conformation at the outset of the experiment. ITAM Activation of Syk—In the cell Syk binds dual-phosphorylated regions within receptors known as ITAM motifs. To explore if binding of phosphorylated ITAM sequences activates Syk in vitro, we studied three dually phosphorylated ITAM peptides based on Syk docking sites on the B cell receptor (BCR), FcϵRIγ, and FcγRIIa receptor (Fig. 2A). Incubation of dephosphorylated Syk with either 1 μm BCR, FcϵRIγ, or FcγRIIa peptide eliminated the lag phase in PTP1B-treated Syk activity and resulted in a linear rate of product formation (see Fig. 2B and supplemental Fig. 2, A and B), indicating that ITAM binding activates Syk. The product formation rate under each condition was determined to be 50 ± 1, 56 ± 1, and 51 ± 1 min-1 for the BCR, FcϵRIγ, and FcγRIIa peptides, respectively (Table 1). To ascertain the potency of Syk for the ITAM peptides, the concentration of each peptide was varied, whereas the initial component of the reaction velocity was monitored. Fitting the data to a sigmoidal dose-response function ascertained that BCR, FcϵRIγ, and FcγRIIa peptides had EC50 values of 72 ± 6, 5 ± 1, and 14 ± 1 nm, respectively (See Table 2). This finding indicates that Syk is potently activated by binding of dually phosphorylated ITAM sequences and that the sequence context of the ITAM peptide can modulate Syk potency. It should be noted that above 10 μm ITAM concentration we observed that each peptide began to inhibit Syk activity (see Fig. 2C and below).TABLE 2Potency of ITAM phosphopeptides for SykITAM peptideEnzyme assay EC50Surface plasmon resonanceKd1Kd2μmμmμmBCR0.072 ± 0.0060.64 ± 0.2732 ± 26FCϵRIγ0.005 ± 0.0010.038 ± 0.0210.64 ± 0.19FCγRIIa0.014 ± 0.0010.040 ± 0.0141.9 ± 0.15 Open table in a new tab To directly characterize the binding interaction between ITAM peptides and Syk, surface plasmon resonance was employed (see supplemental Fig. 3). In these experiments all 3 ITAM peptides were observed to bind to PTP1B-treated Syk with 2 binding modes; a potent binding mode and a weak binding mode. For the more potent binding mode, a potency pattern similar to the enzyme activation assay was obtained; the Kd values of FcϵRIγ and FcγRIIa were similar (Kd ∼ 40 nm), whereas the BCR peptide was 10-fold weaker (Table 2). Potent binding presumably occurs between the ITAM peptide and the tandem SH2 domains of Syk. The weaker binding mode observed in the surface plasmon resonance experiments demonstrated Kd values in the range of 0.64–32 μm (Table 2). The observation of a weak binding mode between Syk and ITAM peptides in surface plasmon resonance experiments (Table 2) and the finding of inhibition of Syk activity at high ITAM peptide concentrations (Fig. 3C) together suggest that ITAM peptides can weakly interact with Syk in a previously uncharacterized way that inhibits enzyme activity. Syk Activation Is an OR-gate Switch—The experiments above indicate that autophosphorylation and ITAM binding each activate Syk by the same magnitude (Table 1). However, they do not reveal whether phosphorylation and ITAM binding activate through the same mechanism or different mechanisms. If phosphorylation and ITAM binding operate through different mechanisms, it would be expected that simultaneous application of both stimuli could further increase Syk activity. In contrast, if both stimuli worked through the same mechanism, application of both autophosphorylation and ITAM binding would be expected to result in little further increase in activity. The latter type of mechanism is referred to as an OR-gate switch as either one stimulus OR the other is sufficient to cause full activation (17Dueber J.E. Yeh B.J. Chak K. Lim W.A. Science. 2003; 301: 1904-1908Crossref PubMed Scopus (243) Google Scholar, 24Anderson J.C. Voigt C.A. Arkin A.P. Mol. Syst. Biol. 2007; 3: 133Crossref PubMed Scopus (273) Google Scholar, 25Bhattacharyya R.P. Remenyi A. Yeh B.J. Lim W.A. Annu. Rev. Biochem. 2006; 75: 655-680Crossref PubMed Scopus (365) Google Scholar). An OR-gate switch is distinguished from an "AND-gate" switch where multiple stimuli must be present simultaneously to get activation of the molecular switch (25Bhattacharyya R.P. Remenyi A. Yeh B.J. Lim W.A. Annu. Rev. Biochem. 2006; 75: 655-680Crossref PubMed Scopus (365) Google Scholar, 26Prehoda K.E. Lim W.A. Curr. Opin. Cell Biol. 2002; 14: 149-154Crossref PubMed Scopus (50) Google Scholar). To ascertain the relationship between autophosphorylation and ITAM binding, the activity of autophosphorylated Syk in the presence of ITAM peptide was evaluated. The activity in the presence of both stimuli was observed to be identical to either stimuli individually (Fig. 3A, Table 1). This finding indicates that Syk activation is an OR-gate switch and implies that ITAM binding and autophosphorylation activate through the same molecular mechanism (see "Discussion"). Identical results were obtained with each ITAM studied and using multiple autophosphorylation conditions (data not shown). The ITAM-stimulated increase in Syk activity could potentially be due to 2 different molecular mechanisms; that is, direct stimulation of Syk activity by ITAM or rapid ITAM-mediated autophosphorylation. To address if ITAM binding directly stimulates Syk activity, we monitored the time course of autophos
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