Auto-thiophosphorylation activity of Src tyrosine kinase
© The Author(s). 2016
Received: 5 April 2016
Accepted: 29 June 2016
Published: 7 July 2016
Intermolecular autophosphorylation at Tyr416 is a conserved mechanism of activation among the members of the Src family of nonreceptor tyrosine kinases. Like several other tyrosine kinases, Src can catalyze the thiophosphorylation of peptide and protein substrates using ATPγS as a thiophosphodonor, although the efficiency of the reaction is low.
Here, we have characterized the ability of Src to auto-thiophosphorylate. Auto-thiophosphorylation of Src at Tyr416 in the activation loop proceeds efficiently in the presence of Ni2+, resulting in kinase activation. Other tyrosine kinases (Ack1, Hck, and IGF1 receptor) also auto-thiophosphorylate in the presence of Ni2+. Tyr416-thiophosphorylated Src is resistant to dephosphorylation by PTP1B phosphatase.
Src and other tyrosine kinases catalyze auto-thiophosphorylation in the presence of Ni2+. Thiophosphorylation of Src occurs at Tyr416 in the activation loop, and results in enhanced kinase activity. Tyr416-thiophosphorylated Src could serve as a stable, persistently-activated mimic of Src.
Autophosphorylation is a common mechanism by which the activities of eukaryotic protein kinases are controlled [1, 2]. The canonical protein kinase fold consists of two lobes separated by a deep cleft into which ATP binds . Protein and peptide substrates bind in an extended conformation at the entrance to this cleft. A flexible protein segment between the lobes called the “activation loop” interacts with protein and peptide substrates. In many serine/threonine kinases (e.g., PKA), and tyrosine kinases (e.g., Src), this loop contains one or more phosphorylation sites. Autophosphorylation within the activation loop stabilizes a conformation that allows substrate binding, and promotes kinase activity [1–3]. In principle, autophosphorylation can be either an intermolecular reaction between two kinase molecules (also called autophosphorylation “in trans”) or an intramolecular reaction within one kinase (“cis”). For tyrosine kinases where this has been examined explicitly, autophosphorylation is intermolecular [4–6]. This mode of kinase regulation appears to be evolutionarily ancient, as (for example) Src kinases from unicellular choanoflagellates and filastereans are activated by autophosphorylation at the position corresponding to Tyr416 of c-Src [7, 8] (chicken Src numbering is used throughout this paper). The number and positioning of phosphate residues within the activation loop varies from kinase to kinase.
Several serine/threonine and tyrosine protein kinases have the ability to catalyze thiophosphorylation of peptide and protein substrates (by the use of ATPγS rather than ATP as cosubstrate) [9–12]. Thiophosphorylated proteins are more metabolically stable than their phosphorylated counterparts; in particular, they are resistant to cellular phosphatases [13, 14]. This has facilitated their use in proteomic investigations of kinase activity, where some phosphoproteins are intrinsically unstable, or are modified at low stoichiometry . The thiophosphoryl group can also be further functionalized for proteomic studies , or for specific “caging” reactions to produce molecules that are released upon photolysis .
Although a number of protein kinases have the capacity to use ATPγS as a phosphodonor, the kinetic efficiency of protein kinase reactions with ATPγS is typically much lower than that for comparable reactions with ATP. This is particularly true for tyrosine kinases. To circumvent this problem, we and others have shown that tyrosine kinases can catalyze thiophosphorylation of peptide substrates in the presence of divalent transition metals (e.g., Co2+ or Ni2+) in the reaction buffer rather than magnesium [10, 16]. This is thought to be due to the increased relative affinity of Co2+ or Ni2+ toward sulfur in nucleotide complexes, as compared with Mg2+, which has a strong preference for binding oxygen over sulfur [17, 18]. Thus, for Csk tyrosine kinase, kcat for substrate phosphorylation was comparable for ATP vs. ATPγS in the presence of thiophilic divalent metals, but kcat for ATPγS was greatly reduced in the presence of magnesium or manganese. This was attributed to the important role of γ-phosphoryl bonding and salt bridging in the Csk reaction transition state . Similarly, we showed that an SH2-binding peptide could be thiophosphorylated by Hck kinase in the presence of cobalt .
Previous work in this area has focused primarily on the kinase-catalyzed thiophosphorylation of peptide substrates. Gel-based methods have been used to demonstrate incorporation of thiophosphate into kinase substrates and kinases, including Src and Abl [19, 20]. Here, we have characterized the ability of Src to catalyze intermolecular auto-thiophosphorylation. We report that auto-thiophosphorylation of Src at Tyr416 in the activation loop proceeds efficiently in the presence of Ni2+, stabilizing the active conformation and resulting in kinase activation. Tyr416-thiophosphorylated Src is resistant to dephosphorylation by PTP1B phosphatase, and could serve as a stable, persistently-activated mimic of Src.
Next, we wished to determine whether Src kinase could catalyze intermolecular auto-thiophosphorylation (as opposed to phosphorylation of an exogenous peptide substrate). The Src kinase used for these experiments was purified from bacteria, and contains very low levels of phosphorylation . Using the continuous assay, we detected auto-thiophosphorylation activity in the presence of Ni2+ (Fig. 1b). The initial rate of auto-thiophosphorylation (0–100 s) was similar to that observed for Src autophosphorylation in the presence of ATP and Mg2+ (Fig. 1b); after 600 s, the overall rate of the NiATPγS reaction was roughly one-half that of the MgATP reaction. We observed minimal activity using ATPγS as cosubstrate with Mg2+ as the divalent cation. This is consistent with earlier studies in which MgATPγS did not support autoactivation of Src, and acted as a competitive inhibitor versus MgATP with Ki = 23 μM .
We report here that Src and other tyrosine kinases (including examples of receptor and nonreceptor tyrosine kinases) can catalyze auto-thiophosphorylation. The reaction is greatly enhanced in the presence of Ni2+. For Src, thiophosphorylation takes place primarily at Tyr416 within the activation loop, and produces a form of Src that is active and relatively resistant to the action of tyrosine phosphatases.
The stoichiometry of thiophosphorylation after 30 min was 0.91 mol/mol (Fig. 3b). The structure of thiophosphorylated Src suggests that this modification should increase enzymatic activity, and we have confirmed that this is the case. The activity of auto-thiophosphorylated Src is significantly higher than that of unphosphorylated Src, as measured toward a synthetic peptide substrate (Fig. 5). Thiophosphorylated Src had activity that was roughly one half that of phosphorylated Src (Fig. 5a). This may reflect subtle differences in the conformation of phosphorylated vs. thiophosphorylated Tyr416 that result in changes in catalytic efficiency.
Thiophosphorylated Src was resistant to dephosphorylation by PTP1B tyrosine phosphatase (Fig. 6). This is consistent with earlier reports on a wide variety of tyrosine phosphatases; PTPs are able to bind peptides and proteins containing thiophosphorylated tyrosine, but the catalytic rates of dephosphorylation are sluggish [13, 22, 23]. Thiophosphotyrosyl analogs of substrates bind to the active sites of PTPs, and act as competitive inhibitors. Acidic residues, such as those found N-terminal to Tyr416 of Src (sequence: Glu-Asp-Asn-Glu-Tyr) are often important specificity determinants for binding to PTPs [23, 28].
We and others have previously observed that the inclusion of thiophilic divalent cations such as Co2+ or Ni2+ enhances the thiophosphorylation activity of protein kinases [10, 16]. Inclusion of Mn2+ together with Mg2+ resulted in high levels of Abl thiophosphorylation, even in the presence of micromolar concentrations of ATP, a development that could allow the study of thiophosphorylation in cell extracts . Previous studies of tyrosine kinases focused on the ability of kinases to thiophosphorylate exogenous substrates. There is one previous study of the functional consequences of auto-thiophosphorylation by a eukaryotic protein kinase. The Ser/Thr kinase calmodulin-dependent protein kinase II (CaM-kinase II) is thiophosphorylated at Thr286 and Thr287 upon reaction with ATPγS . The kinetic properties of thiophosphorylated CaM-kinase II were found to be similar to those of the phosphorylated enzyme. The stability of the thiophosphate linkage allowed the investigators to show that autophosphorylation is required for full enzyme activation . In a similar manner, thiophosphorylated derivatives could serve as stable, persistently-activated mimics of tyrosine kinases.
We show that: (1) In the presence of Ni2+, Src and other tyrosine kinases catalyze auto-thiophosphorylation using ATPγS as a phosphodonor; (2) Auto-thiophosphorylation of Src occurs predominantly at Tyr416 in the activation loop; (3) Src auto-thiophosphorylation increases the enzyme’s catalytic activity; (4) Tyr416-thiophosphorylated Src is resistant to dephosphorylation by PTP1B phosphatase, and could serve as a stable, persistently-activated mimic of Src.
The catalytic domains of Src and Hck were expressed in bacteria and purified as previously described by Seeliger, Kuriyan, and colleagues . The catalytic domains of Ack1 and IGF1R kinases were expressed in Sf9 cells using recombinant baculoviruses, as previously described [29, 30]. ATP, adenosine 5′-(3-thiotriphosphate) (ATPγS), and PK/LDH were purchased from Sigma. The anti-Src (pY419; equivalent to chicken c-Src pY416) antibody was from Biosource, and anti-pTyr antibody (4G10) was from Millipore. [35S]-labeled ATPγS was from Perkin-Elmer. Dithiothreitol (DTT), acetonitrile (ACN), ammonium bicarbonate, trifluoroacetic acid (TFA), and iodoacetamide (IAA) were from Thermo Fisher Scientific (Waltham, MA). Trypsin Gold, mass spectrometry grade, was from Promega (Madison, WI). Tris-HCl (10 %) non-denaturing gels were purchased from Bio-Rad.
Two kinase assays were employed : (1) Continuous kinase assays were performed by a coupled spectrophotometric assay . In this assay, the production of ADP is coupled to the oxidation of NADH measured as a reduction in absorbance at 340 nm. All experiments were carried out at 30 °C. Reactions were performed in buffer containing 100 mM Tris pH 7.5, 1 mM phosphoenolpyruvate, 0.28 mM NADH, 89 units/ml pyruvate kinase and 124 units/ml lactate dehydrogenase, with varying concentrations of enzyme and divalent cations. In some experiments, a peptide substrate (AEEEIYGEFEAKKKKG) was included. (2) Peptide phosphorylation was also measured using [γ-32P]-ATP and a phosphocellulose paper binding assay . Reactions were performed in 20 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 0.25 mM ATP, varying concentrations of peptide substrate, and [γ-32P] -ATP (100–500 cpm/pmol).
Src kinase (3.2 μM) was incubated with 2 mM ATPγS and 3 mM NiCl2 for 1 h, 45 min at 30°. A control sample was prepared by carrying out a similar reaction without ATPγS. Both samples were analyzed by SDS-PAGE. Gel bands were cut out, destained and digested with trypsin essentially as described  with minor modifications. The gel bands were not reduced and alkylated as iodoacetamide also reacted with the thiol group in the thiophosphate and produced a more complicated fragmentation pattern in the mass spectrometer.
The dried peptide mix was reconstituted in a solution of 2 % acetonitrile (ACN), 2 % formic acid (FA) for MS analysis. Peptides were loaded with the autosampler directly onto a 2 cm C18 PepMap pre-column (Thermo Scientific, San Jose, CA) which was attached to a 50 cm EASY-Spray C18 column (Thermo Scientific). Peptides were eluted from the column using a Dionex Ultimate 3000 Nano LC system with a 10 min gradient from 2 % buffer B to 35 % buffer B (100 % acetonitrile, 0.1 % formic acid). The gradient was switched from 35 to 85 % buffer B over 1 min and held constant for 2 min. Finally, the gradient was changed from 85 % buffer B to 98 % buffer A (100 % water, 0.1 % formic acid) over 1 min, and then held constant at 98 % buffer A for 5 more minutes. The application of a 2.0 kV distal voltage electrosprayed the eluting peptides directly into the Thermo Fusion Tribrid mass spectrometer equipped with an EASY-Spray source (Thermo Scientific). Initial experiments were performed to identify potential phosphorylation and thiophosphorylation sites by running each digest separately with a data dependent method to acquire as many MS/MS in a 3 s span. This data was analyzed for the presence of phosphorylation and thiophosphorylation and subsequently the samples were rerun in which the mass spectrometer was set to a targeted analysis method to only acquire CID MS/MS of the expected unphosphorylated (m/z 612.29 and 617.81, for peptide LIEDNEYTAR and WTAPEAALYGR, respectively) and phosphorylated (m/z 652.28 and 657.79) and thiophosphorylated (m/z 660.27 and 665.79) peptides. These MS/MS scans were acquired in the Orbitrap at 15,000 resolution with a scan range of m/z 200–1300 and 200–1200, respectively. Mass spectrometer-scanning functions and HPLC gradients were controlled by the Xcalibur data system (Thermo Scientific). The acquired MS data were analyzed manually to confirm the precursor mass, fragmentation ions, and phosphorylations and thiophosphorylations in the targeted peptides.
Reactions were separated on 10 % SDS-PAGE, and transferred onto a polyvinylidene difluoride membrane. Proteins were detected by Western blotting with antiphosphotyrosine and anti-pY419 antibodies.
Native gel analysis
Src kinase (8.5 μM) was incubated in 20 mM Tris-HCl pH 7.5 at 30 °C with either (1) 5 mM ATPγS, 10 mM NiCl2 or (2) 5 mM ATP, 10 mM MgCl2. Reactions were stopped at various time points by addition of 100 mM EDTA and analyzed by native PAGE using 10 % Tris–HCl gels. The thiophosphorylated and phosphorylated forms of Src were visualized by staining with Coomassie blue.
Reactions (30 °C) contained Src kinase (1 μM), 30 mM Tris-HCl (pH 7.5), 10 mM NiCl2, and [35S]-labeled ATPγS (30 pmol). Aliquots of the reactions were withdrawn at various time points, and 35S-labeled Src was spotted onto Whatman 3MM paper, washed with 5 % trichloroacetic acid at 55 °C, and counted by liquid scintillation counting .
Ack1, activated Cdc42-associated kinase; ACN, acetonitrile; ATPγS, adenosine 5′-O-(3-thio)triphosphate; CaM, calcium/calmodulin; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; EGFR, epidermal growth factor receptor; FA, formic acid; Hck, hematopoietic cell kinase; HPLC, high pressure liquid chromatography; IAA, iodoacetamide; IGF1R, insulin-like growth factor 1 receptor; MS, mass spectrometry; NADH, nicotinamide adenine dinucleotide; PK/LDH, pyruvate kinase/lactate dehydrogenase; PKA, protein kinase A; PTP1B, protein tyrosine phosphatase 1B; TFA, trifluoroacetic acid
We thank Dr. Markus Seeliger (Stony Brook University) for helpful discussions.
This work was supported by NIH grant CA58530 (to W.T.M.).
Availability of data and materials
The data supporting the conclusions of this article are included within the article and supporting files. Materials are available upon request.
Availability of data and materials
The data supporting the conclusions of this article are included within the article and supporting files. Materials are available upon request.
MZC carried out thiophosphorylation experiments and assisted in drafting the manuscript. EIC and AK carried out mass spectrometry experiments, analyzed MS data, and assisted in drafting the manuscript. WTM conceived the study, carried out thiophosphorylation experiments, coordinated the study, and drafted the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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- Huse M, Kuriyan J. The conformational plasticity of protein kinases. Cell. 2002;109(3):275–82.View ArticlePubMedGoogle Scholar
- Johnson LN, Noble ME, Owen DJ. Active and inactive protein kinases: structural basis for regulation. Cell. 1996;85(2):149–58.View ArticlePubMedGoogle Scholar
- Taylor SS, Kornev AP. Protein kinases: evolution of dynamic regulatory proteins. Trends Biochem Sci. 2011;36(2):65–77.View ArticlePubMedGoogle Scholar
- Moarefi I, LaFevre-Bernt M, Sicheri F, Huse M, Lee CH, Kuriyan J, Miller WT. Activation of the Src-family tyrosine kinase Hck by SH3 domain displacement. Nature. 1997;385(6617):650–3.View ArticlePubMedGoogle Scholar
- Favelyukis S, Till JH, Hubbard SR, Miller WT. Structure and autoregulation of the insulin-like growth factor 1 receptor kinase. Nat Struct Biol. 2001;8(12):1058–63.View ArticlePubMedGoogle Scholar
- Barker SC, Kassel DB, Weigl D, Huang X, Luther MA, Knight WB. Characterization of pp60c-src tyrosine kinase activities using a continuous assay: autoactivation of the enzyme is an intermolecular autophosphorylation process. Biochemistry. 1995;34(45):14843–51.View ArticlePubMedGoogle Scholar
- Li W, Young SL, King N, Miller WT. Signaling Properties of a Non-metazoan Src Kinase and the Evolutionary History of Src Negative Regulation. J Biol Chem. 2008;283(22):15491–501.View ArticlePubMedPubMed CentralGoogle Scholar
- Schultheiss KP, Suga H, Ruiz-Trillo I, Miller WT. Lack of Csk-Mediated Negative Regulation in a Unicellular Src Kinase. Biochemistry. 2012;51:8267–77.View ArticlePubMedPubMed CentralGoogle Scholar
- Allen JJ, Lazerwith SE, Shokat KM. Bio-orthogonal affinity purification of direct kinase substrates. J Am Chem Soc. 2005;127(15):5288–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Grace MR, Walsh CT, Cole PA. Divalent ion effects and insights into the catalytic mechanism of protein tyrosine kinase Csk. Biochemistry. 1997;36(7):1874–81.View ArticlePubMedGoogle Scholar
- Jeong S, Nikiforov TT. Kinase assay based on thiophosphorylation and biotinylation. Biotechniques. 1999;27(6):1232–8.PubMedGoogle Scholar
- Ikeda A, Okuno S, Fujisawa H. Studies on the generation of Ca2+/calmodulin-independent activity of calmodulin-dependent protein kinase II by autophosphorylation. Autothiophosphorylation of the enzyme. J Biol Chem. 1991;266(18):11582–8.PubMedGoogle Scholar
- Cassel D, Glaser L. Resistance to phosphatase of thiophosphorylated epidermal growth factor receptor in A431 membranes. Proc Natl Acad Sci U S A. 1982;79(7):2231–5.View ArticlePubMedPubMed CentralGoogle Scholar
- Ishida A, Kitani T, Fujisawa H. Evidence that autophosphorylation at Thr-286/Thr-287 is required for full activation of calmodulin-dependent protein kinase II. Biochim Biophys Acta. 1996;1311(3):211–7.View ArticlePubMedGoogle Scholar
- Parker LL, Schilling AB, Kron SJ, Kent SB. Optimizing thiophosphorylation in the presence of competing phosphorylation with MALDI-TOF-MS detection. J Proteome Res. 2005;4(5):1863–6.View ArticlePubMedPubMed CentralGoogle Scholar
- Zou K, Miller WT, Givens RS, Bayley H. Caged thiophosphotyrosine peptides. Angew Chem Int Ed Engl. 2001;40(16):3049–51.View ArticlePubMedGoogle Scholar
- Cohn M, Shih N, Nick J. Reactivity and metal-dependent stereospecificity of the phosphorothioate analogs of ATP in the arginine kinase reaction. Structure of the metal-nucleoside triphosphate substrate. J Biol Chem. 1982;257(13):7646–9.PubMedGoogle Scholar
- Pecoraro VL, Hermes JD, Cleland WW. Stability constants of Mg2+ and Cd2+ complexes of adenine nucleotides and thionucleotides and rate constants for formation and dissociation of MgATP and MgADP. Biochemistry. 1984;23(22):5262–71.View ArticlePubMedGoogle Scholar
- Garber KC, Carlson EE. Thiol-ene enabled detection of thiophosphorylated kinase substrates. ACS Chem Biol. 2013;8(8):1671–6.View ArticlePubMedPubMed CentralGoogle Scholar
- Allen JJ, Li M, Brinkworth CS, Paulson JL, Wang D, Hubner A, Chou WH, Davis RJ, Burlingame AL, Messing RO. A semisynthetic epitope for kinase substrates. Nat Methods. 2007;4(6):511–6.Google Scholar
- Seeliger MA, Young M, Henderson MN, Pellicena P, King DS, Falick AM, Kuriyan J. High yield bacterial expression of active c-Abl and c-Src tyrosine kinases. Protein Sci. 2005;14(12):3135–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Tonks NK, Diltz CD, Fischer EH. Purification of the major protein-tyrosine-phosphatases of human placenta. J Biol Chem. 1988;263(14):6722–30.PubMedGoogle Scholar
- Hiriyanna KT, Baedke D, Baek KH, Forney BA, Kordiyak G, Ingebritsen TS. Thiophosphorylated substrate analogs are potent active site-directed inhibitors of protein-tyrosine phosphatases. Anal Biochem. 1994;223(1):51–8.View ArticlePubMedGoogle Scholar
- Bjorge JD, Jakymiw A, Fujita DJ. Selected glimpses into the activation and function of Src kinase. Oncogene. 2000;19(49):5620–35.View ArticlePubMedGoogle Scholar
- Roskoski Jr R. Src kinase regulation by phosphorylation and dephosphorylation. Biochem Biophys Res Commun. 2005;331(1):1–14.View ArticlePubMedGoogle Scholar
- Azam M, Seeliger MA, Gray NS, Kuriyan J, Daley GQ. Activation of tyrosine kinases by mutation of the gatekeeper threonine. Nat Struct Mol Biol. 2008;15(10):1109–18.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhu X, Kim JL, Newcomb JR, Rose PE, Stover DR, Toledo LM, Zhao H, Morgenstern KA. Structural analysis of the lymphocyte-specific kinase Lck in complex with non-selective and Src family selective kinase inhibitors. Structure. 1999;7(6):651–61.View ArticlePubMedGoogle Scholar
- Zhang ZY, Walsh AB, Wu L, McNamara DJ, Dobrusin EM, Miller WT. Determinants of substrate recognition in the protein-tyrosine phosphatase, PTP1. J Biol Chem. 1996;271(10):5386–92.View ArticlePubMedGoogle Scholar
- Prieto-Echague V, Gucwa A, Craddock BP, Brown DA, Miller WT. Cancer-associated mutations activate the nonreceptor tyrosine kinase Ack1. J Biol Chem. 2010;285(14):10605–15.View ArticlePubMedPubMed CentralGoogle Scholar
- Cabail MZ, Li S, Lemmon E, Bowen ME, Hubbard SR, Miller WT. The insulin and IGF1 receptor kinase domains are functional dimers in the activated state. Nat Commun. 2015;6:6406.View ArticlePubMedPubMed CentralGoogle Scholar
- Casnellie JE. Assay of protein kinases using peptides with basic residues for phosphocellulose binding. Methods Enzymol. 1991;200:115–20.View ArticlePubMedGoogle Scholar
- Shevchenko A, Jensen ON, Podtelejnikov AV, Sagliocco F, Wilm M, Vorm O, Mortensen P, Shevchenko A, Boucherie H, Mann M. Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels. Proc Natl Acad Sci U S A. 1996;93(25):14440–5.Google Scholar