Cabail et al. BMC Biochemistry (2016) 17:13 DOI 10.1186/s12858-016-0071-z RESEARCH ARTICLE Open Access Auto-thiophosphorylation activity of Src tyrosine kinase M. Zulema Cabail1,2, Emily I. Chen3,4, Antonius Koller3 and W. Todd Miller1* Abstract Background: 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. Results: 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. Conclusions: 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. Keywords: Tyrosine kinase, Src, Thiophosphate, Autophosphorylation, Phosphatase Background appears to be evolutionarily ancient, as (for example) Src Autophosphorylation is a common mechanism by which kinases from unicellular choanoflagellates and filaster- the activities of eukaryotic protein kinases are controlled eans are activated by autophosphorylation at the position [1, 2]. The canonical protein kinase fold consists of two corresponding to Tyr416 of c-Src [7, 8] (chicken Src lobes separated by a deep cleft into which ATP binds numbering is used throughout this paper). The number [3]. Protein and peptide substrates bind in an extended and positioning of phosphate residues within the activa- conformation at the entrance to this cleft. A flexible pro- tion loop varies from kinase to kinase. tein segment between the lobes called the “activation Several serine/threonine and tyrosine protein kinases loop” interacts with protein and peptide substrates. In have the ability to catalyze thiophosphorylation of pep- many serine/threonine kinases (e.g., PKA), and tyrosine tide and protein substrates (by the use of ATPγS rather kinases (e.g., Src), this loop contains one or more phos- than ATP as cosubstrate) [9–12]. Thiophosphorylated phorylation sites. Autophosphorylation within the activa- proteins are more metabolically stable than their phos- tion loop stabilizes a conformation that allows substrate phorylated counterparts; in particular, they are resistant binding, and promotes kinase activity [1–3]. In principle, to cellular phosphatases [13, 14]. This has facilitated autophosphorylation can be either an intermolecular their use in proteomic investigations of kinase activity, reaction between two kinase molecules (also called auto- where some phosphoproteins are intrinsically unstable, phosphorylation “in trans”) or an intramolecular reaction or are modified at low stoichiometry [15]. The thiopho- within one kinase (“cis”). For tyrosine kinases where this sphoryl group can also be further functionalized for prote- has been examined explicitly, autophosphorylation is omic studies [9], or for specific “caging” reactions to intermolecular [4–6]. This mode of kinase regulation produce molecules that are released upon photolysis [16]. Although a number of protein kinases have the * Correspondence: [email protected] capacity to use ATPγS as a phosphodonor, the kinetic 1 Department of Physiology and Biophysics, School of Medicine, Stony Brook efficiency of protein kinase reactions with ATPγS is typ- University, Stony Brook, NY 11794, USA Full list of author information is available at the end of the article ically much lower than that for comparable reactions © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Cabail et al. BMC Biochemistry (2016) 17:13 Page 2 of 10 with ATP. This is particularly true for tyrosine kinases. substrates and kinases, including Src and Abl [19, 20]. To circumvent this problem, we and others have shown Here, we have characterized the ability of Src to catalyze that tyrosine kinases can catalyze thiophosphorylation of intermolecular auto-thiophosphorylation. We report that peptide substrates in the presence of divalent transition auto-thiophosphorylation of Src at Tyr416 in the activa- metals (e.g., Co2+ or Ni2+) in the reaction buffer rather tion loop proceeds efficiently in the presence of Ni2+, than magnesium [10, 16]. This is thought to be due to stabilizing the active conformation and resulting in the increased relative affinity of Co2+ or Ni2+ toward kinase activation. Tyr416-thiophosphorylated Src is sulfur in nucleotide complexes, as compared with Mg2+, resistant to dephosphorylation by PTP1B phosphatase, which has a strong preference for binding oxygen over and could serve as a stable, persistently-activated mimic sulfur [17, 18]. Thus, for Csk tyrosine kinase, kcat for of Src. substrate phosphorylation was comparable for ATP vs. ATPγS in the presence of thiophilic divalent metals, but Results kcat for ATPγS was greatly reduced in the presence of In our previous studies [16], we showed that the Src magnesium or manganese. This was attributed to the family kinase Hck can catalyze thiophosphorylation of a important role of γ-phosphoryl bonding and salt bridging peptide substrate in the presence of CoCl2. In those in the Csk reaction transition state [10]. Similarly, we studies, an endpoint assay was used; we analyzed ali- showed that an SH2-binding peptide could be thiopho- quots from reactions by analytical HPLC, and confirmed sphorylated by Hck kinase in the presence of cobalt [16]. peptide thiophosphorylation by mass spectrometry. Previous work in this area has focused primarily on Here, we tested whether a continuous assay could be the kinase-catalyzed thiophosphorylation of peptide used to monitor peptide thiophosphorylation. Using a substrates. Gel-based methods have been used to dem- coupled spectrophotometric assay that measures NADH onstrate incorporation of thiophosphate into kinase consumption, we found that Src catalyzed peptide A B Fig. 1 Thiophosphorylation and auto-thiophosphorylation catalyzed by Src kinase. a. The activity of Src kinase domain (450 nM) toward a syn- thetic peptide substrate (AEEEIYGEFEAKKKKG, 800 μM) was measured using the continuous spectrophotometric assay. The reactions contained no ion/nucleotide (squares), 10 mM MgCl2 and 2 mM ATPγS(circles), or 10 mM NiCl2 and 2 mM ATPγS(triangles). Absorbance data were recorded every 8 s. b. The activity of Src (2.2 μM) was measured in the absence of peptide substrate using the continuous spectrophotometric assay. The reactions contained 10 mM divalent cation (Mg2+ or Ni2+) and 2 mM nucleotide (ATP or ATPγS), as indicated Cabail et al. BMC Biochemistry (2016) 17:13 Page 3 of 10 thiophosphorylation in the presence of ATPγS and Ni2+, was purified from bacteria, and contains very low levels but not Mg2+ (Fig. 1a). Co2+ was much less efficient in of phosphorylation [21]. Using the continuous assay, we these experiments (data not shown). We did not observe detected auto-thiophosphorylation activity in the pres- any consumption of NADH in the absence of Src, or in ence of Ni2+ (Fig. 1b). The initial rate of auto- Src reactions without divalent cations (Fig. 1a). thiophosphorylation (0–100 s) was similar to that Next, we wished to determine whether Src kinase observed for Src autophosphorylation in the presence of could catalyze intermolecular auto-thiophosphorylation ATP and Mg2+ (Fig. 1b); after 600 s, the overall rate of (as opposed to phosphorylation of an exogenous peptide the NiATPγS reaction was roughly one-half that of the substrate). The Src kinase used for these experiments MgATP reaction. We observed minimal activity using A B C Fig. 2 Auto-thiophosphorylation activity of other tyrosine kinases. Conditions for the continuous spectrophotometric assay were similar to Fig. 1. The reactions contained enzyme alone (circles), enzyme with 2 mM ATPγS and 10 mM MgCl2 (squares) or enzyme with 2 mM ATPγS and 10 mM NiCl2 (triangles). a Hck kinase (0.6 μM). b Ack1 (kinase-SH3 construct), 1.0 μM. c IGF1R kinase domain, 1.4 μM Cabail et al. BMC Biochemistry (2016) 17:13 Page 4 of 10 ATPγS as cosubstrate with Mg2+ as the divalent cation. 20 min (Fig. 3a, right-hand lanes). This increased mobil- This is consistent with earlier studies in which MgATPγS ity is consistent with the behavior of Src and other tyro- did not support autoactivation of Src, and acted as a sine kinases after activation loop phosphorylation. Src competitive inhibitor versus MgATP with Ki =23μM [6]. also showed a pronounced shift in mobility upon We tested whether other tyrosine kinases can catalyze incubation