Characterization of the interactions between the active site of a protein tyrosine kinase and a divalent metal activator
© Lin and Sun; licensee BioMed Central Ltd. 2005
Received: 31 August 2005
Accepted: 23 November 2005
Published: 23 November 2005
Protein tyrosine kinases are important enzymes for cell signalling and key targets for anticancer drug discovery. The catalytic mechanisms of protein tyrosine kinase-catalysed phosphorylation are not fully understood. Protein tyrosine kinase Csk requires two Mg2+ cations for activity: one (M1) binds to ATP, and the other (M2) acts as an essential activator.
Experiments in this communication characterize the interaction between M2 and Csk. Csk activity is sensitive to pH in the range of 6 to 7. Kinetic characterization indicates that the sensitivity is not due to altered substrate binding, but caused by the sensitivity of M2 binding to pH. Several residues in the active site with potential of binding M2 are mutated and the effect on metal activation studied. An active mutant of Asn319 is generated, and this mutation does not alter the metal binding characteristics. Mutations of Glu236 or Asp332 abolish the kinase activity, precluding a positive or negative conclusion on their role in M2 coordination. Finally, the ability of divalent metal cations to activate Csk correlates to a combination of ionic radius and the coordination number.
These studies demonstrate that M2 binding to Csk is sensitive to pH, which is mainly responsible for Csk activity change in the acidic arm of the pH response curve. They also demonstrate critical differences in the metal activator coordination sphere in protein tyrosine kinase Csk and a protein Ser/Thr kinase, the cAMP-dependent protein kinase. They shed light on the physical interactions between a protein tyrosine kinase and a divalent metal activator.
Protein tyrosine kinases (PTK)1 are a large family of enzymes that transfer the γ-phosphate of ATP to tyrosine hydroxyl groups in proteins. By phosphorylation, PTKs regulate the conformation and function of their protein substrates . This covalent modification is a fundamental mechanism of signal transduction in mammalian cells. Aberrant activation of many specific protein tyrosine kinases causes mishaps in cell signalling, and results in proliferative diseases, such as cancer . Many protein tyrosine kinases are considered as important targets for drug development against such diseases . For full understanding of phosphorylation-mediated signalling and to provide a knowledge base for anti-PTK drug discovery, it is important to understand the catalytic mechanisms of protein tyrosine kinases.
C-terminal Src kinase (Csk) is a cytoplasmic PTK that phosphorylates Src family kinases (SFKs) and down-regulates their kinase activities [4, 5]. The mechanistic basis of catalysis by Csk and PTKs in general is still poorly understood. Csk-catalyzed phosphorylation reaction obeys a ternary complex mechanism, likely with rapid and random binding of ATP-Mg and the phosphate-accepting substrate . In addition to a Mg2+ cation (M1) as part of the ATP-Mg complex, Csk requires another Mg2+ ion (M2) for optimal kinase activity [7, 8]. Kinetic studies demonstrate that M2 is an essential activator . Because the affinity of Csk for the metal activator at 2.3 mM falls within the range of the cellular Mg2+ concentration, this activation may play a regulatory role in the kinase function [7, 9].
Even though Mg2+ is likely the physiological activator, several other divalent metal cations can substitute for Mg2+ and activate Csk to various levels [8, 10]. For example, Mn2+ can replace Mg2+ and results in higher activity of Csk, while Co2+, Ni2+ are not as effective as Mg2+ as an activator. Zn2+ can also substitute for Mg2+ in binding to the M2 binding site, but it cannot serve as an activator. Thus, Zn2+ acts as an inhibitor of Csk activity competitive against M2 . Another intriguing property of the Csk-metal interaction is that these substitution metals all bind to Csk considerably stronger than the physiological activator, Mg2+. While Csk binds to Mg2+ with an AC50 of 2.3 mM, the other metal cations all bind to Csk with AC50 or IC50 in the low μM range. Among all divalent metal cations tested, Zn2+ has the highest affinity for Csk, with an IC50 of 0.5 μM .
The requirement of two divalent metal cations for full activity by Csk appears to represent a general catalytic requirement by all PTKs. Several PTKs from different families, such as v-Fps , Yes , Src , Lck , insulin receptor kinase  and epidermal growth factor receptor , all require two Mg2+ cations for full activity. The insulin receptor kinase has been co-crystallized with both a peptide substrate and an ATP analog . In the active site, two Mg2+ are observed, providing direct structural evidence for the presence of two Mg2+ ions in PTK catalysis. Kinetic analysis reveals that the metal cation activator might participate in catalysis by different mechanisms for different PTKs. For example, M2 activates Csk and Src by increasing the kcat without affecting the Km for ATP . However, M2 activates IRK  and v-Fps  by decreasing the Km for ATP without affecting the kcat. The mechanistic basis for such kinetic differences has not been determined.
Interestingly, a protein Ser/Thr kinase, the cAMP-dependent protein kinase (PKA), also binds to two divalent metal cations in the active site during catalysis . However, the second Mg2+ inhibits the kinase activity . Crystallization of PKA complexed with catalytic ligands reveals that two Mg2+ cations are present in the active site .
In the current study, we characterized the parameters for the interactions between Csk and M2, such as activity sensitivity to pH, required physical parameters of the divalent metal cation activators, and potential M2 coordinating residues. Mutagenic studies eliminated a residue as a potential ligand for M2, but could not determine if two other residues are involved due to lack of activity in all mutants varying these residues.
Csk activity is sensitive to pH in the range of 6 to 7
M2 binding to Csk is sensitive to pH
Characterization of potential metal-coordinating residues in the active site of Csk
Only one protein tyrosine kinase, the insulin receptor kinase, has been co-crystallized with substrate analogs and divalent metal activators . We compared the structures of Csk and IRK to identify Csk residues potentially involved in M2 coordination. In IRK, three residues are involved in metal cation coordination, Asp1150, Glu1047, and Asn1137. All three residues are conserved among PTKs, corresponding to Asp332, Glu236 and Asn319 in Csk . Even though Csk and IRK displayed some differences in the kinetic patterns of Mg2+ activation, it is likely that the conserved residues are playing similar roles in Mg2+ coordination. We performed site-specific mutagenesis on these residues to determine if they are involved in metal activator coordination in Csk.
Catalytic parameters of Csk and Asn319Ser mutant
160 ± 10
0.01 ± 0.007
140 ± 12
150 ± 25
82 ± 12
0.01 ± 0.001
Km-polyE4Y (μg ml-1)
156 ± 30
220 ± 48
109 ± 3
0.01 ± 0.001
6.4 ± 0.1
3.4 ± 0.1
Mutation of Glu236 and Asp332 to a number of residues, Ala, Asp, Gln for Glu236, Ala, Asn, Glu for Asp332, produced inactive mutants, thus kinetic analysis of their role in M2 binding is precluded. These two residues remain likely candidates for coordinating M2, but confirmation awaits further study by other methods.
Divalent Metal cations of certain size bind to and activate Csk
In this communication, we investigated the molecular basis of a commonly observed catalytic property of Csk. First, Csk activity is sensitive to pH change in the range of 6 to 7. Steady state kinetics demonstrates that the sensitivity is not due to the binding of Csk to either ATP-Mg or the protein substrate. The sensitivity is due to the sensitivity of M2 binding to pH in this range. Second, several residues that have the potential for M2 binding were studied by mutagenesis. These studies eliminated Asn319 in the active site as a potential ligand for M2 binding, but were inconclusive about the role of Asp332 and Glu236, because mutants at these two positions were inactive. Third, commercially available divalent metal cations were surveyed for their ability to support Csk activity. A strong correlation between the ability of divalent metal cation to support Csk activity and its physical parameters (ionic radius and the coordination number) was identified. Divalent metal cations with a coordination number of 6 and an ionic radius of 0.65–0.8 Å were able to support the activity while ions outside of this range were not. Overall this investigation provided insights into the kinase-divalent metal interaction in the active site.
The sensitivity of kinase activity to pH has been previously investigated in the cAMP-dependent protein kinase . Interestingly, the binding of ATP was sensitive to pH for PKA while the binding of the second Mg2+ to PKA is not sensitive to pH. This pattern is the opposite of that observed for Csk, likely reflecting different coordination patterns for M1 and M2 in Csk and PKA. This is also consistent with the structural information on IRK and PKA. Although in both PKA and IRK, three conserved residues (Glu1047, Asn1137, and Asp1150 in IRK, Glu91, Asn171 and Asp184 in PKA) are involved in coordinating M1 and M2, the positions of M1 and M2 are switched in the two kinases. In IRK, M1 is coordinated with Asn1127, while M2 is coordinated with Asp1150 directly and Glu1047 through two water molecules. In PKA, M2 is coordinated with Asn171, while M1 is coordinated with Asp184. Because M1 binding to PKA is sensitive to pH, it is likely due to deprotonation of Asp184. In this case, M2 binding to IRK would likely be sensitive to pH. This pattern is observed in Csk. This suggests that Csk and IRK likely uses a similar M2 binding site. In this case, Glu236 and Asp332 would be expected to be key ligands for M2 coordination.
Our effort to pinpoint the residues for coordinating M2 in Csk by mutagenic and kinetic studies is not fully successful. We were able to eliminate Asn319 as responsible for binding to M2, but our results are inconclusive regarding Asp332 and Glu236 due to the inability to generate active mutants at these two positions. This highlights the limitation of mutagenic approach to study catalytically essential residues. Further studies by other tools are required to solve these issues.
Generation of Csk mutants
Glutathione S transferase (GST)-Csk fusion proteins were generated and purified as previously described . Csk point mutants were generated using QuikChange (Stratagene) in the parental plasmid and were confirmed by DNA sequencing. Kinase-defective Src (kdSrc) was produced as described previously [23, 24].
Bacteria harbouring appropriate plasmids were cultured in LB medium at 37°C with shaking at 250 rpm overnight. The overnight culture was then mixed with an equal volume of fresh LB medium, cooled down to about 20°C. IPTG (0.2 mM) was added to the culture to induce recombinant protein expression at 20°C for 12 hours. The GST fusion proteins were purified by glutathione affinity chromatography as previously described . The purified enzymes were desalted on a Sephadex G25 column equilibrated with the storage buffer (100 mM Tris-Cl, pH 8.0, and 0.1% β-mercaptoethanol). Glycerol was added to the purified fractions to 30% and the enzymes were stored at -20°C. Protein concentration was determined by the Bradford assay and the purity of purified proteins was assessed by SDS-PAGE with coomassie blue staining.
Kinase activity assay
For assaying PTK activity, phosphorylation of polyE4Y and kdSrc was measured using the acid precipitation assay as previously described . Standard kinase assay buffer contains 100 mM EPPS, pH 8, 10% glycerol, 0.1% triton X-100 and 0.1% β-mercaptoethanol. Reaction time for the assays was 10 min. Standard assays used polyE4Y at 1 mg ml-1, or kdSrc at 10 μM as the phosphate-accepting substrate and ATP at 0.2 mM as the phosphate-donating substrate. To determine the kinase activity at different pH, the kinase buffer contained all the standard buffer components except EPPS was replaced by 100 mM MES or Tris at designated pH. When Km and kcat were determined with regard to one substrate, the kinase activity was determined at various concentrations of that substrate in the range of 20 to 200 μg ml-1 for polyE4Y, 1 to 10 μM for kdSrc or 20 to 200 μM for ATP. When the phosphate-accepting substrate (either polyE4Y or kdSrc) was the variable substrate, ATP concentration was 0.2 mM. PolyE4Y at 1 mg ml-1 was used when ATP was the variable substrate. The kcat and Km values were determined by Lineweaver-Burk plots with linear regression using Microsoft Excel. All steady state kinetic assays were performed in duplicate, and repeated at least once. Standard errors were calculated if an assay was performed at least three times.
the concentration of a divalent metal cation that activates Csk to 50% of its full activity
C-terminal Src kinase
insulin receptor kinase
the camp-dependent protein kinase
protein tyrosine kinase(s)
Src family kinase(s).
This work was supported by grants from NIH (1RO1CA111687 and 1 P20 RR16457), and the American Cancer Society (RSG-04-247-01-CDD). DNA sequencing was performed at URI Genomics and Sequencing Center.
#G. Sun is an American Cancer Society Research Scholar.
- Hubbard SR, Till JH: Protein tyrosine kinase structure and function. Annu Rev Biochem. 2000, 69: 373-398. 10.1146/annurev.biochem.69.1.373.View ArticlePubMedGoogle Scholar
- Hunter T: The Croonian Lecture 1997. The phosphorylation of proteins on tyrosine: its role in cell growth and disease. Philos Trans R Soc Lond B Biol Sci. 1998, 353: 583-605. 10.1098/rstb.1998.0228.PubMed CentralView ArticlePubMedGoogle Scholar
- Krause DS, Van Etten RA: Tyrosine kinases as targets for cancer therapy. N Engl J Med. 2005, 353: 172-187. 10.1056/NEJMra044389.View ArticlePubMedGoogle Scholar
- Okada M, Nada S, Yamanashi Y, Yamamoto T, Nakagawa H: CSK: a protein-tyrosine kinase involved in regulation of src family kinases. J Biol Chem. 1991, 266: 24249-24252.PubMedGoogle Scholar
- Sun G, Sharma AK, Budde RJ: Autophosphorylation of Src and Yes blocks their inactivation by Csk phosphorylation. Oncogene. 1998, 17: 1587-1595. 10.1038/sj.onc.1202076.View ArticlePubMedGoogle Scholar
- Cole PA, Burn P, Takacs B, Walsh CT: Evaluation of the catalytic mechanism of recombinant human Csk (C-terminal Src kinase) using nucleotide analogs and viscosity effects. J Biol Chem. 1994, 269: 30880-30887.PubMedGoogle Scholar
- Sun G, Budde RJ: Requirement for an additional divalent metal cation to activate protein tyrosine kinases. Biochemistry. 1997, 36: 2139-2146. 10.1021/bi962291n.View ArticlePubMedGoogle Scholar
- Sun G, Budde RJ: Substitution studies of the second divalent metal cation requirement of protein tyrosine kinase CSK. Biochemistry. 1999, 38: 5659-5665. 10.1021/bi982793w.View ArticlePubMedGoogle Scholar
- Maguire ME: Magnesium: a regulated and regulatory cation. Metal Ions Biol Syst. 1990, 26: 135-153.Google Scholar
- Grace MR, Walsh CT, Cole PA: Divalent ion effects and insights into the catalytic mechanism of protein tyrosine kinase Csk. Biochemistry. 1997, 36: 1874-1881. 10.1021/bi962138t.View ArticlePubMedGoogle Scholar
- Saylor P, Wang C, Hirai TJ, Adams JA: A second magnesium ion is critical for ATP binding in the kinase domain of the oncoprotein v-Fps. Biochemistry. 1998, 37: 12624-12630. 10.1021/bi9812672.View ArticlePubMedGoogle Scholar
- Sun G, Budde JA: Expression, purification, and initial characterization of human Yes protein tyrosine kinase from a bacterial expression system. Arch Biochem Biophys. 1997, 345: 135-142. 10.1006/abbi.1997.0236.View ArticlePubMedGoogle Scholar
- Budde JA, Ramdas L, Ke S: Recombinant pp60c-src from baculovirus-infected insect cells: purification and characterization. Prep Biochem. 1993, 23: 493-515.PubMedGoogle Scholar
- Wang QM, Srinivas PR, Harrison ML, Geahlen RL: Partial purification and characterization of the lck protein-tyrosine kinase from bovine thymus. Biochem J. 1991, 279 (Pt 2): 567-574.PubMed CentralView ArticlePubMedGoogle Scholar
- White MF, Haring HU, Kasuga M, Kahn CR: Kinetic properties and sites of autophosphorylation of the partially purified insulin receptor from hepatoma cells. J Biol Chem. 1984, 259: 255-264.PubMedGoogle Scholar
- Koland JG, Cerione RA: Activation of the EGF receptor tyrosine kinase by divalent metal ions: comparison of holoreceptor and isolated kinase domain properties. Biochim Biophys Acta. 1990, 1052: 489-498. 10.1016/0167-4889(90)90160-F.View ArticlePubMedGoogle Scholar
- Hubbard SR: Crystal structure of the activated insulin receptor tyrosine kinase in complex with peptide substrate and ATP analog. EMBO J. 1997, 16: 5572-5581. 10.1093/emboj/16.18.5572.PubMed CentralView ArticlePubMedGoogle Scholar
- Vicario PP, Saperstein R, Bennun A: Role of divalent metals in the kinetic mechanism of insulin receptor tyrosine kinase. Arch Biochem Biophys. 1988, 261: 336-345. 10.1016/0003-9861(88)90349-9.View ArticlePubMedGoogle Scholar
- Mildvan AS, Rosevear PR, Fry DC, Bramson HN, Kaiser ET: NMR studies of the mechanism of action and regulation of protein kinase. Curr Top Cell Regul. 1985, 27: 133-144.View ArticlePubMedGoogle Scholar
- Armstrong RN, Kondo H, Granot J, Kaiser ET, Mildvan AS: Magnetic resonance and kinetic studies of the manganese(II) ion and substrate complexes of the catalytic subunit of adenosine 3',5'-monophosphate dependent protein kinase from bovine heart. Biochemistry. 1979, 18: 1230-1238. 10.1021/bi00574a018.View ArticlePubMedGoogle Scholar
- Zheng J, Knighton DR, ten Eyck LF, Karlsson R, Xuong N, Taylor SS, Sowadski JM: Crystal structure of the catalytic subunit of cAMP-dependent protein kinase complexed with MgATP and peptide inhibitor. Biochemistry. 1993, 32: 2154-2161. 10.1021/bi00060a005.View ArticlePubMedGoogle Scholar
- Yoon MY, Cook PF: Chemical mechanism of the adenosine cyclic 3',5'-monophosphate dependent protein kinase from pH studies. Biochemistry. 1987, 26: 4118-4125. 10.1021/bi00387a056.View ArticlePubMedGoogle Scholar
- Wang D, Huang XY, Cole PA: Molecular determinants for Csk-catalyzed tyrosine phosphorylation of the Src tail. Biochemistry. 2001, 40: 2004-2010. 10.1021/bi002342n.View ArticlePubMedGoogle Scholar
- Lee S, Lin X, Nam NH, Parang K, Sun G: Determination of the substrate-docking site of protein tyrosine kinase C-terminal Src kinase. Proc Natl Acad Sci USA. 2003, 100: 14707-14712. 10.1073/pnas.2534493100.PubMed CentralView ArticlePubMedGoogle Scholar
- Shaffer J, Sun G, Adams JA: Nucleotide release and associated conformational changes regulate function in the COOH-terminal Src kinase, Csk. Biochemistry. 2001, 40: 11149-11155. 10.1021/bi011029y.View ArticlePubMedGoogle Scholar
- Sondhi D, Xu W, Songyang Z, Eck MJ, Cole PA: Peptide and protein phosphorylation by protein tyrosine kinase Csk: insights into specificity and mechanism. Biochemistry. 1998, 37: 165-172. 10.1021/bi9722960.View ArticlePubMedGoogle Scholar
- Ogawa A, Takayama Y, Sakai H, Chong KT, Takeuchi S, Nakagawa A, Nada S, Okada M, Tsukihara T: Structure of the carboxyl-terminal Src kinase, Csk. J Biol Chem. 2002, 277: 14351-14354. 10.1074/jbc.C200086200.View ArticlePubMedGoogle Scholar
- Glusker JP: Structural aspects of metal liganding to functional groups in proteins. Adv Protein Chem. 1991, 42: 1-75.View ArticlePubMedGoogle Scholar
- Sun G, Budde RJ: A modified pGEX expression system that eliminates degradation products and thrombin from the recombinant protein. Anal Biochem. 1995, 231: 458-460. 10.1006/abio.1995.0081.View ArticlePubMedGoogle Scholar
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