Phosphorylation of the transcription factor Ets-1 by ERK2: rapid dissociation of ADP and phospho-Ets-1.

ERK2, a major effector of the BRAF oncogene, is a promiscuous protein kinase that has a strong preference for phosphorylating substrates on Ser-Pro or Thr-Pro motifs. As part of a program to understand the fundamental basis for ERK2 substrate recognition and catalysis, we have studied the mechanism by which ERK2 phosphorylates the transcription factor Ets-1 at Thr-38. A feature of the mechanism in the forward direction is a partially rate-limiting product release step (koff = 59 +/- 6 s(-1)), which is significant because to approach maximum efficiency substrates for ERK2 may evolve to ensure that ADP dissociation is rate-limiting. To improve our understanding of the mechanism of product release, the binding of the products to ERK2 was assessed and the reaction was examined in the reverse direction. These studies demonstrated that phospho-Ets-1 (p-Ets) binds >20-fold more tightly to ERK2 than ADP (Kd = 7.3 and 165 microM, respectively) and revealed that the products exhibit little interaction energetically while bound to ERK2 and that they can dissociate ERK2 in a random order. The overall equilibrium for the reaction in solution (Keq = 250 M(-1)) was found to be similar to that with the substrate bound to the enzyme (Kint = 525 M(-1)). To determine what limits koff, several pre-steady-state experiments were performed. A catalytic trapping approach furnished a rate constant (k-ADPa) of 61 +/- 12 s(-1) for the dissociation of ADP from the abortive ternary complex, ERK2.Ets.ADP. To examine p-Ets dissociation, the binding of a fluorescent derivative (p-Ets-F), which binds ERK2 with an affinity similar to that of p-Ets, was examined by stopped-flow kinetics. Using this approach, p-Ets-F was found to bind through a single-step mechanism, with the following parameters: k-p-Ets-F = 121 +/- 3.8 s(-1), and kp-Ets-F = (9.4 +/- 0.3) X 10(6) M(-1) s(-1). Similar results were found in the presence of a saturating ADP concentration. These data suggest that koff may be limited by the dissociation of both products and are consistent with the notion that Ets-1 has evolved to be an efficient substrate for ERK2, where ADP release is, at least, partially rate-limiting. A molecular mechanics model of the complex formed between ERK2 and residues 28-138 of Ets-1 provides insight into the role of substrate docking interactions.

Evidence that tRNA synthetase-directed proton transfer stops mistranslation.

To prevent mistranslation, aminoacyl-tRNA synthetases (AARSs) discriminate against noncognate amino acids and cellular metabolites. Defects in specificity produce statistical proteins which, in mammalian cells, lead to activation of the unfolded protein response and cell death. Because of inherent limitations in amino acid discrimination by a single active site, AARSs evolved a separate domain to clear mischarged amino acids. Although the structure of a widely distributed editing domain for ThrRS and AlaRS is known, the mechanism of amino acid clearance remains elusive. This domain has two motifs that together have four conserved residues in the pocket used to clear serine from mischarged tRNAs. Here, using ThrRS as an example, rapid single-turnover kinetics, mutagenesis, and solvent isotope analysis show that a strictly conserved histidine (between ThrRS and AlaRS) extracts a proton in the chemical step of the editing reaction. Three other conserved residues, and two additional residues in the editing pocket, are not directly implicated in the chemical step. These results are relevant to the previously reported mutagenesis of the homologous editing pocket of alanyl-tRNA synthetase, where even a mild defect in editing causes neurodegeneration in the mouse. Thus, a single proton-transfer event needed to prevent mistranslation can have profound implications for disease.

Role of a tRNA base modification and its precursors in frameshifting in eukaryotes.

Little is known about the role of specific base modifications of transfer RNAs. Wyosine bases are tRNA(Phe)-specific modifications that are distinguished by differentiated, lateral side chains and base methylations appended to the core ring structure of a universally conserved G37, adjacent to the anticodon of Phe tRNAs. Based on previous data, we hypothesized that this modification was needed for -1 frameshifting. Using a reporter system incorporating a SCV-LA yeast virus slippery site for detecting -1 frameshifts in vivo, yeast strains were created that enabled chemical-genetic dissection of the role of different functional groups of wyebutosine that are added in a three-step post-transcriptional set of reactions. With this system, hypomodification increased Phe-specific frameshifting, with incremental changes in frameshift efficiency after specific intermediates in the progression of wyebutosine synthesis. These data combined with investigations of wild-type and hypomodified tRNA binding to ribosomes suggest that frameshift efficiency is kinetically and not thermodynamically controlled. The progressive nature of frameshift efficiency with the stage of modification is consistent with a stepwise evolution and tuning of frameshift potential. The stepwise tuning of frameshift efficiency could explain why tRNA(Phe) in some eukaryotes is not fully modified but, rather, hypomodified to capture a specific frameshift potential.

Genetic code ambiguity confers a selective advantage on Acinetobacter baylyi.

A primitive genetic code, composed of a smaller set of amino acids, may have expanded via recursive periods of genetic code ambiguity that were followed by specificity. Here we model a step in this process by showing how genetic code ambiguity could result in an enhanced growth rate in Acinetobacter baylyi.

A universal plate format for increased throughput of assays that monitor multiple aminoacyl transfer RNA synthetase activities.

Aminoacyl transfer RNA (tRNA) synthetases are intensely studied enzymes because of their importance in the establishment of the genetic code and their connection to disease and medicine. During the advancement of this field, several assays were developed. Despite many innovations, the sensitivity, simplicity, and reliability of the radiometric assays (which were among the first to be developed) have ensured their continued use. Four activities are measured by these assays: active site titration, amino acid activation, aminoacylation, and posttransfer editing (deacylation). In an effort to maintain the advantage of these assays while enhancing throughput, reducing waste, and improving data quality, a universal 96-well filter plate format was developed. This format facilitates the assays for all four of the widely studied activities.

The expression and purification of the N-terminal activation domain of the transcription factor c-Myc: a model substrate for exploring ERK2 docking interactions.

ERK2 is a mitogen-activated protein kinase (MAPK) that plays pivotal roles in cell signal transduction, where it mediates effects on proliferation and differentiation by growth factors and hormones. An important substrate of ERK2 is the transcription factor c-Myc, which mediates cell cycle progression. The phosphorylation of Ser-62 on c-Myc by ERK2 is thought to contribute to the increased stability of c-Myc during the cell cycle and is thus a critical cellular event. However, the mode of c-Myc recognition by ERK2 is not understood. Early studies by Gupta and Davis concluded that ERK2 specificity determinants are located in residues 1-100 of c-Myc, its activation domain. To pursue both structural and kinetic studies a rapid, but efficient purification method, for the production of the activation domain of c-Myc from an Escherichia coli source, was developed. We chose the minimal number of high-resolution steps to maximize both yield and efficiency without sacrificing purity. Thus, GST-(c-MycDelta2-99)-His(6) was expressed in E. coli, and purified using glutathione-agarose affinity chromatography. Cleavage of the GST fusion protein by thrombin and subsequent purification by nickel-agarose affinity chromatography yielded 8 mg of purified (c-MycDelta2-99)-His(6) from one liter of LB culture. Rigorous characterization demonstrated that under standard assay conditions (c-MycDelta2-99)-His(6) is phosphorylated by ERK2 with the following Michaelis parameters: k(cat)=10.4s(-1), K(M)(c-Myc)=57.4 microM. In summary, a rapid procedure is outlined for the preparation of (c-MycDelta2-99)-His(6) that will be useful for mechanistic and biophysical studies of ERK2.

Discovery of a gene family critical to wyosine base formation in a subset of phenylalanine-specific transfer RNAs.

A large number of post-transcriptional base modifications in transfer RNAs have been described (Sprinzl, M., Horn, C., Brown, M., Ioudovitch, A., and Steinberg, S. (1998) Nucleic Acids Res. 26, 148-153). These modifications enhance and expand tRNA function to increase cell viability. The intermediates and genes essential for base modifications in many instances remain unclear. An example is wyebutosine (yW), a fluorescent tricyclic modification of an invariant guanosine situated on the 3'-side of the tRNA(Phe) anticodon. Although biosynthesis of yW involves several reaction steps, only a single pathway-specific enzyme has been identified (Kalhor, H. R., Penjwini, M., and Clarke, S. (2005) Biochem. Biophys. Res. Commun. 334, 433-440). We used comparative genomics analysis to identify a cluster of orthologous groups (COG0731) of wyosine family biosynthetic proteins. Gene knock-out and complementation studies in Saccharomyces cerevisiae established a role for YPL207w, a COG0731 ortholog that encodes an 810-amino acid polypeptide. Further analysis showed the accumulation of N(1)-methylguanosine (m(1)G(37)) in tRNA from cells bearing a YPL207w deletion. A similar lack of wyosine base and build-up of m(1)G(37) is seen in certain mammalian tumor cell lines. We proposed that the 810-amino acid COG0731 polypeptide participates in converting tRNA(Phe)-m(1)G(37) to tRNA(Phe)-yW.

A kinetic approach towards understanding substrate interactions and the catalytic mechanism of the serine/threonine protein kinase ERK2: identifying a potential regulatory role for divalent magnesium.

We are interested in the mechanism and regulation of the extracellular regulated protein kinases, ERK1 and ERK2, due to their key roles in cellular signal transduction and disease. Both enzymes phosphorylate a large number of structurally disparate proteins upon activation by phorbol esters, serum and growth factors, and are activated through a protein kinase cascade, termed the mitogen activated protein kinase (MAPK) pathway. ERK2 catalyses the transfer of the gamma-phosphate of adenosine triphosphate to serine or threonine residues found in Ser-Pro or Thr-Pro motifs on proteins. Its catalytic mechanism is intriguing, because it appears to predominantly rely on interactions outside of the active site cleft to specify a substrate. To study ERK2, we developed a recombinant protein called EtsDelta138, which comprises residues 1-138 of the transcription factor Ets-1, an excellent substrate of ERK2. Here we review several steady-state kinetic experiments that reveal details of the ERK2 mechanism and a hitherto unknown process of ERK2 activation by free magnesium. The physiological relevance of this mechanism is discussed.

Crystal structure of a human peptidyl-tRNA hydrolase reveals a new fold and suggests basis for a bifunctional activity.

Peptidyl-tRNA hydrolase (Pth) activity releases tRNA from the premature translation termination product peptidyl-tRNA. Two different enzymes have been reported to encode such activity, Pth present in bacteria and eukaryotes and Pth2 present in archaea and eukaryotes. Here we report the crystallographic structure of the Homo sapiens Pth2 at a 2.0-A resolution as well as its catalytic properties. In contrast to the structure of Escherichia coli Pth, H. sapiens Pth2 has an alpha/beta fold with a four-stranded antiparallel beta-sheet in its core surrounded by two alpha-helices on each side. This arrangement of secondary structure elements generates a fold not previously reported. Its catalytic efficiency is comparable with that reported for the archaeal Sulfolobus solfataricus Pth2 and higher than that of the bacterial E. coli Pth. Several lines of evidence target the active site to two close loops with highly conserved residues. This active site architecture is unrelated to that of E. coli Pth. In addition, intermolecular contacts in the crystal asymmetric unit cell suggest a likely surface for protein-protein interactions related to the Pth2-mediated apoptosis.

Two rate-limiting steps in the kinetic mechanism of the serine/threonine specific protein kinase ERK2: a case of fast phosphorylation followed by fast product release.

Extracellular regulated protein kinase 2 (ERK2) is a eukaryotic protein kinase whose activity is regulated by mitogenic stimuli. To gain insight into the catalytic properties of ERK2 and to complement structure-function studies, we undertook a pre-steady state kinetic analysis of the enzyme. To do this, ERK2 was quantitatively activated by MAPKK1 in vitro by monitoring the stoichiometry and site specificity of phosphorylation using a combination of protein mass spectrometry, tryptic peptide analysis, and (32)P radiolabeling. Using a quench-flow apparatus, MgATP(2-) was rapidly mixed (<1 ms) with both ERK2 and the protein substrate EtsDelta138 in the presence of a saturating total concentration (20 mM) of magnesium ion at 27 degrees C and pH 7.5. An exponential burst of product was observed over the first few milliseconds that followed mixing. This burst had an amplitude alpha of 0.44 and was followed by a slower linear phase. The pre-steady state burst is consistent with two partially rate-limiting enzymatic steps, which have the following rate constants: k(2) = 109 +/- 9 s(-1) and k(3) = 56 +/- 4 s(-1). These are attributed to rapid phosphorylation of EtsDelta138 and the process of product release, respectively. Single-turnover experiments provided an independent determination of k(2) (106 +/- 25 s(-1)). The observed catalytic constant (k(cat)(obs)) was found to be sensitive to the concentration of ERK2. The data fit a model in which ERK2 monomers form dimers and suggest that both the monomeric and dimeric forms of ERK2 are active with catalytic constants (k(cat)) of 25 and 37 s(-1), respectively. In addition, the model suggests that in the presence of saturating concentrations of both magnesium and substrates ERK2 subunits dissociate with a dissociation constant (K(d)) of 32 +/- 16 nM.

Physiological concentrations of divalent magnesium ion activate the serine/threonine specific protein kinase ERK2.

Extracellular regulated protein kinase 2 (ERK2) is a eukaryotic protein kinase whose activity is regulated by phorbol esters, serum, and growth factors, and displays enhanced activity in several human tumors. Despite its important biological function, its mechanism of catalysis and mode of regulation are poorly understood. Recently, we showed that in the presence of 10 mM magnesium chloride, ERK2 phosphorylates the transcription factor Ets-1 through a random-ordered ternary-complex mechanism [Waas, W. F., and Dalby, K. N. (2002) J. Biol. Chem. 277, 12532]. Now we provide kinetic evidence that ERK2 must bind two divalent magnesium ions to facilitate catalysis at a physiologically relevant rate, because a second magnesium ion promotes both MgATP2- binding and phosphoryl transfer. The velocity dependence on magnesium at saturating concentrations of the protein substrate, Ets138, over a range of ATP4- and Mg2+ ion concentrations, supports the notion that magnesium is an essential activator of ERK2. At high (> or = 1 mM) concentrations of ATP4-, the velocity dependence on total Mg2+ is sigmoidal, but plateaus at high concentrations of free Mg2+, where the enzyme is fully activated. At concentrations of Mg2+ of < or = 4 mM, the velocity dependence on ATP4- displays a peak when the concentration of ATP4- approaches that of total Mg2+ and tends to zero at high concentrations of ATP4-, where the enzyme is predominantly unactivated. The observed velocity dependencies are consistent with the notion that ERK2*Etsdelta138 complexes and ATP4- compete for the same pool of Mg2+ ions in solution. No binding of ATP4- (0-2.5 mM) by ERK2 (65 microM) can be detected using isothermal titration calorimetry at 27 degrees C, pH 8.0, and an ionic strength of 0.15 M (KCl), suggesting that the complex, MgATP2-, is the true substrate for ERK2. In contrast, 5-iodotubericidin binds ERK2 tightly (K(d) = 1 microM) and displays a competitive inhibition pattern toward MgATP2- and a mixed pattern toward free Mg2+, suggesting that the binding of Mg2+ before MgATP2- is not compulsory.

Transient protein-protein interactions and a random-ordered kinetic mechanism for the phosphorylation of a transcription factor by extracellular-regulated protein kinase 2.

No thorough mechanistic study of extracellular signal-regulated protein kinase 2 (ERK2) has appeared in the literature. A recombinant protein termed EtsDelta138, which comprises of residues 1-138 of the transcription factor Ets-1 is an excellent substrate of ERK2 (Waas W. F., and Dalby, K. N. (2001) Protein Exp. Purif. 23, 191-197). The kinetic mechanism of ERK2 was examined, with excess magnesium, by initial velocity measurements, both in the absence and presence of products at 27 degrees C, pH 7.5, and ionic strength 0.1 m (KCl). The velocity data are consistent with a steady-state random-ordered ternary complex mechanism, where both substrates have unhindered access to binding sites on the enzyme. The mechanism and magnitude of product inhibition by monophosphorylated EtsDelta138 is consistent with, but does not prove, the notion that ERK2 forms a discrete interaction with EtsDelta138 in the absence of active site interactions, and that this "docking complex" facilitates intramolecular phosphorylation of the substrate. The approximation of the steady-state data to a rapid equilibrium model strongly suggests that the formation of ERK2.Ets138 complexes are transient in nature with dissociation constants of greater magnitude than the catalytic constant, of k(cat) = 17 s(-1).