Phosphorylation of the Transcription Factor Ets-1 by ERK2: Rapid Dissociation of ADP and Phospho-Ets-1
Open accessAbstract
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.
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
The SAM domain of Ets-1 binds the FRS or FxF docking site on ERK2, which is located 15 Å away from the ERK2 KIM binding groove.
“For example, the SAM domain of Ets-1 binds the FRS or FxF docking site on ERK2, which is located 15 A ˚away from the ERK2 KIM binding groove [Fig.4(C), lower right].70,74”
From Molecular basis of MAP kinase regulation (2013) · cites this paper as [74]
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
The self-check needs the paper’s full text.
Cites
Jorgensen, W. L., Maxwell, D. S., & Tirado-Rives, J. (1996) · Journal of the American Chemical Society · doi:10.1021/ja9621760 · Cited by 14,078 · see citing works
Mutations of the BRAF gene in human cancer
Davies, H., Bignell, G. R., Cox, C., Stephens, P., Edkins, S., Clegg, S., Teague, J., Woffendin, H., Garnett, M. J., Bottomley, W., et al. (2002) · Nature · doi:10.1038/nature00766 · Cited by 10,674 · see citing works
ras oncogenes in human cancer: a review.
Bos, J. L. (1989) · PubMed · Cited by 5,061 · see citing works
Enzyme kinetics; behavior and analysis of rapid equilibrium and steady state enzyme systems
(1994) · International Journal of Biochemistry · doi:10.1016/0020-711x(94)90021-3 · Cited by 2,978 · see citing works
AP-1: a double-edged sword in tumorigenesis
Eferl, R., & Wagner, E. F. (2003) · Nature reviews. Cancer · doi:10.1038/nrc1209 · Cited by 2,218 · see citing works
Weinberg, R. A., & Weinberg, R. A. (2006) · W.W. Norton eBooks · doi:10.1201/9780203852569 · Cited by 1,806 · see citing works
BRAF mutation predicts sensitivity to MEK inhibition
Solit, D. B., Garraway, L. A., Pratilas, C. A., Sawai, A., Getz, G. A., Basso, A., Ye, Q., Lobo, J. M., She, Y., Osman, I., et al. (2005) · Nature · doi:10.1038/nature04304 · Cited by 1,317 · see citing works
Tinker 8: Software Tools for Molecular Design
Rackers, J. A., Wang, Z., Lu, C. T., Laury, M. L., Lagardère, L., Schnieders, M. J., Piquemal, J., Ren, P. Y., & Ponder, J. W. (2018) · Journal of Chemical Theory and Computation · doi:10.1021/acs.jctc.8b00529 · Cited by 1,194 · see citing works
Self-citationGlobal Kinetic Explorer: A new computer program for dynamic simulation and fitting of kinetic data
Johnson, K. A., Simpson, Z. B., & Blom, T. (2008) · Analytical Biochemistry · doi:10.1016/j.ab.2008.12.024 · Cited by 596 · see citing works
Brown, N. R., Noble, M. E. M., Endicott, J., & Johnson, L. N. (1999) · Nature Cell Biology · doi:10.1038/15674 · Cited by 546 · see citing works
Hyperexpression of mitogen-activated protein kinase in human breast cancer.
Sivaraman, V., Wang, H., Nuovo, G. J., et al. (1997) · Journal of Clinical Investigation · doi:10.1172/jci119309 · Cited by 455 · see citing works
Targeting the ERK signaling pathway in cancer therapy
Kohno, M., & Pouysségur, J. (2006) · Annals of Medicine · doi:10.1080/07853890600551037 · Cited by 422 · see citing works
FitSpace Explorer: An algorithm to evaluate multidimensional parameter space in fitting kinetic data
Johnson, K. A., Simpson, Z. B., & Blom, T. (2008) · Analytical Biochemistry · doi:10.1016/j.ab.2008.12.025 · Cited by 344 · see citing works
Signalling specificity of Ser/Thr protein kinases through docking-site-mediated interactions
BIONDI, R. M., & NEBREDA, A. R. (2003) · Biochemical Journal · doi:10.1042/bj20021641 · Cited by 334 · see citing works
Development of anticancer drugs targeting the MAP kinase pathway
Sebolt–Leopold, J. S. (2000) · Oncogene · doi:10.1038/sj.onc.1204083 · Cited by 321 · see citing works
Qiao, F., & Bowie, J. U. (2005) · Science s STKE · doi:10.1126/stke.2862005re7 · Cited by 280 · see citing works
Fos Family Members Induce Cell Cycle Entry by Activating Cyclin D1
Brown, J. R., Nigh, E. A., Lee, R. J., Ye, H., Thompson, M. A., Saudou, F., Pestell, R. G., & Greenberg, M. E. (1998) · Molecular and Cellular Biology · doi:10.1128/mcb.18.9.5609 · Cited by 253 · see citing works
An ERK2 docking site in the Pointed domain distinguishes a subset of ETS transcription factors
Seidel, J. J., & Graves, B. J. (2002) · Genes & Development · doi:10.1101/gad.950902 · Cited by 182 · see citing works
Marampon, F., Ciccarelli, C., & ZANI, B. M. (2006) · Molecular Cancer · doi:10.1186/1476-4598-5-31 · Cited by 148 · see citing works
Gysin, S., Lee, S., Dean, N. M., & McMahon, M. (2005) · Cancer Research · doi:10.1158/0008-5472.can-04-2848 · Cited by 147 · see citing works
Pharmacological inhibitors of the ERK signaling pathway: application as anticancer drugs.
Kohno, M., & Pouysségur, J. (2003) · PubMed · Cited by 116 · see citing works
Identification of Novel Point Mutations in ERK2 That Selectively Disrupt Binding to MEK1
Robinson, F. L., Whitehurst, A. W., Raman, M., & Cobb, M. H. (2002) · Journal of Biological Chemistry · doi:10.1074/jbc.m107776200 · Cited by 99 · see citing works
A Bipartite Mechanism for ERK2 Recognition by Its Cognate Regulators and Substrates
Zhang, J., Zhou, B., Zheng, C.-F., & Zhang, Z. (2003) · Journal of Biological Chemistry · doi:10.1074/jbc.m303909200 · Cited by 92 · see citing works
Measurement of the dead time of a fluorescence stopped-flow instrument
Peterman, B. F. (1979) · Analytical Biochemistry · doi:10.1016/s0003-2697(79)80176-1 · Cited by 91 · see citing works
A potential smoothing algorithm accurately predicts transmembrane helix packing.
Ponder, J. W., Pappu, R. V., & Marshall, G. R. (1999) · Nature Structural Biology · doi:10.1038/4922 · Cited by 80 · see citing works
PEA-15 Binding to ERK1/2 MAPKs Is Required for Its Modulation of Integrin Activation
Chou, F.-L., Hill, J. M., Hsieh, J.-C., Pouysségur, J., Brunet, A., Glading, A., Überall, F., Ramos, J. W., Werner, M. H., & Ginsberg, M. H. (2003) · Journal of Biological Chemistry · doi:10.1074/jbc.m309322200 · Cited by 59 · see citing works
Shaffer, J., & Adams, J. A. (1999) · Biochemistry · doi:10.1021/bi991109q · Cited by 54 · see citing works
Callaway, K., Abramczyk, O., Martin, L., & Dalby, K. N. (2007) · Biochemistry · doi:10.1021/bi700206u · Cited by 54 · see citing works
Self-citationProximity-Induced Catalysis by the Protein Kinase ERK2
Rainey, M. A., Callaway, K., Barnes, R., Wilson, B., & Dalby, K. N. (2005) · Journal of the American Chemical Society · doi:10.1021/ja052915p · Cited by 52 · see citing works
Self-citationWaas, W. F., & Dalby, K. N. (2002) · Journal of Biological Chemistry · doi:10.1074/jbc.m110523200 · Cited by 48 · see citing works
Self-citationCatalytic Reaction Pathway for the Mitogen-Activated Protein Kinase ERK2
Prowse, C. N., Hagopian, J. C., Cobb, M. H., et al. (2000) · Biochemistry · doi:10.1021/bi000277b · Cited by 47 · see citing works
Kong, C., & Cook, P. (1988) · Biochemistry · doi:10.1021/bi00413a032 · Cited by 46 · see citing works
Properties and Regulation of a Transiently Assembled ERK2·Ets-1 Signaling Complex
Callaway, K., Rainey, M. A., Riggs, A., Abramczyk, O., & Dalby, K. N. (2006) · Biochemistry · doi:10.1021/bi0610451 · Cited by 45 · see citing works
Self-citationMEK Wars, a new front in the battle against cancer
Duesbery, N. S., Webb, C. P., & Woude, G. F. V. (1999) · Nature Medicine · doi:10.1038/10457 · Cited by 41 · see citing works
Waas, W. F., Rainey, M. A., Szafranska, A. E., & Dalby, K. N. (2003) · Biochemistry · doi:10.1021/bi0348617 · Cited by 36 · see citing works
Self-citationKwiatkowski, A. P., Huang, C., & King, M. M. (1990) · Biochemistry · doi:10.1021/bi00453a019 · Cited by 34 · see citing works
Abramczyk, O., Rainey, M. A., Barnes, R., Martin, L., & Dalby, K. N. (2007) · Biochemistry · doi:10.1021/bi7002058 · Cited by 34 · see citing works
Self-citationThottassery, J. V., Sun, Y., Westbrook, L., et al. (2004) · Cancer Research · doi:10.1158/0008-5472.can-03-2645 · Cited by 33 · see citing works
Shaffer, J., Sun, G., & Adams, J. A. (2001) · Biochemistry · doi:10.1021/bi011029y · Cited by 27 · see citing works
Milella, M., Kornblau, S. M., & Andreeff, M. (2003) · PubMed · Cited by 25 · see citing works
Phosphoryl Transfer Step in the C-terminal Src Kinase Controls Src Recognition
Lieser, S. A., Shindler, C., Aubol, B. E., Lee, S., Sun, G., & Adams, J. A. (2004) · Journal of Biological Chemistry · doi:10.1074/jbc.m411736200 · Cited by 24 · see citing works
Purification of a Model Substrate for Transcription Factor Phosphorylation by ERK2
Waas, W. F., & Dalby, K. N. (2001) · Protein Expression and Purification · doi:10.1006/prep.2001.1491 · Cited by 24 · see citing works
Self-citationKinetic Mechanism of Fully Activated S6K1 Protein Kinase
Keshwani, M. M., & Harris, T. K. (2008) · Journal of Biological Chemistry · doi:10.1074/jbc.m800114200 · Cited by 24 · see citing works
Qamar, R., Yoon, M. Y., & Cook, P. F. (1992) · Biochemistry · doi:10.1021/bi00156a018 · Cited by 22 · see citing works
Caffrey, D. R., Shields, D. C., & O'Neill, L. A. J. (2000) · Protein Science · doi:10.1110/ps.9.4.655 · Cited by 20 · see citing works
The Complete Pathway for ERK2-catalyzed Reaction
Wang, Z., & Wu, J. (2007) · Journal of Biological Chemistry · doi:10.1074/jbc.m703161200 · Cited by 12 · see citing works
Flagged first, then by citations
Cited by
Sorted by citations
Genomic and biochemical insights into the specificity of ETS transcription factors
Hollenhorst, P. C., McIntosh, L. P., & Graves, B. J. (2011) · Annual Review of Biochemistry · doi:10.1146/annurev.biochem.79.081507.103945 · Cited by 490 · see citing works
Molecular basis of MAP kinase regulation
Peti, W., & Page, R. (2013) · Protein Science · doi:10.1002/pro.2374 · Cited by 302 · see citing works
“4(C), lower right].(70,74) Similarly, previous studies showed that PEA-15 binds the ERK2 KIM binding groove in the non-canonical C->N direction [Fig.”
ResultSolution NMR Insights into Docking Interactions Involving Inactive ERK2
Piserchio, A., Warthaka, M., Devkota, A. K., Kaoud, T. S., Lee, S., Abramczyk, O., Ren, P., Dalby, K. N., & Ghose, R. (2011) · Biochemistry · doi:10.1021/bi2000559 · Cited by 41 · see citing works
Self-citationLee, S., Warthaka, M., Yan, C., Kaoud, T. S., Piserchio, A., Ghose, R., Ren, P., & Dalby, K. N. (2011) · PLoS ONE · doi:10.1371/journal.pone.0018594 · Cited by 26 · see citing works
Self-citationRole of Intrinsically Disordered Regions in Acceleration of Protein–Protein Association
Misiura, M. M., & Kolomeisky, A. B. (2020) · The Journal of Physical Chemistry B · doi:10.1021/acs.jpcb.9b08793 · Cited by 10 · see citing works
- Authors
- Callaway, K., Waas, W. F., Rainey, M. A., Ren, P., & Dalby, K. N.
- Affiliations
- The University of Texas at Austin (US)
- Cellular and Molecular Biology
- Corresponding author
- Dalby, Kevin N.
- Venue
- Biochemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2010-04-12
- Volume
- 49
- Issue
- 17
- Pages
- 3619-3630
- ISSN
- 0006-2960 (ISSN-L)
- 1520-4995
- PMC
- PMC2868174
- OpenAlex
- W2021976525
- Cited by
- 32
- References
- 46
- Citations by year
- 2024: 3 · 2023: 3 · 2020: 3 · 2019: 4 · 2018: 1 · 2017: 2 · 2015: 2 · 2013: 3 · 2012: 6
- FWCI
- 1.82
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Biochemistry (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Institute of General Medical Sciences (R01GM059802, GM59802, GM79686, R01GM079686)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Melanoma and MAPK Pathways
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Tyrosine Phosphatases
- Keywords
- ERK2, ETS1, protein phosphorylation, product launch, ADP dissociation, substrate recognition, catalytic mechanism, ternary complex, pre-steady-state kinetics, reverse reaction, transcription factor regulation, stopped-flow fluorescence, kinetic isotope effects
- MeSH terms
- Adenosine Diphosphate, Animals, Catalysis, Fluorescence Polarization, Kinetics, Models, Molecular, Magnetic Resonance Spectroscopy, Phosphorylation, Protein Conformation, Mitogen-Activated Protein Kinase 1, Spectrometry, Mass, Electrospray Ionization, Proto-Oncogene Protein c-ets-1, Mice
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review
