Site-specific NMR mapping and time-resolved monitoring of serine and threonine phosphorylation in reconstituted kinase reactions and mammalian cell extracts

Theillet, François‐Xavier; Rose, Honor May; Liokatis, Stamatios; Binolfi, Andrés; Thongwichian, Rossukon; Stuiver, Marchel; Selenko, Philipp

doi:10.1038/nprot.2013.083

Cited by 94 publications

(111 citation statements)

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“…2, A and B; Table 1). ERK2 phosphorylated Thr-50, Thr-153, Thr-175, Thr-181, Thr-205, Thr-231, Ser-235, Ser-404, and Ser-422 with a stoichiometry of Ͼ50%; estimation was based on peak integration (45) (Fig. 2B, Table 1).…”

Section: Identification Of Tau Amino Acids Phosphorylated Inmentioning

confidence: 99%

Characterization of Neuronal Tau Protein as a Target of Extracellular Signal-regulated Kinase

Prabakaran

Cantrelle

et al. 2016

Journal of Biological Chemistry

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Tau neuronal protein has a central role in neurodegeneration and is implicated in Alzheimer disease development. Abnormal phosphorylation of Tau impairs its interaction with other proteins and is associated with its dysregulation in pathological conditions. Molecular mechanisms leading to hyperphosphorylation of Tau in pathological conditions are unknown. Here, we characterize phosphorylation of Tau by extracellular-regulated kinase (ERK2), a mitogen-activated kinase (MAPK) that responds to extracellular signals. Analysis of in vitro phosphorylated Tau by activated recombinant ERK2 with nuclear magnetic resonance spectroscopy (NMR) reveals phosphorylation of 15 Ser/Thr sites. In vitro phosphorylation of Tau using rat brain extract and subsequent NMR analysis identifies the same sites. Phosphorylation with rat brain extract is known to transform Tau into an Alzheimer disease-like state. Our results indicate that phosphorylation of Tau by ERK2 alone is sufficient to produce the same characteristics. We further investigate the mechanism of ERK2 phosphorylation of Tau. Kinases are known to recognize their protein substrates not only by their specificity for a targeted Ser or Thr phosphorylation site but also by binding to linear-peptide motifs called docking sites. We identify two main ERK2 docking sites in Tau sequence using NMR. Our results suggest that ERK2 dysregulation in Alzheimer disease could lead to abnormal phosphorylation of Tau resulting in the pathology of the disease.Tau is an intrinsically disordered protein whose primary sequence is divided into several functional domains: an N-terminal region, a proline-rich domain (PRD), 2 a microtubule binding domain (MTBD) constituted of partially repeated sequences R1 to R4, and a C-terminal region (see Fig. 1A). Both PRD and MTBD are involved in microtubule stabilizing activity of Tau (1, 2).Phosphorylation of Tau is an essential process to regulate its physiological function(s); however, abnormal phosphorylation of Tau has been linked to neuronal dysfunction (3-5). In Alzheimer disease (AD) Tau is hyperphosphorylated and aggregated (6). The longest Tau protein isoform (441 residues) has 80 threonine (Thr) or serine (Ser) residues. These residues are exposed as Tau is an intrinsically disordered protein subject to modification by numerous kinases (7). Mass spectrometry (MS) analyses have identified ϳ45 phosphorylated sites on Tau aggregates extracted from AD patients with a typical paired helical filament (PHF) morphology (8, 9) ERK2 belongs to the mitogen-activated protein kinase (MAPKs) family and is activated by dual phosphorylation on a Thr-Xaa-Tyr sequence in its activation loop by MAP kinase kinases (MKK) (21-23). Human ERK2 is phosphorylated on Thr-183 and Tyr-185 by MEK kinase. MAP kinases have a classic kinase structure with an N-terminal lobe folded as a ␤ sheet associated to a larger C-terminal domain constituted of ␣-helices (24). The activation loop is located at the hinge between these domains and shows a large conformational change upon phosphor...

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Section: Identification Of Tau Amino Acids Phosphorylated Inmentioning

confidence: 99%

Characterization of Neuronal Tau Protein as a Target of Extracellular Signal-regulated Kinase

Prabakaran

Cantrelle

et al. 2016

Journal of Biological Chemistry

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“…29. All experiments were carried out on a Bruker 750 MHz spectrometer equipped with a cryogenically cooled triple-resonance probe.…”

Section: Time-resolved Nmr Profiling Of Wt and Mutant P53tad Phosphormentioning

confidence: 99%

“…Experiments were performed in duplicates. Comparative lysate phosphorylation rates were measured using quenched reaction setups 29 . Extracts were prepared from MCF7 cells containing shRNA-reduced levels of endogenous p53 or stably integrated WT p53 in the shRNA background in the presence of phosphatase inhibitors.…”

Section: Time-resolved Nmr Profiling Of Wt and Mutant P53tad Phosphormentioning

confidence: 99%

Disorder and residual helicity alter p53-Mdm2 binding affinity and signaling in cells

et al. 2014

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Levels of residual structure in disordered interaction domains determine in vitro bindingaffinities, but whether they exert similar roles in cells is not known. Here, we show that increasing residual p53 helicity results in stronger Mdm2 binding, altered p53 dynamics, impaired target gene expression and failure to induce cell cycle arrest upon DNA damage.These results establish that residual structure is an important determinant of signaling fidelity in cells.Intrinsically disordered protein domains often mediate protein-protein interactions that result in disorder-to-order transitions via coupled folding and binding reactions 1 . In addition, many disordered interaction domains exhibit defined levels of transient secondary structure resembling their complex-bound states when free in solution 2 . These levels of residual structure affect binding energies, also by reducing the loss of conformational entropy associated with disorderto-order transitions 3 . Accordingly, higher levels of residual structure in disordered interaction domains increase in vitro binding affinities 4,5 . Here we ask to what extent residual structure contributes to protein binding affinities in cells and whether engineered changes to residual structure affect protein function at the cellular level. To answer these questions, we designed p53 mutants with higher residual helicity within their disordered, N-terminal transactivation domains (TADs) and investigated their effects on cellular Mdm2 binding and p53's ability to induce 2 target gene expression and cell cycle arrest. p53 is activated by many forms of cellular stress, including DNA double-strand breaks (DSBs) and functions as a major tumor suppressor and cell cycle regulator 6 . In the absence of DNA damage, cellular p53 levels are kept low by targeted proteasomal degradation mediated by the E3 ubiquitin ligase Mdm2, which interacts with p53TAD and subsequently ubiquitinates p53's C-terminal regulatory domain 7 . Upon DNA damage, post-translational modifications of p53TAD and Mdm2, together with Mdm2 degradation, disrupt the p53-Mdm2 complex. This leads to p53 accumulation and the expression of p53 target genes that regulate DNA repair, cell cycle arrest, senescence or apoptosis 8,9 . One of these target genes is Mdm2, whose expression establishes a negative feedback loop that shapes cellular p53 dynamics and thereby controls cell fate decisions 10 .In its free form, p53TAD exists in equilibrium between disordered and partially helical conformations 11-13 , whereas residues 19-25 form a stable amphipathic α-helix in the Mdm2 complex 14 (Fig. 1a). To increase the binding affinity between p53 and Mdm2 without altering the binding interface, we designed p53TAD mutants with higher levels of residual helicity by mutating conserved proline residues flanking the Mdm2 binding site (i.e., Pro12, Pro13 or Pro27) to alanines (Supplementary Results, Supplementary Fig. 1a). Using NMR spectroscopy, we determined that wild-type (WT) p53TAD helicity (28%) increased to 64% when we replaced Pro27 with ...

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“…To obtain atomic-resolution insights into phosphorylation of the Elk-1 TAD, we used nuclear magnetic resonance (NMR) spectroscopy (20) to monitor its modification by recombinant ERK2 in vitro ( Fig. 1B; fig.…”

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Opposing effects of Elk-1 multisite phosphorylation shape its response to ERK activation

Mylona

Theillet

Foster

et al. 2016

Science

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Multisite phosphorylation regulates many transcription factors, including the Serum Response Factor partner Elk-1. Phosphorylation of the transcriptional activation domain (TAD) of Elk-1 by the protein kinase ERK at multiple sites potentiates recruitment of the Mediator transcriptional coactivator complex and transcriptional activation, but the roles of individual phosphorylation events remained unclear. Using time-resolved nuclear magnetic resonance spectroscopy, we found that ERK2 phosphorylation proceeds at markedly different rates at eight TAD sites in vitro, which we classified as fast, intermediate and slow. Mutagenesis experiments showed that phosphorylation of fast and intermediate sites promoted Mediator interaction and transcriptional activation, whereas modification of slow sites counteracted both functions, thereby limiting Elk-1 output. Progressive Elk-1 phosphorylation thus ensures a self-limiting response to ERK activation, which occurs independently of antagonizing phosphatase activity. Results and DiscussionMultisite protein phosphorylation increases the complexity of functional signaling outputs that can be generated from single protein kinase inputs. It can set thresholds for activity, or transform graded signals into switch-like responses (1-4). Many transcription factors and their interacting regulatory proteins are subject to multisite phosphorylation, which allows distinct aspects of protein function, including protein turnover, nuclear import and export, and specific protein interactions, to be controlled independently (5). However, in general, the † The ternary complex factor (TCF) subfamily of Ets-domain transcription factors, consisting of Elk-1, SAP-1, and Net, provides an example of multisite phosphorylation in transcriptional activation. TCFs, together with their partner protein SRF, function in many biological processes by coupling SRF target genes to mitogen-activated protein kinase (MAP kinase) signaling (5). Mitogenic and stress stimuli induce phosphorylation of TCF Cterminal transcriptional activation domains (TADs) at multiple S/T-P (Ser-or Thr-Pro) phosphorylation sequences, of which eight are conserved across the family ( Fig. 1A; fig. S1) (6-11). Two MAP kinase-docking sites, the D-box and the Phe-Gln-Phe-Pro (FQFP) motif, control phosphorylation of these sites (12-15). Multisite phosphorylation triggers transcriptional activation by TCFs, facilitating their interaction with the Mediator transcriptional co-activator complex (16-19) but the kinetics with which the different sites are phosphorylated, and whether they serve distinct functions, remain unclear.To obtain atomic-resolution insights into phosphorylation of the Elk-1 TAD, we used nuclear magnetic resonance (NMR) spectroscopy (20) Fig. 2A). In the fast site mutant Elk-1F, phosphorylation rates of intermediate and slow sites increased, whereas those of the fast and slow sites increased in the intermediate-site mutant Elk-1I; in both cases the altered kinetics fit well with those predicted by the model (Fig 2B,...

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Site-specific NMR mapping and time-resolved monitoring of serine and threonine phosphorylation in reconstituted kinase reactions and mammalian cell extracts

Cited by 94 publications

References 84 publications

Characterization of Neuronal Tau Protein as a Target of Extracellular Signal-regulated Kinase

Characterization of Neuronal Tau Protein as a Target of Extracellular Signal-regulated Kinase

Disorder and residual helicity alter p53-Mdm2 binding affinity and signaling in cells

Opposing effects of Elk-1 multisite phosphorylation shape its response to ERK activation

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