Structural assembly of the signaling competent ERK2–RSK1 heterodimeric protein kinase complex

Alexa, Anita; Gógl, Gergő; Glatz, Gábor; Garai, Ágnes; Zeke, András; Várga, J.; Dudás, Erika; Jeszenői, Norbert; Bodor, Andrea; Hetényi, Csaba; Reményi, Attila

doi:10.1073/pnas.1417571112

Cited by 31 publications

(36 citation statements)

References 41 publications

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“…These approaches identified a ‘hotspot’ mutation in ERK2 (E322K) that displays GOF properties in validation experiments (Figure 4B, 4C, 4E). Orthogonal computational approaches, including CLUMPS, additionally identified enrichment ( p =0.0053) of mutant residues (including E322K) within ERK2 effector recruitment domains (Alexa et al, 2015; Kamburov et al, 2015; Lawrence et al, 2014). This observation was buttressed by the finding that additional ERK2 tumor-associated mutations not found within the CLUMPS mutational search space were are also enriched ( p = 4.303 × 10 −5 , Fishers exact test) in ERK2 effector recruitment domains (Figure 5A) (Lee et al, 2004; Zhang et al, 2003).…”

Section: Resultsmentioning

confidence: 99%

Phenotypic Characterization of a Comprehensive Set of MAPK1 /ERK2 Missense Mutants

et al. 2016

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SUMMARY Tumor-specific genomic information has the potential to guide therapeutic strategies and revolutionize patient treatment. Currently, this approach is limited by an abundance of disease-associated mutants whose biological functions and impacts on therapeutic response are uncharacterized. To begin to address this limitation, we functionally characterized nearly all (99.84%) missense mutants of MAPK1/ERK2, an essential effector of oncogenic RAS and RAF. Using this approach, we discovered rare gain- and loss-of-function ERK2 mutants found in human tumors, revealing that, in the context of this assay, mutational frequency alone cannot identify all functionally impactful mutants. Gain-of-function ERK2 mutants induced variable responses to RAF-, MEK- and ERK-directed therapies, providing a reference for future treatment decisions. Tumor-associated mutations spatially clustered in two ERK2 effector-recruitment domains, yet produced mutants with opposite phenotypes. This approach articulates an allele-characterization framework that can be scaled to meet the goals of genome-guided oncology.

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Section: Resultsmentioning

confidence: 99%

Phenotypic Characterization of a Comprehensive Set of MAPK1 /ERK2 Missense Mutants

et al. 2016

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“…Recently, the structures of ERK in complex with protein substrates PEA15 and RSK1 were solved and found to involve a DRS-D site interaction using a reverse-orientation D site (28,33). The structures of peptides corresponding to the MNK1 and RSK1 reverse D sites in complex with ERK have also been solved (32).…”

Section: Resultsmentioning

confidence: 99%

“…Crystal structures of ERK in complex with its binding partners (28)(29)(30)(31)(32)(33) provide invaluable insights, many of which formed the basis of this study. However, the ability to form crystals of a complex is not guaranteed, and most of the structures of ERK complexes use linear peptides derived from the ERK-interacting molecules instead of structured proteins.…”

Section: Discussionmentioning

confidence: 99%

Mapping the binding interface of ERK and transcriptional repressor Capicua using photocrosslinking

Futran

Kyin

Shvartsman

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Extracellular signal-regulated kinase (ERK) coordinates cellular responses to a range of stimuli by phosphorylating its numerous substrates. One of these substrates, Capicua (Cic), is a transcriptional repressor that was first identified in Drosophila and has been implicated in a number of human diseases. Here we use a chemical biology approach to map the binding interface of ERK and Cic. The noncanonical amino acid p-azidophenylalanine (AzF) was introduced into the ERK-binding region of Drosophila Cic, and photocrosslinking and tandem mass spectrometry were used to pinpoint its binding site on ERK. We also identified the ERK-binding region of human Cic and showed that it binds to the same site on ERK despite lacking conservation with the Drosophila Cic binding region. Finally, we mapped the amino acids involved in human Cic binding to ERK using AzF-labeled ERK. These results reveal the molecular details of the ERK-Cic interaction and demonstrate that the photocrosslinking approach is complementary to existing methods for mapping kinase-substrate binding interfaces.photocrosslinking | protein-protein interactions | signal transduction

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“…A). ERK2 forms a complex with RSK1 in which the activation loop of the CTKD becomes phosphorylated at Thr573 . Importantly, there is an autophosphorylation site at the RSK1 CTT (Ser732), which has a role in the dissociation of the ERK–RSK binary complex .…”

Section: Introductionmentioning

confidence: 99%

Dynamic control of RSK complexes by phosphoswitch‐based regulation

et al. 2017

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Assembly and disassembly of protein-protein complexes needs to be dynamically controlled and phosphoswitches based on linear motifs are crucial in this process. Extracellular signal-regulated kinase 2 (ERK2) recognizes a linearbinding motif at the C-terminal tail (CTT) of ribosomal S6 kinase 1 (RSK1), leading to phosphorylation and subsequent activation of RSK1. The CTT also contains a classical PDZ domain-binding motif which binds RSK substrates (e.g. MAGI-1). We show that autophosphorylation of the disordered CTT promotes the formation of an intramolecular charge clamp, which efficiently masks critical residues and indirectly hinders ERK binding. Thus, RSK1 CTT operates as an autoregulated phosphoswitch: its phosphorylation at specific sites affects its protein-binding capacity and its conformational dynamics. These biochemical feedbacks, which form the structural basis for the rapid dissociation of ERK2-RSK1 and RSK1-PDZ substrate complexes under sustained epidermal growth factor (EGF) stimulation, were structurally characterized and validated in living cells. Overall, conformational changes induced by phosphorylation in disordered regions of protein kinases, coupled to allosteric events occurring in the kinase domain cores, may provide mechanisms that contribute to the emergence of complex signaling activities. In addition, we show that phosphoswitches based on linear motifs can be functionally classified as ON and OFF protein-protein interaction switches or dimmers, depending on the specific positioning of phosphorylation target sites in relation to functional linear-binding motifs. Moreover, interaction of phosphorylated residues with positively charged residues in disordered regions is likely to be a common mechanism of phosphoregulation.

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Structural assembly of the signaling competent ERK2–RSK1 heterodimeric protein kinase complex

Cited by 31 publications

References 41 publications

Phenotypic Characterization of a Comprehensive Set of MAPK1 /ERK2 Missense Mutants

Phenotypic Characterization of a Comprehensive Set of MAPK1 /ERK2 Missense Mutants

Mapping the binding interface of ERK and transcriptional repressor Capicua using photocrosslinking

Dynamic control of RSK complexes by phosphoswitch‐based regulation

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