14-3-3 proteins inactivate DAPK2 by promoting its dimerization and protecting key regulatory phosphosites

Horvath, Matej; Petrvalská, Olívia; Heřman, Petr; Obšilová, Veronika; Obšil, Tomáš

doi:10.1038/s42003-021-02518-y

Cited by 24 publications

(25 citation statements)

References 78 publications

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Section: Discussionsupporting

confidence: 93%

“…4f,g). This is in line with previous observations where 14-3-3 protected other phospho-client proteins from dephosphorylation 44,45 and suggests that 14-3-3 binding modulates the phosphoregulation of N. SARS-CoV-2 N fragments 193-200 and 201-210 co-crystallized with 14-3-3 provide a valuable structural framework for analyzing the effect of the widespread mutations and testing new hypotheses. Collectively, our analyses based on structural, interactomic and bioinformatic data support the hypothesis 17 that N mutations improving SARS-CoV-2 fitness positively affect its binding to 14-3-3 (Fig.…”

Section: Discussionsupporting

confidence: 90%

“…Since the improved replicative fitness of the virus correlates well with the increased levels of N phosphorylation 20,24 , factors promoting N phosphorylation or postponing its dephosphorylation appear particularly relevant for the success of the virus. Noteworthily, 14-3-3 proteins play a well-recognized role in both the replication of many viruses 30 and in protecting their bound phospho-clients from dephosphorylation 44,45 , which is likely the case for phosphorylated SARS-CoV-2 N when bound to 14-3-3. We have shown that multiply phosphorylated SARS-CoV-2 N bearing several physiologicallyrelevant phosphosites 4,18 can be successfully reconstituted in vitro to induce its interaction with 14-3-3 26 .…”

Section: Discussionmentioning

confidence: 99%

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Human 14-3-3 proteins site-selectively bind the mutational hotspot region of SARS-CoV-2 nucleoprotein modulating its phosphoregulation

Tugaeva

Sysoev

Smith

et al. 2021

Preprint

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The SARS-CoV-2 nucleocapsid protein (N) is responsible for viral genome packaging and virion assembly. Being highly abundant in the host cell, N interacts with numerous human proteins and undergoes multisite phosphorylation in vivo. When phosphorylated within its Ser/Arg-rich region, a tract highly prone to mutations as exemplified in the Omicron and Delta variants, N recruits human 14-3-3 proteins, potentially hijacking their functions. Here, we show that in addition to phosphorylated Ser197, an alternative, less conserved phosphosite at Thr205 not found in SARS-CoV N binds 14-3-3 with micromolar affinity and is in fact preferred over pS197. Fluorescence anisotropy reveals a distinctive pT205/pS197 binding selectivity towards the seven human 14-3-3 isoforms. Crystal structures explain the structural basis for the discovered selectivity towards SARS-CoV-2 N phosphopeptides, and also enable prediction for how N variants interact with 14-3-3, suggesting a link between the strength of this interaction and replicative fitness of emerging coronavirus variants.

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Section: Discussionsupporting

confidence: 93%

Section: Discussionsupporting

confidence: 90%

Section: Discussionmentioning

confidence: 99%

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Human 14-3-3 proteins site-selectively bind the mutational hotspot region of SARS-CoV-2 nucleoprotein modulating its phosphoregulation

Tugaeva

Sysoev

Smith

et al. 2021

Preprint

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“…(B) The canonical mode II phosphopeptide (sequence RLYH(pS)LPA) bound to 14-3-3ζ (PDB ID: 1QJA) (Rittinger et al, 1999). (C) The canonical mode III phosphopeptide derived from the C-terminus of human DAPK2 (sequence RRSS(pT)S) bound to 14-3-3γ (PDB ID: 7A6R) (Horvath et al, 2021). (D) The non-phosphorylated ExoS peptide (sequence GHGQGLLDALDLAS) bound to 14-3-3ζ (PDB ID: 2O02) (Ottmann et al, 2007b).…”

Section: Homo-and Heterodimerization Of 14-3-3 Proteinsmentioning

confidence: 99%

Structural insights into the functional roles of 14-3-3 proteins

Obšilová

Obšil

2022

Front. Mol. Biosci.

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Signal transduction cascades efficiently transmit chemical and/or physical signals from the extracellular environment to intracellular compartments, thereby eliciting an appropriate cellular response. Most often, these signaling processes are mediated by specific protein-protein interactions involving hundreds of different receptors, enzymes, transcription factors, and signaling, adaptor and scaffolding proteins. Among them, 14-3-3 proteins are a family of highly conserved scaffolding molecules expressed in all eukaryotes, where they modulate the function of other proteins, primarily in a phosphorylation-dependent manner. Through these binding interactions, 14-3-3 proteins participate in key cellular processes, such as cell-cycle control, apoptosis, signal transduction, energy metabolism, and protein trafficking. To date, several hundreds of 14-3-3 binding partners have been identified, including protein kinases, phosphatases, receptors and transcription factors, which have been implicated in the onset of various diseases. As such, 14-3-3 proteins are promising targets for pharmaceutical interventions. However, despite intensive research into their protein-protein interactions, our understanding of the molecular mechanisms whereby 14-3-3 proteins regulate the functions of their binding partners remains insufficient. This review article provides an overview of the current state of the art of the molecular mechanisms whereby 14-3-3 proteins regulate their binding partners, focusing on recent structural studies of 14-3-3 protein complexes.

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“…Presumably, increased dynamics associated with such architectures might have prevented structure determination in other complexes. Interestingly, solution studies designed to characterize dynamic protein-protein interactions, such as small angle X-Ray scattering (SAXS), have previously pointed to similarly asymmetric binding poses between 14-3-3 and the CaMKK2 and DAPK2 kinases 34,35 . While in our experiments 14-3-3 binding clearly reduces the overall diversity of PEAK3’s interactome, the unique architecture of the PEAK3/14-3-3 complex captured in our structure leaves the unstructured PEAK3 N-termini accessible.…”

Section: Discussionmentioning

confidence: 99%

Structural insights into regulation of the PEAK3 pseudokinase scaffold by 14-3-3

Torosyan

Paul²,

Forget³

et al. 2022

Preprint

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The three members of the PEAK family of pseudokinases (PEAK1, PEAK2, and PEAK3) are molecular scaffolds that have recently emerged as important regulatory nodes in signaling pathways that control cell migration, morphology, and proliferation, and they are increasingly found to be mis-regulated in human cancers. While no structures of PEAK3 have been solved to date, crystal structures of the PEAK1 and PEAK2 pseudokinase domains revealed their dimeric organization. It remains unclear how dimerization plays a role in PEAK scaffolding functions, as no structures of PEAK family members in complex with their binding partners have been solved. Here, we report the cryo-EM structure of the PEAK3 pseudokinase, also adopting a dimeric state, and in complex with an endogenous 14-3-3 heterodimer purified from mammalian cells. Our structure reveals an asymmetric binding mode between PEAK3 and 14-3-3 stabilized by one pseudokinase domain and the Split HElical Dimerization (SHED) domain of the PEAK3 dimer. The binding interface is comprised of a canonical primary interaction involving two phosphorylated 14-3-3 consensus binding sites located in the N-terminal domains of the PEAK3 monomers docked in the conserved amphipathic grooves of the 14-3-3 dimer, and a unique secondary interaction between 14-3-3 and PEAK3 that has not been observed in any previous structures of 14-3 3/client complexes. Disruption of these interactions results in the relocation of PEAK3 to the nucleus and changes its cellular interactome. Lastly, we identify Protein Kinase D as the regulator of the PEAK3/14-3-3 interaction, providing a mechanism by which the diverse functions of the PEAK3 scaffold might be fine-tuned in cells.

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14-3-3 proteins inactivate DAPK2 by promoting its dimerization and protecting key regulatory phosphosites

Cited by 24 publications

References 78 publications

Human 14-3-3 proteins site-selectively bind the mutational hotspot region of SARS-CoV-2 nucleoprotein modulating its phosphoregulation

Human 14-3-3 proteins site-selectively bind the mutational hotspot region of SARS-CoV-2 nucleoprotein modulating its phosphoregulation

Structural insights into the functional roles of 14-3-3 proteins

Structural insights into regulation of the PEAK3 pseudokinase scaffold by 14-3-3

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