The Functional Interaction of 14-3-3 Proteins with the ERK1/2 Scaffold KSR1 Occurs in an Isoform-specific Manner

Jagemann, Lucas; Pérez-Rivas, Luis Gustavo; Ruiz, E. Josué; Ranea, Juan A. G.; Sánchez‐Jiménez, Francisca; Nebreda, Ángel R.; Alba, Emilio; Lozano, José

doi:10.1074/jbc.m709185200

Cited by 33 publications

(32 citation statements)

References 49 publications

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“…However, only 14-3-3␥, but neither 14-3-3␤ nor 14-3-3, could bind RapE63. Similarly, only 14-3-3␥, but neither 14-3-3␤ nor 14-3-3, can bind KSR (45). This is consistent with the contribution of KSR to 14-3-3 binding to phosphorylated Rap1.…”

Section: Ksr Is Required For B-raf To Bind Rap1 and Signal To Erks-supporting

confidence: 75%

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Phosphorylation of Rap1 by cAMP-dependent Protein Kinase (PKA) Creates a Binding Site for KSR to Sustain ERK Activation by cAMP

Takahashi

Dillon

et al. 2017

Journal of Biological Chemistry

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Edited by Jeffrey E. PessinCyclic adenosine monophosphate (cAMP) is an important mediator of hormonal stimulation of cell growth and differentiation through its activation of the extracellular signal-regulated kinase (ERK) cascade. Two small G proteins, Ras and Rap1 have been proposed to mediate this activation. Using HEK293 cells as a model system, we have recently shown that both Ras and Rap1 are required for cAMP signaling to ERKs. However, cAMP-dependent Ras signaling to ERKs is transient and rapidly terminated by PKA phosphorylation of the Raf isoforms C-Raf and B-Raf. In contrast, cAMP-dependent Rap1 signaling to ERKs and Rap1 is potentiated by PKA. We show that this is due to sustained binding of B-Raf to Rap1. One of the targets of PKA is Rap1 itself, directly phosphorylating Rap1a on serine 180 and Rap1b on serine 179. We show that these phosphorylations create potential binding sites for the adaptor protein 14-3-3 that links Rap1 to the scaffold protein KSR. These results suggest that Rap1 activation of ERKs requires PKA phosphorylation and KSR binding. Because KSR and B-Raf exist as heterodimers within the cell, this binding also brings B-Raf to Rap1, allowing Rap1 to couple to ERKs through B-Raf binding to Rap1 independently of its Ras-binding domain.Hormones that are coupled to the second messenger cyclic adenosine monophosphate (cAMP) signal to multiple intracellular pathways. One of these, the MAP kinase, or ERK (extracellular signal-regulated kinase) cascade, mediates many physiological functions of cAMP in development (1), metabolism (2, 3), cognition (4 -6), and cell growth (7-12). In all cases, cAMP signaling to ERKs requires the recruitment of the MAP kinase kinase kinase Raf to one of two small G proteins, Ras or Rap1. The ability to signal to both Ras and Rap1 allows cAMP to trigger the sustained activation of ERKs, which is required for many physiological functions, including the transcription/stabilization of many transcription factors (13,14).In addition to its ability to activate both Ras and Rap1, cAMP can inhibit signaling to Ras. PKA antagonizes Ras signaling through its phosphorylation of the Raf isoform C-Raf on serine 259 (Ser-259) (15). This phosphorylation and subsequent recruitment of 14-3-3 result in the release of C-Raf from active Ras (15, 16). We have recently shown that PKA phosphorylation of the homologous site in B-Raf (Ser-365) similarly prevents the direct binding of B-Raf to Ras (16). Despite this phosphorylation of B-Raf, B-Raf is still capable of binding to Rap1. This is physiologically relevant, as Rap1/B-Raf links PKA to ERKs (17, 18). How Rap1 is able to couple to B-Raf following PKA phosphorylation of B-Raf is unknown.We and others have identified Rap1 as a target for phosphorylation by PKA (19 -21). Here, we show that Rap1 phosphorylation plays a role in the ability of B-Raf to remain bound to Rap1 following cAMP stimulation. Rap1 phosphorylation creates a novel 14-3-3 binding site that connects Rap1 to the scaffold KSR. Because KSR can exist as a dimer with B-...

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Section: Ksr Is Required For B-raf To Bind Rap1 and Signal To Erks-supporting

confidence: 75%

“…5C). It has been shown that KSR binds selectively to 14-3-3␥, but not 14-3-3␤ or 14-3-3 (45). Only 14-3-3␥, but neither 14-3-3␤ nor 14-3-3, could bind RapE63 (Fig.…”

Section: -3-3 Binding To Rap1mentioning

confidence: 99%

Phosphorylation of Rap1 by cAMP-dependent Protein Kinase (PKA) Creates a Binding Site for KSR to Sustain ERK Activation by cAMP

Takahashi

Dillon

et al. 2017

Journal of Biological Chemistry

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“…20 The Lys50 residue is located on the charged face of the binding groove (together with Arg57, Arg61, Arg132, and Tyr133) ( Figure 1) and is essential for ligand binding. [61][62][63] All four subjects (B and D-F) from this study are females with seizure onset in the first year of life, although a precise age of onset for subject F is unknown. Subjects B, E, and F have the same mutation and similar epilepsy phenotype, which includes generalized epilepsy with myoclonic, (atypical) absence, and generalized tonic-clonic (GTC) seizures.…”

mentioning

confidence: 99%

De Novo Mutations in YWHAG Cause Early-Onset Epilepsy

Guella

McKenzie

Evans

et al. 2017

The American Journal of Human Genetics

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Massively parallel sequencing has revealed many de novo mutations in the etiology of developmental and epileptic encephalopathies (EEs), highlighting their genetic heterogeneity. Additional candidate genes have been prioritized in silico by their co-expression in the brain. Here, we evaluate rare coding variability in 20 candidates nominated with the use of a reference gene set of 51 established EE-associated genes. Variants within the 20 candidate genes were extracted from exome-sequencing data of 42 subjects with EE and no previous genetic diagnosis. We identified 7 rare non-synonymous variants in 7 of 20 genes and performed Sanger sequence validation in affected probands and parental samples. De novo variants were found only in SLC1A2 (aka EAAT2 or GLT1) (c.244G>A [p.Gly82Arg]) and YWHAG (aka 14-3-3g) (c.394C>T [p.Arg132Cys]), highlighting the potential cause of EE in 5% (2/42) of subjects. Seven additional subjects with de novo variants in SLC1A2 (n ¼ 1) and YWHAG (n ¼ 6) were subsequently identified through online tools. We identified a highly significant enrichment of de novo variants in YWHAG, establishing their role in early-onset epilepsy, and we provide additional support for the prior assignment of SLC1A2. Hence, in silico modeling of brain co-expression is an efficient method for nominating EE-associated genes to further elucidate the disorder's etiology and genotype-phenotype correlations.

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“…Isoformspecific functions of 14-3-3 proteins are difficult to explain, because these molecules share a high degree of structural homology, particularly in their binding regions. Nevertheless, some studies have begun to clarify the structural basis for their specific roles (20,62). Mouse transgenic approaches provide an important tool with which to define the different roles of 14-3-3 isoforms in vivo while awaiting additional insights from structural studies.…”

Section: Discussionmentioning

confidence: 99%

“…(g to i) Expression of 14-3-3ε (green), examined by immunofluorescence staining on frozen E18.5 sections. (48) 23 (20) a No significant differences between the genotypes are evident.…”

Section: -3-3 Is Expressed In the Developing Mouse Heart And Embryomentioning

confidence: 99%

14-3-3ε Plays a Role in Cardiac Ventricular Compaction by Regulating the Cardiomyocyte Cell Cycle

Kosaka

Cieslik

et al. 2012

Molecular and Cellular Biology

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f Trabecular myocardium accounts for the majority of the ventricles during early cardiogenesis, but compact myocardium is the primary component at later developmental stages. Elucidation of the genes regulating compact myocardium development is essential to increase our understanding of left ventricular noncompaction (LVNC), a cardiomyopathy characterized by increased ratios of trabecular to compact myocardium. 14-3-3 is an adapter protein expressed in the lateral plate mesoderm, but its in vivo cardiac functions remain to be defined. Here we show that 14-3-3 is expressed in the developing mouse heart as well as in cardiomyocytes. 14-3-3 deletion did not appear to induce compensation by other 14-3-3 isoforms but led to ventricular noncompaction, with features similar to LVNC, resulting from a selective reduction in compact myocardium thickness. Abnormal compaction derived from a 50% decrease in cardiac proliferation as a result of a reduced number of cardiomyocytes in G 2 /M and the accumulation of cardiomyocytes in the G 0 /G 1 phase of the cell cycle. These defects originated from downregulation of cyclin E1 and upregulation of p27 Kip1 , possibly through both transcriptional and posttranslational mechanisms. Our work shows that 14-3-3 regulates cardiogenesis and growth of the compact ventricular myocardium by modulating the cardiomyocyte cell cycle via both cyclin E1 and p27Kip1 . These data are consistent with the long-held view that human LVNC may result from compaction arrest, and they implicate 14-3-3 as a new candidate gene in congenital human cardiomyopathies.

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The Functional Interaction of 14-3-3 Proteins with the ERK1/2 Scaffold KSR1 Occurs in an Isoform-specific Manner

Cited by 33 publications

References 49 publications

Phosphorylation of Rap1 by cAMP-dependent Protein Kinase (PKA) Creates a Binding Site for KSR to Sustain ERK Activation by cAMP

Phosphorylation of Rap1 by cAMP-dependent Protein Kinase (PKA) Creates a Binding Site for KSR to Sustain ERK Activation by cAMP

De Novo Mutations in YWHAG Cause Early-Onset Epilepsy

14-3-3ε Plays a Role in Cardiac Ventricular Compaction by Regulating the Cardiomyocyte Cell Cycle

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