14-3-3 protein directly interacts with the kinase domain of calcium/calmodulin-dependent protein kinase kinase (CaMKK2)

Pšenáková, Katarína; Petrvalská, Olívia; Kylarová, Salome; Santo, Domenico Lentini; Kalabova, Dana; Heřman, Petr; Obšilová, Veronika; Obšil, Tomáš

doi:10.1016/j.bbagen.2018.04.006

Cited by 31 publications

(82 citation statements)

References 57 publications

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“…This discrepancy can perhaps be explained on the ground that the reaction time for PKA phosphorylation used in this study was apparently short ( Fig. 4B and C, at 30 o C for 60 min) as compared to that in a recent report (at 30 °C for 3 h and then overnight at 4 °C [34]) and/or the kinase activity of truncated form of CaMKKβ (93-517) used in a recent report [34] might be more sensitive to PKA-mediated phosphorylation than that of wild-type enzyme. It is noteworthy that CaMKKα has demonstrated to be phosphorylated at Thr108 by PKA that is equivalent to Thr144 in CaMKKβ, resulting in suppression of Ca 2+ /CaM-dependent kinase activity [20,21] unlike CaMKKβ.…”

Section: Regulatory Phosphorylation Of Camkkβ By Pkacontrasting

confidence: 74%

“…(kinase-dead mutant) as substrates, respectively [34]. This discrepancy can perhaps be explained on the ground that the reaction time for PKA phosphorylation used in this study was apparently short ( Fig.…”

Section: Regulatory Phosphorylation Of Camkkβ By Pkamentioning

confidence: 76%

“…It is noteworthy that CaMKKα has demonstrated to be phosphorylated at Thr108 by PKA that is equivalent to Thr144 in CaMKKβ, resulting in suppression of Ca 2+ /CaM-dependent kinase activity [20,21] unlike CaMKKβ. Furthermore, it has been reported that the catalytic activity of phosphorylated CaMKK2 (CaMKKβ) by PKA was not inhibited by 14-3-3γ binding [34] whereas the 14-3-3 protein binding suppressed the activity of phosphorylated CaMKKα [22]. Therefore, cAMP/PKA signaling may differentially regulate CaMKK isoforms in intact cells.…”

Section: Regulatory Phosphorylation Of Camkkβ By Pkamentioning

confidence: 99%

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Regulation of Ca2+/calmodulin-dependent protein kinase kinase β by cAMP signaling

Takabatake

Ohtsuka

Sugawara

et al. 2019

Biochimica et Biophysica Acta (BBA) - General Subjects

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BACKGROUND: Ca 2+ /calmodulin-dependent protein kinase kinase (CaMKK) is a pivotal activator of CaMKI, CaMKIV and 5'-AMP-activated protein kinase (AMPK), controlling Ca 2+-dependent intracellular signaling including various neuronal, metabolic and pathophysiological responses. Recently, we demonstrated that CaMKKβ is feedback phosphorylated at Thr144 by the downstream AMPK, resulting in the conversion of CaMKKβ into Ca 2+ /CaM-dependent enzyme. However, the regulatory phosphorylation of CaMKKβ at Thr144 in intact cells and in vivo remains unclear. METHODS: Anti-phosphoThr144 antibody was used to characterize the site-specific phosphorylation of CaMKKβ in immunoprecipitated samples from mouse cerebellum and in transfected mammalian cells that were treated with various agonists and protein kinase inhibitors. CaMKK activity assay and LC-MS/MS analysis were used for biochemical characterization of phosphorylated CaMKKβ. RESULTS: Our data suggest that the phosphorylation of Thr144 in CaMKKβ is rapidly induced by cAMP/cAMP-dependent protein kinase (PKA) signaling in CaMKKβ-transfected HeLa cells, that is physiologically relevant in mouse cerebellum. We confirmed that the catalytic subunit of PKA was capable of directly phosphorylating CaMKKβ at Thr144 in vitro and in transfected cells. In addition, the basal phosphorylation of CaMKKβ at Thr144 in transfected HeLa cells was suppressed by AMPK inhibitor (compound C). PKA-catalyzed phosphorylation reduced the autonomous activity of CaMKKβ in vitro without significant effect on the Ca 2+ /CaM-dependent activity, resulting in the conversion of CaMKKβ into Ca 2+ /CaM-dependent enzyme. CONCLUSION: cAMP/PKA signaling may confer Ca 2+-dependency to the CaMKKβ-mediated signaling pathway through direct phosphorylation of Thr144 in intact cells. GENERAL SIGNIFICANCE: Our results suggest a novel cross-talk between cAMP/PKA and Ca 2+ /CaM/ CaMKKβ signaling through regulatory phosphorylation.

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Section: Regulatory Phosphorylation Of Camkkβ By Pkacontrasting

confidence: 74%

Section: Regulatory Phosphorylation Of Camkkβ By Pkamentioning

confidence: 76%

Section: Regulatory Phosphorylation Of Camkkβ By Pkamentioning

confidence: 99%

See 1 more Smart Citation

Regulation of Ca2+/calmodulin-dependent protein kinase kinase β by cAMP signaling

Takabatake

Ohtsuka

Sugawara

et al. 2019

Biochimica et Biophysica Acta (BBA) - General Subjects

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“…Unlike the pT 594 ERα phosphosite from which phosphopeptide 3 is derived, the N‐terminal domain of CaMKK2 pS 100 phosphosite is not a canonical mode III 14‐3‐3 binder . However, the crystal structure of 14‐3‐3ζ in complex with a phosphopeptide derived from the CaMKK2 pS 100 site (sequence RKLpS 100 LQER, PDB ID: 6EWW) indicates that it mimics a canonical mode III binder and that the FC pocket in this complex is largely unoccupied and available for potential binding of stabilizers such as ( R )‐ 2 …”

Section: Resultsmentioning

confidence: 99%

“…We determined the binding affinity of CaMKK2 pS 100 13‐mer phosphopeptide 4 to 14‐3‐3ζ by SPR (estimated K d 112±14 μ m , Figure S12) and found it significantly lower than for 14‐3‐3ζ/ 3 ( K d 227±14 n m ). An FP assay was then developed based on a FAM‐labeled derivative of 4 (FAM‐ 4 ).…”

Section: Resultsmentioning

confidence: 99%

Design of Drug‐Like Protein–Protein Interaction Stabilizers Guided By Chelation‐Controlled Bioactive Conformation Stabilization

Bosica

Andrei

Neves

et al. 2020

Chemistry A European J

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Protein-protein interactions( PPIs) of 14-3-3 proteins are am odel system for studyingPPI stabilization. The complex natural product Fusicoccin As tabilizes many 14-3-3 PPIs but is not amenable for use in SAR studies, motivating the search for more drug-like chemical matter.H owever, drug-like 14-3-3PPI stabilizers enablingsuch studies have remainede lusive. An X-ray crystal structure of aP PI in complex with an extremelyl ow potencys tabilizer uncovered an unexpected non-protein interacting, ligand-chelated Mg 2 + leading to the discovery of metal-ion-dependent 14-3-3 PPI stabilization potency.T his originates from an ovel chelationcontrolled bioactive conformation stabilization effect. Metal chelation hasb een associated with pan-assayi nterference compounds (PAINS) and frequent hitter behavior,b ut chelation can evidently also lead to true potencyg ains and find use as am edicinal chemistry strategy to guidec ompound optimization. To demonstrate this, we exploited the effect to designt he first potent, selective, and drug-like 14-3-3 PPI stabilizers.Supporting information and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.10 mm Mg 2 + ."Fold increase" (y-axis)i st he mean fold-increase of signal over baseline (no (R)-2). (e)Ca 2 + (red triangles) and Mn 2 + (purple squares)a lso increase apparente fficacy of (R)-2 in 14-3-3/ERa(pT 594 )FPa ssay.

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14‐3‐3 Protein‐Protein Interactions: From Mechanistic Understanding to Their Small‐Molecule Stabilization

Somsen,

Cossar,

Arkin

et al. 2024

ChemBioChem

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Protein‐protein interactions (PPIs) are of utmost importance for maintenance of cellular homeostasis. Herein, a central role can be found for 14‐3‐3 proteins. These hub‐proteins are known to bind hundreds of interaction partners, thereby regulating their activity, localization, and/or stabilization. Due to their ability to bind a large variety of client proteins, studies of 14‐3‐3 protein complexes flourished over the last decades, aiming to gain greater molecular understanding of these complexes and their role in health and disease. Because of their crucial role within the cell, 14‐3‐3 protein complexes are recognized as highly interesting therapeutic targets, encouraging the discovery of small molecule modulators of these PPIs. We discuss various examples of 14‐3‐3‐mediated regulation of its binding partners on a mechanistic level, highlighting the versatile and multi‐functional role of 14‐3‐3 within the cell. Furthermore, an overview is given on the development of stabilizers of 14‐3‐3 protein complexes, from initially used natural products to fragment‐based approaches. These studies show the potential of 14‐3‐3 PPI stabilizers as novel agents in drug discovery and as tool compounds to gain greater molecular understandings of the role of 14‐3‐3‐based protein regulation.

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14-3-3 protein directly interacts with the kinase domain of calcium/calmodulin-dependent protein kinase kinase (CaMKK2)

Cited by 31 publications

References 57 publications

Regulation of Ca2+/calmodulin-dependent protein kinase kinase β by cAMP signaling

Regulation of Ca2+/calmodulin-dependent protein kinase kinase β by cAMP signaling

Design of Drug‐Like Protein–Protein Interaction Stabilizers Guided By Chelation‐Controlled Bioactive Conformation Stabilization

14‐3‐3 Protein‐Protein Interactions: From Mechanistic Understanding to Their Small‐Molecule Stabilization

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