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The Ras-like GTPase Rem2 is a potent inhibitor of calcium/calmodulin-dependent kinase II activity.

ABSTRACT: Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is a well-characterized, abundant protein kinase that regulates a diverse set of functions in a tissue-specific manner. For example, in heart muscle, CaMKII regulates Ca²⁺ homeostasis, whereas in neurons, CaMKII regulates activity-dependent dendritic remodeling and long-term potentiation (LTP), a neurobiological correlate of learning and memory. Previously, we identified the GTPase Rem2 as a critical regulator of dendrite branching and homeostatic plasticity in the vertebrate nervous system. Here, we report that Rem2 directly interacts with CaMKII and potently inhibits the activity of the intact holoenzyme, a previously unknown Rem2 function. Our results suggest that Rem2 inhibition involves interaction with both the CaMKII hub domain and substrate recognition domain. Moreover, we found that Rem2-mediated inhibition of CaMKII regulates dendritic branching in cultured hippocampal neurons. Lastly, we report that substitution of two key amino acid residues in the Rem2 N terminus (Arg-79 and Arg-80) completely abolishes its ability to inhibit CaMKII. We propose that our biochemical findings will enable further studies unraveling the functional significance of Rem2 inhibition of CaMKII in cells.

SUBMITTER: Royer L

PROVIDER: S-EPMC6153278 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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The Ras-like GTPase Rem2 is a potent inhibitor of calcium/calmodulin-dependent kinase II activity.

Royer Leandro L Herzog Josiah J JJ Kenny Katelyn K Tzvetkova Boriana B Cochrane Jesse C JC Marr Michael T MT Paradis Suzanne S

The Journal of biological chemistry 20180802 38

Ca<sup>2+</sup>/calmodulin-dependent protein kinase II (CaMKII) is a well-characterized, abundant protein kinase that regulates a diverse set of functions in a tissue-specific manner. For example, in heart muscle, CaMKII regulates Ca<sup>2+</sup> homeostasis, whereas in neurons, CaMKII regulates activity-dependent dendritic remodeling and long-term potentiation (LTP), a neurobiological correlate of learning and memory. Previously, we identified the GTPase Rem2 as a critical regulator of dendrite ...[more]

PMID: 30072381

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Project description:The calcium calmodulin-dependent protein kinase II (CaMKII) is a dodecameric holoenzyme important for encoding memory. Its activation, triggered by binding of calcium-calmodulin, persists autonomously after calmodulin dissociation. One (receiver) kinase captures and subsequently phosphorylates the regulatory domain peptide of a donor kinase forming a chained dimer as the first stage of autonomous activation. Protein dynamics simulations examined the conformational changes triggered by dimer formation and phosphorylation, aimed to provide a molecular rationale for human mutations that result in learning disabilities. Ensembles generated from X-ray crystal structures were characterized by network centrality and community analysis. Mutual information related collective motions to local fragment dynamics encoded with a structural alphabet. Implicit solvent tCONCOORD conformational ensembles revealed the dynamic architecture of inactive kinase domains was co-opted in the activated dimer but the network hub shifted from the nucleotide binding cleft to the captured peptide. Explicit solvent molecular dynamics (MD) showed nucleotide and substrate binding determinants formed coupled nodes in long-range signal relays between regulatory peptides in the dimer. Strain in the extended captured peptide was balanced by reduced flexibility of the receiver kinase C-lobe core. The relays were organized around a hydrophobic patch between the captured peptide and a key binding helix. The human mutations aligned along the relays. Thus, these mutations could disrupt the allosteric network alternatively, or in addition, to altered binding affinities. Non-binding protein sectors distant from the binding sites mediated the allosteric signalling; providing possible targets for inhibitor design. Phosphorylation of the peptide modulated the dielectric of its binding pocket to strengthen the patch, non-binding sectors, domain interface and temporal correlations between parallel relays. These results provide the molecular details underlying the reported positive kinase cooperativity to enrich the discussion on how autonomous activation by phosphorylation leads to long-term behavioural effects.

Project description:BACKGROUND:Sustained ?-adrenergic receptor (?-AR) stimulation causes pathophysiological changes during heart failure (HF), including inhibition of the slow component of the delayed rectifier potassium current (IKs). Aberrant calcium handling, including increased activation of calcium/calmodulin-dependent protein kinase II (CaMKII), contributes to arrhythmia development during HF. OBJECTIVE:The purpose of this study was to investigate CaMKII regulation of KCNQ1 (pore-forming subunit of IKs) during sustained ?-AR stimulation and associated functional implications on IKs. METHODS:KCNQ1 phosphorylation was assessed using liquid chromatography-tandem mass spectrometry after sustained ?-AR stimulation with isoproterenol (ISO). Peptide fragments corresponding to KCNQ1 residues were synthesized to identify CaMKII phosphorylation at the identified sites. Dephosphorylated (alanine) and phosphorylated (aspartic acid) mimics were introduced at identified residues. Whole-cell, voltage-clamp experiments were performed in human endothelial kidney 293 cells coexpressing wild-type or mutant KCNQ1 and KCNE1 (auxiliary subunit) during ISO treatment or lentiviral ?CaMKII overexpression. RESULTS:Novel KCNQ1 carboxy-terminal sites were identified with enhanced phosphorylation during sustained ?-AR stimulation at T482 and S484. S484 peptides demonstrated the strongest ?CaMKII phosphorylation. Sustained ?-AR stimulation reduced IKs activation (P = .02 vs control) similar to the phosphorylated mimic (P = .62 vs sustained ?-AR). Individual phosphorylated mimics at S484 (P = .04) but not at T482 (P = .17) reduced IKs function. Treatment with CN21 (CaMKII inhibitor) reversed the reductions in IKs vs CN21-Alanine control (P < .01). ?CaMKII overexpression reduced IKs similar to ISO treatment in wild type (P < .01) but not in the dephosphorylated S484 mimic (P = .99). CONCLUSION:CaMKII regulates KCNQ1 at S484 during sustained ?-AR stimulation to inhibit IKs. The ability of CaMKII to inhibit IKs may contribute to arrhythmogenicity during HF.

Dataset Information

The Ras-like GTPase Rem2 is a potent inhibitor of calcium/calmodulin-dependent kinase II activity.

Publications

The Ras-like GTPase Rem2 is a potent inhibitor of calcium/calmodulin-dependent kinase II activity.

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