Functional analysis of an R311C variant of Ca²⁺‐calmodulin‐dependent protein kinase kinase‐2 (CaMKK2) found as a de novo mutation in a patient with bipolar disorder

Abstract

Objectives: Loss-of-function mutations in the gene encoding the calcium-calmodulin (Ca 2+-CaM)-dependent protein kinase kinase-2 (CaMKK2) enzyme are linked to bipolar disorder. Recently, a de novo arginine to cysteine (R311C) mutation in CaMKK2 was identified from a whole exome sequencing study of bipolar patients and their unaffected parents. The aim of the present study was to determine the functional consequences of the R311C mutation on CaMKK2 activity and regulation by Ca 2+-CaM. Methods: The effects of the R311C mutation on CaMKK2 activity and Ca 2+-CaM activation were examined using a radiolabeled adenosine triphosphate (ATP) kinase assay. We performed immunoblot analysis to determine whether the R311C mutation impacts threonine-85 (T85) autophosphorylation, an activating phosphorylation site on CaMKK2 that has also been implicated in bipolar disorder. We also expressed the R311C mutant in CaMKK2 knockout HAP1 cells and used immunoblot analysis and an MTS reduction assay to study its effects on Ca 2+-dependent downstream signaling and cell viability, respectively. Results: The R311C mutation maps to the conserved HRD motif within the catalytic loop of CaMKK2 and caused a marked reduction in kinase activity and Ca 2+-CaM activation. The R311C mutation virtually abolished T85 autophosphorylation in response to Ca 2+-CaM and exerted a dominant-negative effect in cells as it impaired the ability of wild-type CaMKK2 to initiate downstream signaling and maintain cell viability. Conclusions: The highly disruptive, loss-of-function impact of the de novo R311C mutation in human CaMKK2 provides a compelling functional rationale for being considered a potential rare monogenic cause of bipolar disorder.

CaMKK2 is inactivated by cAMP-PKA signaling and 14-3-3 adaptor proteins

“…The autoinhibitory sequence obstructs the catalytic site by an intra-steric mechanism that is relieved by Ca 2+ -CaM binding, allowing for maximal kinase activity (8). In human CaMKK2, activation by Ca 2+ -CaM induces Thr85 autophosphorylation, which creates a molecular memory of the Ca 2+ -signal that keeps CaMKK2 in the activated state after the stimulus has diminished (9,10). Once activated, CaMKK2 triggers the downstream actions of the Ca 2+ -CaM dependent protein kinases-1 and -4 (CaMK1 and CaMK4) and the AMP-activated protein kinase (AMPK) signaling pathways (11)(12)(13).…”

Hinge Binder Scaffold Hopping Identifies Potent Calcium/Calmodulin-Dependent Protein Kinase Kinase 2 (CAMKK2) Inhibitor Chemotypes

“…CAMKK1 and CAMKK2 activity was determined by measuring the transfer of radiolabeled phosphate from [γ-32 P]-ATP to a synthetic peptide substrate (CaMKKtide) as previously described. 66 Briefly, purified recombinant CAMKK1 or CAMKK2 (100 pM) was incubated in the assay buffer CAMKK2 NanoBRET Assay. To quantify the cellular activity of these inhibitors, we developed a CAMKK2 NanoBRET target engagement assay.…”

“…CAMKK1 and CAMKK2 activity was determined by measuring the transfer of radiolabeled phosphate from [γ- 32 P]-ATP to a synthetic peptide substrate (CaMKKtide) as previously described. 66 Briefly, purified recombinant CAMKK1 or CAMKK2 (100 pM) was incubated in the assay buffer [50 mM HEPES (pH 7.4), 1 mM dithiothreitol, 0.02% (v/v) Brij-35] containing 200 μM CaMKKtide (Genscript), 100 μM CaCl 2 , 1 μM CaM (Sigma-Aldrich, Castle Hill, NSW, Australia), 200 μM [γ- 32 P]-ATP (Perkin Elmer, Boston, MA, USA), 5 mM MgCl 2 (Sigma-Aldrich, Castle Hill, NSW, Australia), and various concentrations of inhibitors (0–1 μM) in a standard 30 μL assay for 10 min at 30 °C. Reactions were terminated by spotting 15 μl onto P81 phosphocellulose paper (GE Lifesciences, Paramatta, NSW, Australia) and washing extensively in 1% phosphoric acid (Sigma-Aldrich, Castle Hill, NSW, Australia).…”

Molecular Mechanisms Underlying Ca2+/Calmodulin-Dependent Protein Kinase Kinase Signal Transduction

“…In contrast with CaMKKα/1, another CaMKK isoform (CaMKKβ/2) is constitutively active, exhibiting a significant Ca 2+ /CaM-independent activity (60–70% of total activity), attributable to the N-terminal regulatory segment (129–151) because a deletion of the N-terminal segment (residues 129–151) from rat CaMKKβ/2 significantly reduces its Ca 2+ /CaM-independent activity (10% of total activity) without any effect on the Ca 2+ /CaM-dependent activity ( Figure 2 B,C) [ 22 , 31 ]. Although CaMKK α/1 and β/2 had been considered monomeric kinases similar to other CaMKs including CaMKI and CaMKIV, Ling et al recently reported that FLAG-tagged CaMKKβ/2 and HA-tagged CaMKK2 β/2 mutant (Arg311Cys) might form a dimer or larger oligomer [ 74 ]. Consistent with this possibility, we demonstrated the oligomerization of both rat CaMKK isoforms in transfected cells by chemical crosslinking [ 75 ].…”