Regulation of brain-specific kinases 1 and 2 (BRSK1/2) by Ca2+/calmodulin.

Protein-protein interactions are thought to modulate the efficiency and specificity of Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) signaling in specific subcellular compartments. Here we show that the F-actin-binding protein α-actinin targets CaMKIIα to F-actin in cells by binding to the CaMKII regulatory domain, mimicking CaM. The interaction with α-actinin is blocked by CaMKII autophosphorylation at Thr-306, but not by autophosphorylation at Thr-305, whereas autophosphorylation at either site blocks Ca(2+)/CaM binding. The binding of α-actinin to CaMKII is Ca(2+)-independent and activates the phosphorylation of a subset of substrates in vitro. In intact cells, α-actinin selectively stabilizes CaMKII association with GluN2B-containing glutamate receptors and enhances phosphorylation of Ser-1303 in GluN2B, but inhibits CaMKII phosphorylation of Ser-831 in glutamate receptor GluA1 subunits by competing for activation by Ca(2+)/CaM. These data show that Ca(2+)-independent binding of α-actinin to CaMKII differentially modulates the phosphorylation of physiological targets that play key roles in long-term synaptic plasticity.

Muscarinic acetylcholine receptors (mAChRs) are widely expressed in the mammalian brain and are essential for neuronal functions. These receptors are believed to be actively regulated by intracellular signals, although the underlying mechanisms are largely unknown. In this study, we show that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) binds directly and selectively to one of five mAChR subtypes, M4 receptors (M4Rs), at their C-terminal regions of second intracellular loops. This binding relies on Ca(2+) activation of the kinase and leads to the phosphorylation of M4Rs at a specific threonine site (Thr145). Complementary in vivo studies in rat striatal neurons enriched with M4Rs confirm that rising Ca(2+) recruits CaMKIIalpha to M4Rs to potentiate receptor signalling, which controls behavioural sensitivity to dopamine stimulation in an activity-dependent manner. Our data identify a new model of protein-protein interactions. In a Ca(2+)-sensitive manner, CaMKIIalpha regulates M4R efficacy and controls the acetylcholine-dopamine balance in the basal ganglia and also the dynamics of movement.

A hallmark feature of Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) regulation is the generation of Ca(2+)-independent autonomous activity by Thr-286 autophosphorylation. CaMKII autonomy has been regarded a form of molecular memory and is indeed important in neuronal plasticity and learning/memory. Thr-286-phosphorylated CaMKII is thought to be essentially fully active ( approximately 70-100%), implicating that it is no longer regulated and that its dramatically increased Ca(2+)/CaM affinity is of minor functional importance. However, this study shows that autonomy greater than 15-25% was the exception, not the rule, and required a special mechanism (T-site binding; by the T-substrates AC2 or NR2B). Autonomous activity toward regular R-substrates (including tyrosine hydroxylase and GluR1) was significantly further stimulated by Ca(2+)/CaM, both in vitro and within cells. Altered K(m) and V(max) made autonomy also substrate- (and ATP) concentration-dependent, but only over a narrow range, with remarkable stability at physiological concentrations. Such regulation still allows molecular memory of previous Ca(2+) signals, but prevents complete uncoupling from subsequent cellular stimulation.

Liver Kinase B1 (LKB1), encoded by Serine-Threonine Kinase 11 (STK11), is a master kinase that regulates cell migration, polarity, proliferation, and metabolism through downstream adenosine monophosphate-activated protein kinase (AMPK) and AMPK-related kinase signalling. Since genetic screens identified STK11 mutations in Peutz-Jeghers Syndrome, STK11 mutants have been implicated in tumourigenesis labelling it as a tumour suppressor. In support of this, several compounds reduce tumour burden through upregulating LKB1 signalling, and LKB1-AMPK agonists are cytotoxic to tumour cells. However, in certain contexts, its role in cancer is paradoxical as LKB1 promotes tumour cell survival by mediating resistance against metabolic and oxidative stressors. LKB1 deficiency has also enhanced the selectivity and cytotoxicity of several cancer therapies. Taken together, there is a need to develop LKB1-specific pharmacological compounds, but prior to developing LKB1 inhibitors, further work is needed to understand LKB1 activity and regulation. However, investigating LKB1 activity is strenuous as cell/tissue type, mutations to the LKB1 signalling pathway, STE-20-related kinase adaptor protein (STRAD) binding, Mouse protein 25-STRAD binding, splicing variants, nucleocytoplasmic shuttling, post-translational modifications, and kinase conformation impact the functional status of LKB1. For these reasons, guidelines to standardize experimental strategies to study LKB1 activity, associate proteins, spliced isoforms, post-translational modifications, and regulation are of upmost importance to the development of LKB1-specific therapies. Therefore, to assess the therapeutic relevancy of LKB1 inhibitors, this review summarizes the importance of LKB1 in cell physiology, highlights contributors to LKB1 activation, and outlines the benefits and risks associated with targeting LKB1.

RAGE signaling mediates post-injury arterial neointima formation by suppression of liver kinase B1 and AMPK activity

Complex of Calmodulin with a Ryanodine Receptor Target Reveals a Novel, Flexible Binding Mode

N-Methyl-d-aspartic acid receptor-dependent long term potentiation (LTP), a model of memory formation, requires Ca2+·calmodulin-dependent protein kinase II (αCaMKII) activity and Thr286 autophosphorylation via both global and local Ca2+ signaling, but the mechanisms of signal transduction are not understood. We tested the hypothesis that the Ca2+-binding activator protein calmodulin (CaM) is the primary decoder of Ca2+ signals, thereby determining the output, e.g. LTP. Thus, we investigated the function of CaM mutants, deficient in Ca2+ binding at sites 1 and 2 of the N-terminal lobe or sites 3 and 4 of the C-terminal CaM lobe, in the activation of αCaMKII. Occupancy of CaM Ca2+ binding sites 1, 3, and 4 is necessary and sufficient for full activation. Moreover, the N- and C-terminal CaM lobes have distinct functions. Ca2+ binding to N lobe Ca2+ binding site 1 increases the turnover rate of the enzyme 5-fold, whereas the C lobe plays a dual role; it is required for full activity, but in addition, via Ca2+ binding site 3, it stabilizes ATP binding to αCaMKII 4-fold. Thr286 autophosphorylation is also dependent on Ca2+ binding sites on both the N and the C lobes of CaM. As the CaM C lobe sites are populated by low amplitude/low frequency (global) Ca2+ signals, but occupancy of N lobe site 1 and thus activation of αCaMKII requires high amplitude/high frequency (local) Ca2+ signals, lobe-specific sensing of Ca2+-signaling patterns by CaM is proposed to explain the requirement for both global and local Ca2+ signaling in the induction of LTP via αCaMKII.

The activation of Ca2+/calmodulin (CaM)-dependent protein kinase II (CaM-KII) by Ca2+/CaM results in autophosphorylation and the generation of Ca2+/CaM-independent enzyme activity. We postulated that CaM binding and subsequent autophosphorylation alters the conformation of CaM-KII and exposes its substrate-binding and catalytic site(s). Previous peptide mapping studies on CaM-KII demonstrated the close proximity of CaM-binding and autophosphorylation domains. Analyses of the deduced amino acid sequences encoding CaM-KII have allowed the identification of its CaM-binding domain and have revealed two consensus phosphorylation sites that flank this regulatory domain. We report herein the distinct properties of two synthetic peptides modeled after the CaM-binding domain of CaM-KII. The first peptide binds CaM in a Ca2+-dependent manner and is an antagonist of CaM-KII activation (IC50 approximately equal to 75 nM). It does not, however, inhibit CaM-KII activity. A second peptide containing the same CaM-binding domain plus a putative autophosphorylation sequence at its N terminus displayed bifunctional regulatory properties. In addition to being a CaM antagonist, the latter was a potent inhibitor of Ca2+/CaM-independent kinase activity (IC50 approximately equal to 2 microM). We suggest that this bifunctional peptide represents an active site-directed inhibitory element of CaM-KII. The separation of CaM antagonist and active site-directed inhibitory properties of this peptide distinguishes CaM-KII from other CaM-dependent enzymes in which bifunctional regulatory properties appear to reside in the same peptide domain. These results indicate that the definition of site-directed inhibitory peptides should, in some cases, be expanded to include bona fide phosphorylation sites.

High-Speed Atomic Force Microscopy Shows Conformational Dynamics of Ca2+/Calmodulin-Dependent Protein Kinase II

Aberrant cell metabolism is rapidly establishing itself as a critical hallmark of human malignancies. Cancer cells are faced with huge metabolic demands to support rapid tumor growth, yet are commonly starved for nutrients. In response, cancer cells hijack alternative signaling pathways during these times of energy and metabolic stress to sustain viability. Metastatic epithelial ovarian cancer (EOC) cells are faced with additional stressors during transcoelomic spread, such as detachment from a matrix substratum and inhospitable conditions in peritoneal fluid. However, EOC cells have a natural ability to aggregate when in suspension to form multicellular aggregates, or spheroids, which supports a survival advantage for cells when transiting the peritoneal space during metastatic progression. Therefore, we utilize an in vitro spheroid culture model system to investigate signaling pathways altered in EOC cells that may be implicated in ovarian cancer pathobiology and promote metastasis. For example, we have shown that spheroid formation induces cellular quiescence and autophagy, two disparate processes which promote EOC cell survival and resistance to platinum-based chemotherapeutics. In addition, we discovered that EOC spheroids have significantly reduced mitochondrial activity and ATP levels compared with matched proliferating adherent cells. Liver kinase B1 (LKB1) acts as a chief responder to intracellular stress due to reduced energy and nutrients by eliciting general growth suppression during these starvation-like conditions. Thus, we hypothesized that LKB1 activity is increased in EOC spheroids to promote tumor cell dormancy and cell survival. Although the STK11 gene encoding LKB1 is heterozygously deleted in 84% of serous ovarian tumors, we demonstrate that almost all ovarian tumor cells and established EOC cell lines retain intact LKB1 expression. In fact, LKB1 protein expression increases when EOC cells form spheroids and this coincides with induced serine-428 phosphorylation, a modification that is required for LKB1 growth suppressive activity. To address the potential functional requirement of LKB1 in EOC spheroids, we first performed transient knockdown of STK11 to block LKB1 expression. Indeed, reduced LKB1 renders spheroids susceptible to cell death and increases sensitivity to carboplatin. Next, we utilized Cas9-mediated genome editing of the STK11 locus to completely ablate LKB1 expression in HEYA8 and OVCAR8 cells. STK11-knockout HEYA8 and OVCAR8 cells yielded significantly decreased spheroid number and viability compared with parental cell lines. In a reciprocal fashion, forced re-expression of LKB1 in CaOV3 and TOV21G cells—two EOC cell lines which harbor inactivating STK11 mutations—reduces cell growth and colony formation in proliferating adherent culture. Proliferation and dispersion of CaOV3-LKB1 and TOV21G-LKB1 cells from re-attached spheroids are also reduced. Taken together, LKB1 has growth suppressive effects in EOC cells, yet it serves the additional purpose to promote cell survival in spheroids. The canonical target of LKB1 is AMP-activated protein kinase (AMPK), which is thought to elicit the majority of LKB1 growth suppressive effects during stress metabolism signaling. As expected, AMPK phosphorylation at threonine-172 is also induced upon spheroid formation. Using STK11-knockout EOC cells, however, we demonstrate that LKB1 is not required to phosphorylate AMPK in spheroids; more importantly, knockdown of PRKAA1 encoding the catalytic alpha-subunit of AMPK has no effect on spheroid cell viability. Thus, our results suggest that LKB1 utilizes alternative mechanisms to regulate the dormancy phenotype in EOC spheroids. Future work will entail direct experiments testing whether LKB1-mediated stress metabolism signaling has the potential to promote EOC metastasis and recurrence of chemo-resistant disease. Citation Format: Trevor G. Shepherd, Yudith Ramos Valdes, Teresa Peart, Meera Shah, Gabriel E. DiMattia. Stress management: LKB1 controls growth and survival of dormant epithelial ovarian cancer spheroid cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B51.

BackgroundWe reported that zinc neurotoxicity, a key mechanism of ischemic neuronal death, was mediated by poly ADP-ribose polymerase (PARP) over-activation following NAD+/ATP depletion in cortical cultures. Because AMP-activated protein kinase (AMPK) can be activated by ATP depletion, and AMPK plays a key role in excitotoxicity and ischemic neuronal death, we examined whether AMPK could be involved in zinc neurotoxicity in mouse cortical neuronal cultures.ResultsCompound C, an AMPK inhibitor, significantly attenuated zinc-induced neuronal death. Activation of AMPK was detected beginning 2 h after a 10-min exposure of mouse cortical neurons to 300 μM zinc, although a significant change in AMP level was not detected until 4 h after zinc treatment. Thus, AMPK activation might not have been induced by an increase in intracellular AMP in zinc neurotoxicity. Furthermore, we observed that liver kinase B1 (LKB1) but not Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ), was involved in AMPK activation. Although STO-609, a chemical inhibitor of CaMKKβ, significantly attenuated zinc neurotoxicity, zinc-induced AMPK activation was not affected, which suggested that CaMKKβ was not involved in AMPK activation. Knockdown of LKB1 by siRNA significantly reduced zinc neurotoxicity, as well as zinc-induced AMPK activation, which indicated a possible role for LKB1 as an upstream kinase for AMPK activation. In addition, mRNA and protein levels of Bim, a pro-apoptotic Bcl-2 family member, were noticeably increased by zinc in an AMPK-dependent manner. Finally, caspase-3 activation in zinc-induced neuronal death was mediated by LKB1 and AMPK activation.ConclusionsThe results suggested that AMPK mediated zinc-induced neuronal death via up-regulation of Bim and activation of caspase-3. Rapid activation of AMPK was detected after exposure of cortical neuronal cultures to zinc, which was induced by LKB1 activation but not increased intracellular AMP levels or CaMKKβ activation. Hence, blockade of AMPK in the brain may protect against zinc neurotoxicity, which is likely to occur after acute brain injury.

Despite the critical importance of Ca(2+)/calmodulin (CaM)-dependent protein kinase (CaMK) II signaling in neuroplasticity, only a limited amount of work has so far been available regarding the presence and significance of another predominant CaMK subfamily, the CaMKI/CaMKIV family, in the central nervous system. We here searched for kinases with a core catalytic structure similar to CaMKI and CaMKIV. We isolated full-length cDNAs encoding three mouse CaMKI/CaMKIV-related kinases, CLICK-I (CL1)/doublecortin and CaM kinase-Like (DCAMKL)1, CLICK-II (CL2)/DCAMKL2, and CLICK-I,II-related (CLr)/DCAMKL3, the kinase domains of which had an intermediate homology not only to CaMKI/CaMKIV but also to CaMKII. Furthermore, CL1, CL2, and CLr were highly expressed in the central nervous system, in a neuron-specific fashion. CL1alpha and CL1beta were shorter isoforms of DCAMKL1, which lacked the doublecortin-like domain (Dx). In contrast, CL2alpha and CL2beta contained a full N-terminal Dx, whereas CLr only possessed a partial and dysfunctional Dx. Interestingly, despite a large similarity in the kinase domain, CL1/CL2/CLr had an impact on CRE-dependent gene expression distinct from that of the related CaMKI/CaMKIV and CaMKII. Although these were previously shown to activate Ca(2+)/cAMP-response element-binding protein (CREB)-dependent transcription, we here show that CL1 and CL2 were unable to significantly phosphorylate CREB Ser-133 and rather inhibited CRE-dependent gene expression by a dominant mechanism that bypassed CREB and was mediated by phosphorylated TORC2.

Calmodulin (CaM) binds to the skeletal muscle ryanodine receptor Ca(2+) release channel (RyR1) with high affinity, and it may act as a Ca(2+)-sensing subunit of the channel. Apo-CaM increases RyR1 channel activity, but Ca(2+)-CaM is inhibitory. Here we examine the functional effects of CaM oxidation on RyR1 regulation by both apo-CaM and Ca(2+)-CaM, as assessed via determinations of [(3)H]ryanodine and [(35)S]CaM binding to skeletal muscle sarcoplasmic reticulum vesicles. Oxidation of all nine CaM Met residues abolished functional interactions of CaM with RyR1. Incomplete CaM oxidation, affecting 5-8 Met residues, increased the CaM concentration required to modulate RyR1, having a greater effect on the apo-CaM species. Mutating individual CaM Met residues to Gln demonstrated that Met-109 was required for apo-CaM activation of RyR1 but not for Ca(2+)-CaM inhibition of the channel. Furthermore, substitution of Gln for Met-124 increased the apo- and Ca(2+)-CaM concentrations required to regulate RyR1. These results thus identify Met residues critical for the productive association of CaM with RyR1 channels and suggest that oxidation of CaM may contribute to altered regulation of sarcoplasmic reticulum Ca(2+) release during oxidative stress.

Most epithelial ovarian cancer (EOC) patients with metastatic disease initially respond to cytotoxic chemotherapy, yet almost all will relapse with resistant disease. Thus, improving patient outcomes will require novel approaches to limit metastasis and overcome chemo-resistance. Liver Kinase B1 (LKB1), encoded by the STK11 gene, is a key intracellular regulator of metabolic stress and is considered a putative tumor suppressor in some cancers. However, we have demonstrated that LKB1 is intact and required for EOC cell viability and growth in an in vitro spheroid model of ovarian cancer metastasis. We propose that LKB1 signaling enables malignant EOC cells to maintain viability and survive in metabolically challenging environments like that encountered during intraperitoneal metastasis. To further investigate the therapeutic potential of targeting LKB1 activity in metastatic EOC, we generated STK11-knockout cell lines—normal FT190 cells, and EOC cell lines OVCAR8, HeyA8, and iOvCa147—using CRISPR technology. STK11KO resulted in decreased malignant properties of EOC cells in vitro, including clonogenicity and anchorage-independent growth; however, loss of LKB1 in FT190 cells had no effect on cell proliferation, clonogenicity, or anchorage-independent growth, indicating LKB1 does not likely act as a tumor suppressor in EOC. Loss of LKB1 sensitized EOC cells to the growth-inhibiting effects of specific metabolic stresses. OVCAR8-STK11KO cells were more sensitive to nutrient deprivation in adherent culture, and to carboplatin and paclitaxel treatment in spheroid culture, as compared with OVCAR8 cells. Among the three EOC cell lines, STK11KO yielded variable sensitivity to inhibition of mitochondrial ATP production via oligomycin treatment. Interestingly, STK11KO did not affect induction of AMP-activated protein kinase (AMPK) phosphorylation in EOC spheroids, indicating that metabolic stress signaling to support EOC cell survival in spheroids during metastasis may occur via alternative pathways. In support of our previous knockdown results, EOC spheroids completely lacking LKB1 had markedly impaired growth in suspension culture compared to parental cell controls. In contrast, FT190 spheroids exhibited rapid cell attrition in spheroid culture regardless of LKB1 status. These results indicate that LKB1 may be specifically required in EOC cells to evade anoikis during metastatic spread. Finally, to test directly whether loss of LKB1 activity affects the metastatic potential of EOC cells, we performed intraperitoneal injections of OVCAR8-STK11KO and HeyA8-STK11KO cells with their respective parental cell controls. Loss of LKB1 in both aggressive EOC cell lines exhibited a dramatic reduction on tumor burden. STK11KO significantly decreased the establishment of large, solid tumor masses, reduced adhesion of OVCAR8 tumor nodules, and even changed the metastatic trajectory of HeyA8 cells with evidence of tumor cell growth only as a thin layer on peritoneal walls. Histologic analysis revealed evidence of extensive necrosis in STK11KO tumors, which was likely the major contributor to reduced tumor burden. These results strongly indicate that loss of LKB1 activity abrogates the metabolic stress response necessary during EOC metastasis both in spheroids and establishment of intraperitoneal tumors. Overall, LKB1 or its AMPK-independent signaling mediators represent unique yet very potent therapeutic vulnerabilities in metastatic EOC. Citation Format: Adrian Buensuceso, Yudith R. Valdes, Rene Figueredo, Gabriel E. DiMattia, Trevor G. Shepherd. The metabolic stress mediator LKB1 is required for ovarian cancer metastasis. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A12.

The endothelium glycocalyx layer (ECL), presents on the apical surface of endothelial cells, creates a barrier between circulating blood and the vessel wall. Low shear stress (LSS) may accelerate the degradation of the glycocalyx via hyaluronidase2 (Hyal2) and then alter the cell polarity. Yet the liver kinase B1 (LKB1) signaling pathway plays an important role in regulating cell polarity. However, the relationship between LKB1 and glycocalyx during LSS is not clear. In the current study, we demonstrate that LSS attenuates LKB1 and AMP-activated protein kinase activation as well as activated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (p47phox ) and Hyal2 in the human umbilical vein endothelial cell (HUVEC). Pretreatment with 5-Aminoimidazole-4-carboxamide1-β-D-ribofuranoside(AICAR), or diphenyleneiodonium(DPI chloride) and transfection with LKB1 overexpression vector and p47phox small interfering RNA downregulated LSS-induced Hyal2 activation. By coimmunoprecipitation, we discovered the existence of p47phox /Hyal2 complex. LSS induced the dissociation of p47phox /Hyal2 complex, which was inhibited by LKB1 overexpression and AICAR. Furthermore, knockdown of Hyal2 performed a positive feedback on LKB1 activity. In addition, we also show that LSS enhanced LKB1 translocation from the cytosol to the nucleus. Taken together, these data indicate that Hyal2 regulates LSS-induced injury of the glycocalyx via LKB1/AMPK/NADPH oxidase signaling cascades.