Efficacy and safety of endoxifen in bipolar disorder: A systematic review.

BACKGROUND: Endoxifen, a protein kinase C inhibitor and selective estrogen receptor modulator, primarily used in breast cancer treatment, has recently emerged as a potential therapeutic option for managing manic episodes associated with bipolar disorder (BD). This review aims to assess the existing evidence base for endoxifen in BD treatment and evaluate the strengths and limitations of current research findings. METHODS: A systematic search was conducted on Medline, Embase, and Web of Science databases. We included studies published in English that used endoxifen in BD, alongside any relevant studies identified through manual searching and conference papers with full-text availability. Information pertaining to dose, duration, clinical effects, and safety profiles was extracted from the included studies. The Cochrane Risk of Bias 2 tool was used to assess the risk of bias in clinical trials. RESULTS: The final review included seven case reports (including two conference presentations), two clinical trials, and one prospective study. Most studies administered endoxifen 8 mg and reported an improvement in manic symptoms. Several case reports included patients with comorbid substance use, and most patients received mood stabilizers concurrently. Few reports lacked any structured outcome measures. The clinical trials used divalproex 1000 mg as an active comparator, which was deemed sub-therapeutic. Despite being multicentric, the first trial lacked data on center-wise recruitment, and certain methodological concerns were observed across the included trials. There were no serious adverse effects noted, except for a significant elevation in lipid profile within a 3-week period. Limited data were available regarding endoxifen efficacy and safety in mixed episodes, depressive episodes, and maintenance treatment. CONCLUSION: There is a paucity of research on the efficacy and safety of endoxifen in BD. While existing evidence suggests short-term efficacy in manic episodes, significant limitations were identified in most of the included studies. Further research is imperative to establish the efficacy and safety of endoxifen in BD before considering its recommendation as a viable treatment option.

Neuroproteomic mapping of kinases and their substrates downstream of acetylcholine: finding and implications.

INTRODUCTION: Since the emergence of the cholinergic hypothesis of Alzheimer's disease (AD), acetylcholine has been viewed as a mediator of learning and memory. Donepezil improves AD-associated learning deficits and memory loss by recovering brain acetylcholine levels. However, it is associated with side effects due to global activation of acetylcholine receptors. Muscarinic acetylcholine receptor M1 (M1R), a key mediator of learning and memory, has been an alternative target. The importance of targeting a specific pathway downstream of M1R has recently been recognized. Elucidating signaling pathways beyond M1R that lead to learning and memory holds important clues for AD therapeutic strategies. AREAS COVERED: This review first summarizes the role of acetylcholine in aversive learning, one of the outputs used for preliminary AD drug screening. It then describes the phosphoproteomic approach focused on identifying acetylcholine intracellular signaling pathways leading to aversive learning. Finally, the intracellular mechanism of donepezil and its effect on learning and memory is discussed. EXPERT OPINION: The elucidation of signaling pathways beyond M1R by phosphoproteomic approach offers a platform for understanding the intracellular mechanism of AD drugs and for developing AD therapeutic strategies. Clarifying the molecular mechanism that links the identified acetylcholine signaling to AD pathophysiology will advance the development of AD therapeutic strategies.

Elucidating the role of missense SNP of protein kinase C epsilon in HCV-induced hepatocellular carcinoma.

BACKGROUND: The protein kinase C (PKC) family of serine/threonine kinases contains more than ten isozymes that are involved in multiple signaling pathways, including cell cycle regulation and carcinogenesis. The PKCε isozyme is an oncogene known to be upregulated in various signaling pathways involved in hepatitis C virus (HCV)-induced hepatocellular carcinoma (HCC). However, there is no known association of missense SNPs in PKCε with this disease, which can be a potential biomarker for early diagnosis and treatment. This research reveals a novel missense SNP in PKCε that is associated with HCV-induced HCC in the Pakistani population. METHODS: The PKCε SNP with amino acid substitution of E14K was chosen for wet lab analysis. Tetra ARMS-PCR was employed for the identification of high-risk SNP in PKCε of HCV-induced HCC patients. Liver function testing was also performed for comparison between the liver condition of the HCC patient and control group, and the viral load of HCC patient samples was evaluated to determine any alteration in the viral infectivity between different genotypes of the selected high-risk PKCε variant SNP. RESULTS: Frequency distribution of the homozygous GG genotype was found to be highest among HCV-induced HCC patients and was also found to be significantly associated with disease development and progression. The p values of comparative data obtained for the other two genotypes, heterozygous AG and homozygous AA, of the SNP also showed the significance of the data for these alleles. Still, their odds ratio and relative risk analysis did not indicate their association with HCV-induced HCC. CONCLUSION: The distribution of a genotype GG of PKCε has been found in HCV- induced HCC patients. Therefore, these PKCε SNP have the potential to be biomarkers for HCV-induced HCC. Further investigation using a larger sample size would provide additional insight into these initial data and open a new avenue for a better prognosis of this disease.

Pharmacological study on the enhancing effects of U46619 on guinea pig urinary bladder smooth muscle contraction induced by acetylcholine and α,ß-methylene ATP and the possible involvement of protein kinase C.

We examined whether U46619 (a prostanoid TP receptor agonist) could enhance the contractions of guinea pig urinary bladder smooth muscle (UBSM) in response to acetylcholine (ACh) and an ATP analog (α,ß-methylene ATP (αß-MeATP)) through stimulation of the UBSM TP receptor and whether protein kinase C (PKC) is involved. U46619 (10-7 M) markedly enhanced UBSM contractions induced by electrical field stimulation and ACh/αß-MeATP (3 × 10-6 M each), the potentiation of which was completely suppressed by SQ 29,548 (a TP receptor antagonist, 6 × 10-7 M). PKC inhibitors did not attenuate the ACh-induced contractions enhanced by U46619 although they partly suppressed the U46619-enhanced, αß-MeATP-induced contractions. While phorbol 12-myristate 13-acetate (PMA, a PKC activator, 10-6 M) did not enhance ACh-induced contractions, it enhanced αß-MeATP-induced contractions, an effect that was completely suppressed by PKC inhibitors. αß-MeATP-induced contractions, both with and without U46619 enhancement, were strongly inhibited by diltiazem. U46619/PMA enhanced 50 mM KCl-induced contractions, the potentiation of which was partly/completely attenuated by PKC inhibitors. These findings suggest that U46619 potentiates parasympathetic nerve-associated UBSM contractions by stimulating UBSM TP receptors. PKC-increased Ca2+ influx through voltage-dependent Ca2+ channels may partially play a role in purinergic receptor-mediated UBSM contractions enhanced by TP receptor stimulation.

Extracellular Histone-Induced Protein Kinase C Alpha Activation and Troponin Phosphorylation Is a Potential Mechanism of Cardiac Contractility Depression in Sepsis.

Reduction in cardiac contractility is common in severe sepsis. However, the pathological mechanism is still not fully understood. Recently it has been found that circulating histones released after extensive immune cell death play important roles in multiple organ injury and disfunction, particularly in cardiomyocyte injury and contractility reduction. How extracellular histones cause cardiac contractility depression is still not fully clear. In this work, using cultured cardiomyocytes and a histone infusion mouse model, we demonstrate that clinically relevant histone concentrations cause significant increases in intracellular calcium concentrations with subsequent activation and enriched localization of calcium-dependent protein kinase C (PKC) α and ßII into the myofilament fraction of cardiomyocytes in vitro and in vivo. Furthermore, histones induced dose-dependent phosphorylation of cardiac troponin I (cTnI) at the PKC-regulated phosphorylation residues (S43 and T144) in cultured cardiomyocytes, which was also confirmed in murine cardiomyocytes following intravenous histone injection. Specific inhibitors against PKCα and PKCßII revealed that histone-induced cTnI phosphorylation was mainly mediated by PKCα activation, but not PKCßII. Blocking PKCα also significantly abrogated histone-induced deterioration in peak shortening, duration and the velocity of shortening, and re-lengthening of cardiomyocyte contractility. These in vitro and in vivo findings collectively indicate a potential mechanism of histone-induced cardiomyocyte dysfunction driven by PKCα activation with subsequent enhanced phosphorylation of cTnI. These findings also indicate a potential mechanism of clinical cardiac dysfunction in sepsis and other critical illnesses with high levels of circulating histones, which holds the potential translational benefit to these patients by targeting circulating histones and downstream pathways.

An Update on Protein Kinases as Therapeutic Targets-Part I: Protein Kinase C Activation and Its Role in Cancer and Cardiovascular Diseases.

Protein kinases are one of the most significant drug targets in the human proteome, historically harnessed for the treatment of cancer, cardiovascular disease, and a growing number of other conditions, including autoimmune and inflammatory processes. Since the approval of the first kinase inhibitors in the late 1990s and early 2000s, the field has grown exponentially, comprising 98 approved therapeutics to date, 37 of which were approved between 2016 and 2021. While many of these small-molecule protein kinase inhibitors that interact orthosterically with the protein kinase ATP binding pocket have been massively successful for oncological indications, their poor selectively for protein kinase isozymes have limited them due to toxicities in their application to other disease spaces. Thus, recent attention has turned to the use of alternative allosteric binding mechanisms and improved drug platforms such as modified peptides to design protein kinase modulators with enhanced selectivity and other pharmacological properties. Herein we review the role of different protein kinase C (PKC) isoforms in cancer and cardiovascular disease, with particular attention to PKC-family inhibitors. We discuss translational examples and carefully consider the advantages and limitations of each compound (Part I). We also discuss the recent advances in the field of protein kinase modulators, leverage molecular docking to model inhibitor-kinase interactions, and propose mechanisms of action that will aid in the design of next-generation protein kinase modulators (Part II).

Evidence for Angiotensin II as a Naturally Existing Suppressor for the Guanylyl Cyclase A Receptor and Cyclic GMP Generation.

The natriuretic peptide system (NPS) and renin-angiotensin-aldosterone system (RAAS) function oppositely at multiple levels. While it has long been suspected that angiotensin II (ANGII) may directly suppress NPS activity, no clear evidence to date supports this notion. This study was designed to systematically investigate ANGII-NPS interaction in humans, in vivo, and in vitro. Circulating atrial, b-type, and c-type natriuretic peptides (ANP, BNP, CNP), cyclic guanosine monophosphate (cGMP), and ANGII were simultaneously investigated in 128 human subjects. Prompted hypothesis was validated in vivo to determine the influence of ANGII on ANP actions. The underlying mechanisms were further explored via in vitro approaches. In humans, ANGII demonstrated an inverse relationship with ANP, BNP, and cGMP. In regression models predicting cGMP, adding ANGII levels and the interaction term between ANGII and natriuretic peptides increased the predictive accuracy of the base models constructed with either ANP or BNP, but not CNP. Importantly, stratified correlation analysis further revealed a positive association between cGMP and ANP or BNP only in subjects with low, but not high, ANGII levels. In rats, co-infusion of ANGII even at a physiological dose attenuated cGMP generation mediated by ANP infusion. In vitro, we found the suppressive effect of ANGII on ANP-stimulated cGMP requires the presence of ANGII type-1 (AT1) receptor and mechanistically involves protein kinase C (PKC), as this suppression can be substantially rescued by either valsartan (AT1 blocker) or Go6983 (PKC inhibitor). Using surface plasmon resonance (SPR), we showed ANGII has low binding affinity to the guanylyl cyclase A (GC-A) receptor compared to ANP or BNP. Our study reveals ANGII is a natural suppressor for the cGMP-generating action of GC-A via AT1/PKC dependent manner and highlights the importance of dual-targeting RAAS and NPS in maximizing beneficial properties of natriuretic peptides in cardiovascular protection.

Protein kinase C fusion proteins are paradoxically loss of function in cancer.

Within the AGC kinase superfamily, gene fusions resulting from chromosomal rearrangements have been most frequently described for protein kinase C (PKC), with gene fragments encoding either the C-terminal catalytic domain or the N-terminal regulatory moiety fused to other genes. Kinase fusions that eliminate regulatory domains are typically gain of function and often oncogenic. However, several quality control pathways prevent accumulation of aberrant PKC, suggesting that PKC fusions may paradoxically be loss of function. To explore this topic, we used biochemical, cellular, and genome editing approaches to investigate the function of fusions that retain the portion of the gene encoding either the catalytic domain or regulatory domain of PKC. Overexpression studies revealed that PKC catalytic domain fusions were constitutively active but vulnerable to degradation. Genome editing of endogenous genes to generate a cancer-associated PKC fusion resulted in cells with detectable levels of fusion transcript but no detectable protein. Hence, PKC catalytic domain fusions are paradoxically loss of function as a result of their instability, preventing appreciable accumulation of protein in cells. Overexpression of a PKC regulatory domain fusion suppressed both basal and agonist-induced endogenous PKC activity, acting in a dominant-negative manner by competing for diacylglycerol. For both catalytic and regulatory domain fusions, the PKC component of the fusion proteins mediated the effects of the full-length fusions on the parameters examined, suggesting that the partner protein is dispensable in these contexts. Taken together, our findings reveal that PKC gene fusions are distinct from oncogenic fusions and present a mechanism by which loss of PKC function occurs in cancer.

Phosphorylation of a chronic pain mutation in the voltage-gated sodium channel Nav1.7 increases voltage sensitivity.

Mutations in voltage-gated sodium channels (Navs) can cause alterations in pain sensation, such as chronic pain diseases like inherited erythromelalgia. The mutation causing inherited erythromelalgia, Nav1.7 p.I848T, is known to induce a hyperpolarized shift in the voltage dependence of activation in Nav1.7. So far, however, the mechanism to explain this increase in voltage sensitivity remains unknown. In the present study, we show that phosphorylation of the newly introduced Thr residue explains the functional change. We expressed wildtype human Nav1.7, the I848T mutant, or other mutations in HEK293T cells and performed whole-cell patch-clamp electrophysiology. As the insertion of a Thr residue potentially creates a novel phosphorylation site for Ser/Thr kinases and because Nav1.7 had been shown in Xenopus oocytes to be affected by protein kinases C and A, we used different nonselective and selective kinase inhibitors and activators to test the effect of phosphorylation on Nav1.7 in a human system. We identify protein kinase C, but not protein kinase A, to be responsible for the phosphorylation of T848 and thereby for the shift in voltage sensitivity. Introducing a negatively charged amino acid instead of the putative phosphorylation site mimics the effect on voltage gating to a lesser extent. 3D modeling using the published cryo-EM structure of human Nav1.7 showed that introduction of this negatively charged site seems to alter the interaction of this residue with the surrounding amino acids and thus to influence channel function. These results could provide new opportunities for the development of novel treatment options for patients with chronic pain.

PKCα-mediated phosphorylation of the diacylglycerol kinase ζ MARCKS domain switches cell migration modes by regulating interactions with Rac1 and RhoA.

Cells can switch between Rac1 (lamellipodia-based) and RhoA (blebbing-based) migration modes, but the molecular mechanisms regulating this shift are not fully understood. Diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid, forms independent complexes with Rac1 and RhoA, selectively dissociating each from their common inhibitor RhoGDI. DGKζ catalytic activity is required for Rac1 dissociation but is dispensable for RhoA dissociation; instead, DGKζ stimulates RhoA release via a kinase-independent scaffolding mechanism. The molecular determinants that mediate the selective targeting of DGKζ to Rac1 or RhoA signaling complexes are unknown. Here, we show that protein kinase Cα (PKCα)-mediated phosphorylation of the DGKζ MARCKS domain increased DGKζ association with RhoA and decreased its interaction with Rac1. The same modification also enhanced DGKζ interaction with the scaffold protein syntrophin. Expression of a phosphomimetic DGKζ mutant stimulated membrane blebbing in mouse embryonic fibroblasts and C2C12 myoblasts, which was augmented by inhibition of endogenous Rac1. DGKζ expression in differentiated C2 myotubes, which have low endogenous Rac1 levels, also induced substantial membrane blebbing via the RhoA-ROCK pathway. These events were independent of DGKζ catalytic activity, but dependent upon a functional C-terminal PDZ-binding motif. Rescue of RhoA activity in DGKζ-null cells also required the PDZ-binding motif, suggesting that syntrophin interaction is necessary for optimal RhoA activation. Collectively, our results define a switch-like mechanism whereby DGKζ phosphorylation by PKCα plays a role in the interconversion between Rac1 and RhoA signaling pathways that underlie different cellular migration modes.

Muscarinic signaling regulates voltage-gated potassium channel KCNQ2 phosphorylation in the nucleus accumbens via protein kinase C for aversive learning.

The nucleus accumbens (NAc) plays critical roles in emotional behaviors, including aversive learning. Aversive stimuli such as an electric foot shock increase acetylcholine (ACh) in the NAc, and muscarinic signaling appears to increase neuronal excitability and aversive learning. Muscarinic signaling inhibits the voltage-dependent potassium KCNQ current which regulates neuronal excitability, but the regulatory mechanism has not been fully elucidated. Phosphorylation of KCNQ2 at threonine 217 (T217) and its inhibitory effect on channel activity were predicted. However, whether and how muscarinic signaling phosphorylates KCNQ2 in vivo remains unclear. Here, we found that PKC directly phosphorylated KCNQ2 at T217 in vitro. Carbachol and a muscarinic M1 receptor (M1R) agonist facilitated KCNQ2 phosphorylation at T217 in NAc/striatum slices in a PKC-dependent manner. Systemic administration of the cholinesterase inhibitor donepezil, which is commonly used to treat dementia, and electric foot shock to mice induced the phosphorylation of KCNQ2 at T217 in the NAc, whereas phosphorylation was suppressed by an M1R antagonist. Conditional deletion of Kcnq2 in the NAc enhanced electric foot shock induced aversive learning. Our findings indicate that muscarinic signaling induces the phosphorylation of KCNQ2 at T217 via PKC activation for aversive learning.

Dexmedetomidine modulates mitochondrial dynamics to protect against endotoxin-induced lung injury via the protein kinase C-ɑ/haem oxygenase-1 signalling pathway.

BACKGROUND: Endotoxin-induced acute lung injury (ALI) has a high mortality rate, and there are limited effective treatment options available. The aim of the present study was to identify if dexmedetomidine could regulate mitochondrial fusion and fission through the protein kinase C (PKC)-α/haem oxygenase (HO)-1 pathway to protect against endotoxin-induced ALI. MATERIALS AND METHODS: Dexmedetomidine was administered by intraperitoneal injection once daily for three days prior to induction of lung injury to mice. Mice in the PKC-α inhibitor group received dexmedetomidine by intraperitoneal injection 1 h after each chelerythrine injection, and lipopolysaccharide was injected 1 h after the last dose of dexmedetomidine. The lung wet/dry weight ratio, oxidative stress, inflammatory response, and expression levels of PKC-α, Nrf2, HO-1, Mfn1, Mfn2, OPA1, Drp1, and Fis1 were determined. RESULTS: Dexmedetomidine administration attenuated lung oxidative stress, decreased inflammatory cytokines secretion, and downregulated the expression levels of Drp1 and Fis1. Moreover, dexmedetomidine increased levels of Mfn1, Mfn2, and OPA1, and alleviated endotoxin-induced lung injury. Administration of chelerythrine partially reversed the pneumoprotective effects of dexmedetomidine. CONCLUSIONS: Dexmedetomidine may activate the PKC-ɑ/HO-1 pathway to increase the expression of Mfn1, Mfn2, and OPA1, while decreasing Drp1 and Fis1 expression, thereby reduce endotoxin-induced acute lung injury.

Dopamine transporter trafficking and Rit2 GTPase: Mechanism of action and in vivo impact.

Following its evoked release, dopamine (DA) signaling is rapidly terminated by presynaptic reuptake, mediated by the cocaine-sensitive DA transporter (DAT). DAT surface availability is dynamically regulated by endocytic trafficking, and direct protein kinase C (PKC) activation acutely diminishes DAT surface expression by accelerating DAT internalization. Previous cell line studies demonstrated that PKC-stimulated DAT endocytosis requires both Ack1 inactivation, which releases a DAT-specific endocytic brake, and the neuronal GTPase, Rit2, which binds DAT. However, it is unknown whether Rit2 is required for PKC-stimulated DAT endocytosis in DAergic terminals or whether there are region- and/or sex-dependent differences in PKC-stimulated DAT trafficking. Moreover, the mechanisms by which Rit2 controls PKC-stimulated DAT endocytosis are unknown. Here, we directly examined these important questions. Ex vivo studies revealed that PKC activation acutely decreased DAT surface expression selectively in ventral, but not dorsal, striatum. AAV-mediated, conditional Rit2 knockdown in DAergic neurons impacted baseline DAT surface:intracellular distribution in DAergic terminals from female ventral, but not dorsal, striatum. Further, Rit2 was required for PKC-stimulated DAT internalization in both male and female ventral striatum. FRET and surface pulldown studies in cell lines revealed that PKC activation drives DAT-Rit2 surface dissociation and that the DAT N terminus is required for both PKC-mediated DAT-Rit2 dissociation and DAT internalization. Finally, we found that Rit2 and Ack1 independently converge on DAT to facilitate PKC-stimulated DAT endocytosis. Together, our data provide greater insight into mechanisms that mediate PKC-regulated DAT internalization and reveal unexpected region-specific differences in PKC-stimulated DAT trafficking in bona fide DAergic terminals.

The mTOR inhibitor manassantin B reveals a crucial role of mTORC2 signaling in Epstein-Barr virus reactivation.

Lytic replication of Epstein-Barr virus (EBV) is not only essential for its cell-to-cell spread and host-to-host transmission, but it also contributes to EBV-induced oncogenesis. Thus, blocking EBV lytic replication could be a strategy for managing EBV-associated diseases. Previously, we identified a series of natural lignans isolated from the roots of Saururus chinensis (Asian lizard's tail) that efficiently block EBV lytic replication and virion production with low cytotoxicity. In this study, we attempted to elucidate the molecular mechanism by which these lignans inhibit EBV lytic replication. We found that a representative compound, CSC27 (manassantin B), inhibits EBV lytic replication by suppressing the expression of EBV immediate-early gene BZLF1 via disruption of AP-1 signal transduction. Further analysis revealed that manassantin B specifically blocks the mammalian target of rapamycin complex 2 (mTORC2)-mediated phosphorylation of AKT Ser/Thr protein kinase at Ser-473 and protein kinase Cα (PKCα) at Ser-657. Using phosphoinositide 3-kinase-AKT-specific inhibitors for kinase mapping and shRNA-mediated gene silencing, we validated that manassantin B abrogates EBV lytic replication by inhibiting mTORC2 activity and thereby blocking the mTORC2-PKC/AKT-signaling pathway. These results suggest that mTORC2 may have utility as an antiviral drug target against EBV infections and also reveal that manassantin B has potential therapeutic value for managing cancers that depend on mTORC2 signaling for survival.

Annexin A5 is essential for PKCθ translocation during T-cell activation.

T-cell activation is a critical part of the adaptive immune system, enabling responses to foreign cells and external stimulus. In this process, T-cell antigen receptor (TCR) activation stimulates translocation of the downstream kinase PKCθ to the membrane, leading to NF-κB activation and thus transcription of relevant genes. However, the details of how PKCθ is recruited to the membrane remain enigmatic. It is known that annexin A5 (ANXA5), a calcium-dependent membrane-binding protein, has been reported to mediate PKCδ activation by interaction with PKCδ, a homologue of PKCθ, which implicates a potential role of ANXA5 involved in PKCθ signaling. Here we demonstrate that ANXA5 does play a critical role in the recruitment of PKCθ to the membrane during T-cell activation. ANXA5 knockout in Jurkat T cells substantially inhibited the membrane translocation of PKCθ upon TCR engagement and blocked the recruitment of CARMA1-BCL10-MALT1 signalosome, which provides a platform for the catalytic activation of IKKs and subsequent activation of canonical NF-κB signaling in activated T cells. As a result, NF-κB activation was impaired in ANXA5-KO T cells. T-cell activation was also suppressed by ANAX5 knockdown in primary T cells. These results demonstrated a novel role of ANXA5 in PKC translocation and PKC signaling during T-cell activation.

The African natural product knipholone anthrone and its analogue anthralin (dithranol) enhance HIV-1 latency reversal.

A sterilizing or functional cure for HIV is currently precluded by resting CD4+ T cells that harbor latent but replication-competent provirus. The "shock-and-kill" pharmacological ap-proach aims to reactivate provirus expression in the presence of antiretroviral therapy and target virus-expressing cells for elimination. However, no latency reversal agent (LRA) to date effectively clears viral reservoirs in humans, suggesting a need for new LRAs and LRA combinations. Here, we screened 216 compounds from the pan-African Natural Product Library and identified knipholone anthrone (KA) and its basic building block anthralin (dithranol) as novel LRAs that reverse viral latency at low micromolar concentrations in multiple cell lines. Neither agent's activity depends on protein kinase C; nor do they inhibit class I/II histone deacetylases. However, they are differentially modulated by oxidative stress and metal ions and induce distinct patterns of global gene expression from established LRAs. When applied in combination, both KA and anthralin synergize with LRAs representing multiple functional classes. Finally, KA induces both HIV RNA and protein in primary cells from HIV-infected donors. Taken together, we describe two novel LRAs that enhance the activities of multiple "shock-and-kill" agents, which in turn may inform ongoing LRA combination therapy efforts.

Functional and structural characterization of allosteric activation of phospholipase Cε by Rap1A.

Phospholipase Cε (PLCε) is activated downstream of G protein-coupled receptors and receptor tyrosine kinases through direct interactions with small GTPases, including Rap1A and Ras. Although Ras has been reported to allosterically activate the lipase, it is not known whether Rap1A has the same ability or what its molecular mechanism might be. Rap1A activates PLCε in response to the stimulation of ß-adrenergic receptors, translocating the complex to the perinuclear membrane. Because the C-terminal Ras association (RA2) domain of PLCε was proposed to the primary binding site for Rap1A, we first confirmed using purified proteins that the RA2 domain is indeed essential for activation by Rap1A. However, we also showed that the PLCε pleckstrin homology (PH) domain and first two EF hands (EF1/2) are required for Rap1A activation and identified hydrophobic residues on the surface of the RA2 domain that are also necessary. Small-angle X-ray scattering showed that Rap1A binding induces and stabilizes discrete conformational states in PLCε variants that can be activated by the GTPase. These data, together with the recent structure of a catalytically active fragment of PLCε, provide the first evidence that Rap1A, and by extension Ras, allosterically activate the lipase by promoting and stabilizing interactions between the RA2 domain and the PLCε core.

A Tale of two receptors.

Calcium (Ca2+) oscillations in hepatocytes have a wide dynamic range. In particular, recent experimental evidence shows that agonist stimulation of the P2Y family of receptors leads to qualitatively diverse Ca2+ oscillations. We present a new model of Ca2+ oscillations in hepatocytes based on these experiments to investigate the mechanisms controlling P2Y-activated Ca2+ oscillations. The model accounts for Ca2+ regulation of the IP3 receptor (IP3R), the positive feedback from Ca2+ on phospholipase C (PLC) and the P2Y receptor phosphorylation by protein kinase C (PKC). Furthermore, PKC is shown to control multiple cellular substrates. Utilising the model, we suggest the activity and intensity of PLC and PKC necessary to explain the qualitatively diverse Ca2+ oscillations in response to P2Y receptor activation.

Myeloid Lineage Ablation of Phlpp1 Regulates M-CSF Signaling and Tempers Bone Resorption in Female Mice.

Prior work demonstrated that Phlpp1 deficiency alters trabecular bone mass and enhances M-CSF responsiveness, but the cell types and requirement of Phlpp1 for this effect were unclear. To understand the function of Phlpp1 within myeloid lineage cells, we crossed Phlpp1 floxed mice with mice harboring LysM-Cre. Micro-computed tomography of the distal femur of 12-week-old mice revealed a 30% increase in bone volume per total volume of Phlpp1 female conditional knockouts, but we did not observe significant changes within male Phlpp1 cKOLysM mice. Bone histomorphmetry of the proximal tibia further revealed that Phlpp1 cKOLysM females exhibited elevated osteoclast numbers, but conversely had reduced levels of serum markers of bone resorption as compared to littermate controls. Osteoblast number and serum markers of bone formation were unchanged. In vitro assays confirmed that Phlpp1 ablation enhanced osteoclast number and area, but limited bone resorption. Additionally, reconstitution with exogenous Phlpp1 suppressed osteoclast numbers. Dose response assays demonstrated that Phlpp1-/- cells are more responsive to M-CSF, but reconstitution with Phlpp1 abrogated this effect. Furthermore, small molecule-mediated Phlpp inhibition enhanced osteoclast numbers and size. Enhanced phosphorylation of Phlpp substrates-including Akt, ERK1/2, and PKCζ-accompanied these observations. In contrast, actin cytoskeleton disruption occurred within Phlpp inhibitor treated osteoclasts. Moreover, Phlpp inhibition reduced resorption of cells cultured on bovine bone slices in vitro. Our results demonstrate that Phlpp1 deficiency within myeloid lineage cells enhances bone mass by limiting bone resorption while leaving osteoclast numbers intact; moreover, we show that Phlpp1 represses osteoclastogenesis and controls responses to M-CSF.

Protein Kinase C as a Therapeutic Target in Non-Small Cell Lung Cancer.

Driver-directed therapeutics have revolutionized cancer treatment, presenting similar or better efficacy compared to traditional chemotherapy and substantially improving quality of life. Despite significant advances, targeted therapy is greatly limited by resistance acquisition, which emerges in nearly all patients receiving treatment. As a result, identifying the molecular modulators of resistance is of great interest. Recent work has implicated protein kinase C (PKC) isozymes as mediators of drug resistance in non-small cell lung cancer (NSCLC). Importantly, previous findings on PKC have implicated this family of enzymes in both tumor-promotive and tumor-suppressive biology in various tissues. Here, we review the biological role of PKC isozymes in NSCLC through extensive analysis of cell-line-based studies to better understand the rationale for PKC inhibition. PKC isoforms α, ε, η, ι, ζ upregulation has been reported in lung cancer, and overexpression correlates with worse prognosis in NSCLC patients. Most importantly, PKC isozymes have been established as mediators of resistance to tyrosine kinase inhibitors in NSCLC. Unfortunately, however, PKC-directed therapeutics have yielded unsatisfactory results, likely due to a lack of specific evaluation for PKC. To achieve satisfactory results in clinical trials, predictive biomarkers of PKC activity must be established and screened for prior to patient enrollment. Furthermore, tandem inhibition of PKC and molecular drivers may be a potential therapeutic strategy to prevent the emergence of resistance in NSCLC.