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Allosteric regulation of multi-domain protein kinases provides a common mechanism to acutely control kinase activity. Protein kinase C serves as a paradigm for multi-domain proteins whose activity is exquisitely tuned by interdomain conformational changes that keep the enzyme off in the absence of appropriate stimuli, but unleash activity in response to second messenger binding. Allosteric regulation of protein kinase C signaling has been optimized not just for itself: Alessi and colleagues discover that protein kinase C phosphorylates LRRK1, a kinase with even more domains, at sites on its CORB GTPase domain to allosterically activate LRRK1.

Protein kinase C (PKC) isozymes are maintained in a 'ready-to-go' but 'safe' autoinhibited conformation until second messenger binding unleashes an autoinhibitory pseudosubstrate to allow substrate phosphorylation. However, to gain this 'ready-to-go' conformation, PKC must be processed by a series of complex priming phosphorylations, the mechanism of which was enigmatic until now. Recent findings snapped the pieces of the phosphorylation puzzle into place to unveil a process that involves a newly described motif (TOR interaction motif, TIM), a well-described kinase [mechanistic target of rapamycin complex 2 (mTORC2)], and an often-used mechanism (autophosphorylation) to prime PKC to signal. This review highlights new insights into how phosphorylation controls PKC and discusses them in the context of common mechanisms for AGC kinase regulation by phosphorylation and autophosphorylation.

Conventional protein kinase C (PKC) isozymes tune the signaling output of cells, with loss-of-function somatic mutations associated with cancer and gain-of-function germline mutations identified in neurodegeneration. PKC with impaired autoinhibition is removed from the cell by quality-control mechanisms to prevent accumulation of aberrantly active enzyme. Here, we examine how a single residue in the C1A domain of PKCβ, arginine 42 (R42), permits quality-control degradation when mutated to histidine in cancer (R42H) and blocks downregulation when mutated to proline in the neurodegenerative disease spinocerebellar ataxia (R42P). Using FRET-based biosensors, we determined that mutation of R42 to any residue, including lysine, resulted in reduced autoinhibition as indicated by higher basal activity and faster agonist-induced plasma membrane translocation. R42 is predicted to form a stabilizing salt bridge with E655 in the C-tail and mutation of E655, but not neighboring E657, also reduced autoinhibition. Western blot analysis revealed that whereas R42H had reduced stability, the R42P mutant was stable and insensitive to activator-induced ubiquitination and downregulation, an effect previously observed by deletion of the entire C1A domain. Molecular dynamics (MD) simulations and analysis of stable regions of the domain using local spatial pattern (LSP) alignment suggested that P42 interacts with Q66 to impair mobility and conformation of one of the ligand-binding loops. Additional mutation of Q66 to the smaller asparagine (R42P/Q66N), to remove conformational constraints, restored degradation sensitivity to that of WT. Our results unveil how disease-associated mutations of the same residue in the C1A domain can toggle between gain- or loss-of-function of PKC.

Pleckstrin homology domain leucine-rich repeat protein phosphatases (PHLPP) belong to the protein phosphatase magnesium/manganese-dependent family of Ser/Thr phosphatases. Their general role as tumor suppressors has been documented for over a decade. In recent years, accumulating evidence suggests that PHLPP isozymes have key regulatory roles in both innate and adaptive immunity. In macrophages, PHLPP1 dampens signaling through TLR4 and the IFN-γ receptor by altering cytosolic signaling pathways. Additionally, nuclear-localized PHLPP1 inhibits STAT1-mediated inflammatory gene expression by direct dephosphorylation at Ser 727. PHLPP1 also regulates the migratory and inflammatory capacity of neutrophils in vivo. Furthermore, PHLPP1-mediated dephosphorylation of AKT on Ser 473 is required for both the suppressive function of regulatory T cells and for the pro-apoptotic effects of PHLPP1 in B cell chronic lymphocytic leukemia. In the context of immune homeostasis, PHLPP1 expression is modulated in multiple cell types by inflammatory signals, and the dynamics of its expression have varying effects on the pathogenesis of inflammatory bowel disease and septic shock. In this review, we summarize recent findings on the functions of PHLPP in inflammatory and regulatory signaling in the context of both innate and adaptive immunity.

Spinocerebellar ataxia type 14 (SCA14) is a neurodegenerative disease caused by germline variants in the diacylglycerol (DAG)/Ca 2+ -regulated protein kinase Cγ (PKCγ), leading to Purkinje cell degeneration and progressive cerebellar dysfunction. Most of the identified mutations cluster in the DAG-sensing C1 domains. Here, we found with a FRET-based activity reporter that SCA14-associated PKCγ mutations, including a previously undescribed variant, D115Y, enhanced the basal activity of the kinase by compromising its autoinhibition. Unlike other mutations in PKC that impair its autoinhibition but lead to its degradation, the C1 domain mutations protected PKCγ from such down-regulation. This enhanced basal signaling rewired the brain phosphoproteome, as revealed by phosphoproteomic analysis of cerebella from mice expressing a human SCA14-associated H101Y mutant PKCγ transgene. Mutations that induced a high basal activity in vitro were associated with earlier average age of onset in patients. Furthermore, the extent of disrupted autoinhibition, but not agonist-stimulated activity, correlated with disease severity. Molecular modeling indicated that almost all SCA14 variants not within the C1 domain were located at interfaces with the C1B domain, suggesting that mutations in and proximal to the C1B domain are a susceptibility for SCA14 because they uniquely enhance PKCγ basal activity while protecting the enzyme from down-regulation. These results provide insight into how PKCγ activation is modulated and how deregulation of the cerebellar phosphoproteome by SCA14-associated mutations affects disease progression.

Protein kinase signalling, which transduces external messages to mediate cellular growth and metabolism, is frequently deregulated in human disease, and specifically in cancer. As such, there are 77 kinase inhibitors currently approved for the treatment of human disease by the FDA. Due to their historical association as the receptors for the tumour-promoting phorbol esters, PKC isozymes were initially targeted as oncogenes in cancer. However, a meta-analysis of clinical trials with PKC inhibitors in combination with chemotherapy revealed that these treatments were not advantageous, and instead resulted in poorer outcomes and greater adverse effects. More recent studies suggest that instead of inhibiting PKC, therapies should aim to restore PKC function in cancer: cancer-associated PKC mutations are generally loss-of-function and high PKC protein is protective in many cancers, including most notably KRAS-driven cancers. These recent findings have reframed PKC as having a tumour suppressive function. This review focusses on a potential new mechanism of restoring PKC function in cancer — through targeting of its negative regulator, the Ser/Thr protein phosphatase PHLPP. This phosphatase regulates PKC steady-state levels by regulating the phosphorylation of a key site, the hydrophobic motif, whose phosphorylation is necessary for the stability of the enzyme. We also consider whether the phosphorylation of the potent oncogene KRAS provides a mechanism by which high PKC expression may be protective in KRAS-driven human cancers.

Whereas protein kinases have been successfully targeted for a variety of diseases, protein phosphatases remain an underutilized therapeutic target, in part because of incomplete characterization of their effects on signaling networks. The pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP) is a relatively new player in the cell signaling field, and new roles in controlling the balance among cell survival, proliferation, and apoptosis are being increasingly identified. Originally characterized for its tumor-suppressive function in deactivating the prosurvival kinase Akt, PHLPP may have an opposing role in promoting survival, as recent evidence suggests. Additionally, identification of the transcription factor STAT1 as a substrate unveils a role for PHLPP as a critical mediator of transcriptional programs in cancer and the inflammatory response. This review summarizes the current knowledge of PHLPP as both a tumor suppressor and an oncogene and highlights emerging functions in regulating gene expression and the immune system. Understanding the context-dependent functions of PHLPP is essential for appropriate therapeutic intervention.

The family of AGC kinases not only regulates cellular biology by phosphorylating substrates but is itself controlled by phosphorylation. Phosphorylation generally occurs at two conserved regions in these kinases: a loop near the entrance to the active site, termed the activation loop, that correctly aligns residues for catalysis, and a C-terminal tail whose phosphorylation at a site termed the hydrophobic motif stabilizes the active conformation. Whereas phosphorylation of the activation loop is well established to be catalyzed by the phosphoinositide-dependent kinase 1, the mechanism of phosphorylation of the C-tail hydrophobic motif has been controversial. For a subset of AGC kinases, which include most protein kinase C (PKC) isozymes and Akt, phosphorylation of the hydrophobic motif in cells was shown to depend on mTORC2 over 15 years ago, yet whether this was by direct phosphorylation or by another mechanism has remained elusive. The recent identification of a novel and evolutionarily conserved phosphorylation site on the C-tail, termed the TOR interaction motif (TIM), has finally unraveled the mystery of how mTORC2 regulates its client kinases. mTORC2 does not directly phosphorylate the hydrophobic motif; instead, it converts kinases such as PKC and Akt into a conformation that can ultimately autophosphorylate at the hydrophobic motif. Identification of the direct mTOR phosphorylation that facilitates autoregulation of the C-tail hydrophobic motif revises the activation mechanisms of mTOR-regulated AGC kinases. This new twist to an old tail opens avenues for therapeutic intervention. SIGNIFICANCE STATEMENT: The enzyme mTORC2 has been an enigmatic regulator of AGC kinases such as protein kinase C (PKC) and Akt. The recent discovery of a motif named the TOR interaction motif in the C-tail of these kinases solves the mystery: mTORC2 marks these kinases for maturity by, ultimately, facilitating autophosphorylation of another C-tail site, the hydrophobic motif.

Small-molecule inhibitors are a key resource in the cell signaling toolbox. However, because of their global distribution in the cell, they cannot provide a refined understanding of signaling at distinct subcellular locations. Bucko and colleagues have designed a novel tool to localize inhibitors to specific protein scaffolds, opening a new avenue to study localized kinase activity.

Protein Kinase C (PKC) isozymes are tightly regulated kinases that transduce a myriad of signals from receptor-mediated hydrolysis of membrane phospholipids. They play an important role in brain physiology, and dysregulation of PKC activity is associated with neurodegeneration. Gain-of-function mutations in PKCα are associated with Alzheimer’s disease (AD) and mutations in PKCγ cause spinocerebellar ataxia (SCA) type 14 (SCA14). This article presents an overview of the role of the conventional PKCα and PKCγ in neurodegeneration and proposes repurposing PKC inhibitors, which failed in clinical trials for cancer, for the treatment of neurodegenerative diseases.

Exquisitely tuned activity of protein kinase C (PKC) isozymes is essential to maintaining cellular homeostasis. Whereas loss-of-function mutations are generally associated with cancer, gain-of-function variants in one isozyme, PKCα, are associated with Alzheimer’s disease (AD). Here we show that the enhanced activity of one variant, PKCα M489V, is sufficient to rewire the brain phosphoproteome, drive synaptic degeneration, and impair cognition in a mouse model. This variant causes a modest 30% increase in catalytic activity without altering on/off activation dynamics or stability, underscoring that enhanced catalytic activity is sufficient to drive the biochemical, cellular, and ultimately cognitive effects observed. Analysis of hippocampal neurons from the PKCα M489V mice reveals enhanced amyloid-β-induced synaptic depression and reduced spine density compared to wild-type mice. Behavioral studies reveal that this mutation alone is sufficient to impair cognition, and, when coupled to a mouse model of AD, further accelerates cognitive decline. The druggability of protein kinases positions PKCα as a new and promising therapeutic target in AD.

Exquisitely tuned activity of protein kinase C (PKC) isozymes is essential to maintaining cellular homeostasis. Whereas loss-of-function mutations are generally associated with cancer, gain-of-function variants in one isozyme, PKCα, are associated with Alzheimer’s disease (AD). Here we show that the enhanced activity of one variant, PKCα M489V, is sufficient to rewire the brain phosphoproteome, drive synaptic degeneration, and impair cognition in a mouse model. This variant causes a modest 30% increase in catalytic activity without altering on/off activation dynamics or stability, underscoring that enhanced catalytic activity is sufficient to drive the biochemical, cellular, and ultimately cognitive effects observed. Analysis of hippocampal neurons from PKCα M489V mice reveals enhanced amyloid-β-induced synaptic depression and reduced spine density compared to wild-type mice. Behavioral studies reveal that this mutation alone is sufficient to impair cognition, and, when coupled to a mouse model of AD, further accelerates cognitive decline. The druggability of protein kinases positions PKCα as a promising therapeutic target in AD.

Endoplasmic/sarcoplasmic reticulum Ca2+ stores are essential to myriad cellular processes, however, the structure of these stores is largely unknown and existing models neither explain observations made in vivo nor sufficiently account for physiological data. We investigate CASQ1 - the major Ca2+ binding protein of skeletal muscle – and discover Ca2+-dependent liquid-liquid phase separation activity. The intrinsic disorder of CASQ1 underlies this activity and is regulated via phosphorylation by the secretory pathway kinase FAM20C. This novel divalent cation driven condensation demonstrates liquid-liquid phase separation occurs within the endoplasmic/sarcoplasmic reticulum, mechanistically explains efficient Ca2+ buffering and storage, and represents a largely unexplored mechanism of divalent-cation driven protein association.

Spinocerebellar ataxia type 14 (SCA14) is a neurodegenerative disease caused by germline variants in the diacylglycerol (DG)/Ca2+-regulated protein kinase C gamma (PKCγ), leading to Purkinje cell degeneration and progressive cerebellar dysfunction. The majority of the approximately 50 identified variants cluster to the DG-sensing C1 domains. Here, we use a FRET- based activity reporter to show that ataxia-associated PKCγ mutations enhance basal activity by compromising autoinhibition. Although impaired autoinhibition generally leads to PKC degradation, the C1 domain mutations protect PKCγ from phorbol ester-induced downregulation. Furthermore, it is the degree of disrupted autoinhibition, not changes in the amplitude of agonist- stimulated activity, that correlate with disease severity. This enhanced basal signaling rewires the brain phosphoproteome, as assessed by phosphoproteomic analysis of cerebella from mice expressing a human PKCγ transgene harboring a SCA14 C1 domain mutation, H101Y. Validating that the pathology arises from disrupted autoinhibition, we show that the degree of impaired autoinhibition correlates inversely with age of disease onset in patients: mutations that cause high basal activity are associated with early onset, whereas those that only modestly increase basal activity, including a previously undescribed variant, D115Y, are associated with later onset. Molecular modeling indicates that almost all SCA14 variants that are not in the C1 domains are at interfaces with the C1B domain, and bioinformatics analysis reveals that variants in the C1B domain are under-represented in cancer. Thus, clustering of SCA14 variants to the C1B domain provides a unique mechanism to enhance PKCγ basal activity while protecting the enzyme from downregulation, deregulating the cerebellar phosphoproteome.One Sentence SummarySCA14 driver mutations in PKCγ impair autoinhibition, with defect correlating inversely with age of disease onset.

Loss of apical-basal polarity and activation of epithelial-mesenchymal transition (EMT) both contribute to carcinoma progression and metastasis. Here, we report that apical-basal polarity inhibits EMT to suppress metastatic dissemination. Using mouse and human epithelial three-dimensional organoid cultures, we show that the PAR-atypical protein kinase C (aPKC) polarity complex inhibits EMT and invasion by promoting degradation of the SNAIL family protein SNAI1. Under intact apical-basal polarity, aPKC kinases phosphorylate S249 of SNAI1, which leads to protein degradation. Loss of apical-basal polarity prevents aPKC-mediated SNAI1 phosphorylation and stabilizes the SNAI1 protein to promote EMT and invasion. In human breast tumour xenografts, inhibition of the PAR-complex-mediated SNAI1 degradation mechanism promotes tumour invasion and metastasis. Analyses of human breast tissue samples reveal negative correlations between PAR3 and SNAI1 protein levels. Our results demonstrate that apical-basal polarity functions as a critical checkpoint of EMT to precisely control epithelial-mesenchymal plasticity during tumour metastasis.

The kinase complex mTORC2 is widely accepted as controlling phosphorylation of the hydrophobic motif, a key regulatory switch in the C-terminal tail of protein kinase C (PKC), Akt, and other AGC kinases. Yet the biochemical mechanism by which it controls this site and whether mTOR is the direct hydrophobic motif kinase remain controversial. Here we identify a distinct mTOR-mediated phosphorylation site we term the TOR-Interaction Motif (TIM; F-x(3)-F-pT), which controls hydrophobic motif phosphorylation and activity of PKC and Akt. The TIM is invariant in all mTOR-dependent kinases, is evolutionarily conserved, and co-evolved with mTORC2 components. Mutation of this motif alone in Akt1 (Thr(443)) or together with the turn motif in PKCβII (Thr(634)/Thr(641)) abolishes cellular kinase activity by impairing activation loop and hydrophobic motif phosphorylation. mTORC2 directly phosphorylates the PKC TIM in vitro, and its phosphorylation is detected in mouse brain by mass spectrometry. Overexpression of PDK1 in cells lacking mTORC2 rescues hydrophobic motif phosphorylation of PKC and Akt by a mechanism that depends on their intrinsic catalytic, revealing that mTORC2 facilitates the PDK1 phosphorylation step, which in turn permits autophosphorylation. Analysis of a previously reported PKCβII crystal structure reveals a PKC homodimer driven by a helix containing the TIM. Biophysical proximity assays show that unphosphorylated PKC, but not phosphorylated PKC, dynamically dimerizes in cells. Furthermore, disruption of the dimer interface by stapled peptides promotes hydrophobic motif phosphorylation. Our data support a model in which mTORC2 relieves nascent PKC dimerization through TIM phosphorylation, recruiting PDK1 to phosphorylate the activation loop, and triggering intramolecular hydrophobic motif autophosphorylation. Identification of TIM phosphorylation and its role in the regulation of PKC provides the basis for AGC kinase regulation by mTORC2.

PH domain Leucine-Rich Repeat Protein Phosphatase 1 (PHLPP1) is a tumor suppressor that directly dephosphorylates a wide array of substrates, most notably the pro-survival kinase Akt. However, little is known about the molecular mechanisms governing PHLPP1 itself. Here we report that PHLPP1 is dynamically regulated in a cell cycle-dependent manner, and deletion of PHLPP1 results in mitotic delays and increased rates of chromosomal segregation errors. We show that PHLPP1 is hyperphosphorylated during mitosis by Cdk1 in a functionally uncharacterized region known as the PHLPP1 N-terminal extension (NTE). A proximity-dependent biotin identification (BioID) interaction screen revealed that during mitosis PHLPP1 dissociates from plasma membrane scaffolds, such as Scribble, by a mechanism that depends on its NTE, and gains proximity with kinetochore and mitotic spindle proteins such as KNL1 and TPX2. Our data are consistent with a model in which phosphorylation of PHLPP1 during mitosis regulates binding to its mitotic partners and allows accurate progression through mitosis. The finding that PHLPP1 binds mitotic proteins in a cell cycle- and phosphorylation-dependent manner may have relevance to its tumor suppressive function.

Publisher Summary PKC isozymes play pivotal roles in cell signaling by relaying information from lipid mediators to protein substrates. The relay of this information is under exquisite conformational, spatial, and temporal regulation, and extensive studies on the molecular mechanisms of this control have provided much insight into how PKC is regulated. This chapter summarizes the current understanding of the molecular mechanisms of how protein kinase C transduces information from lipid mediators to protein phosphorylation. The normal function of PKC is under the coordinated regulation of five major mechanisms illustrated: phosphorylation, dephosphorylation, membrane targeting modules, degradation, and anchor proteins. The conserved maturation phosphorylations have been best characterized for conventional PKC isozymes and are described. The control of subcellular localization of kinases by scaffold proteins is emerging as a key requirement in maintaining fidelity and specificity in signaling by protein kinases. Protein kinase C activation is integral to an abundance of intracellular signaling pathways; specific isozymes play roles in most key cellular functions, including survival vs apoptotic pathways, proliferative vs quiescent pathways, receptor desensitization, and cytoskeletal architecture, among many others. New understanding of the regulatory inputs of protein kinase C will likely provide novel therapeutic targets in the coming years.

The mechanistic target of rapamycin complex 1 (mTORC1) integrates growth, nutrient and energy status cues to control cell growth and metabolism. While mTORC1 activation at the lysosome is well characterized, it is not clear how this complex is regulated at other subcellular locations. Here, we combine location-selective kinase inhibition, live-cell imaging and biochemical assays to probe the regulation of growth factor-induced mTORC1 activity in the nucleus. Using a nuclear targeted Akt Substrate-based Tandem Occupancy Peptide Sponge (Akt-STOPS) that we developed for specific inhibition of Akt, a critical upstream kinase, we show that growth factor-stimulated nuclear mTORC1 activity requires nuclear Akt activity. Further mechanistic dissection suggests that nuclear Akt activity mediates growth factor-induced nuclear translocation of Raptor, a regulatory scaffolding component in mTORC1, and localization of Raptor to the nucleus results in nuclear mTORC1 activity in the absence of growth factor stimulation. Taken together, these results reveal a mode of regulation of mTORC1 that is distinct from its lysosomal activation, which controls mTORC1 activity in the nuclear compartment., The role of mTORC1 at the lysosome is well established, but mTORC1 is known to be active in other cellular locations. Here, the authors develop Akt-STOPS to inhibit Akt specifically in the nucleus and identify a new regulatory mode for mTORC1 distinct in the nucleus.

Inflammation is an essential aspect of innate immunity but also contributes to diverse human diseases. Although much is known about the kinases that control inflammatory signaling, less is known about the opposing phosphatases. Here we report that deletion of the gene encoding PH domain Leucine-rich repeat Protein Phosphatase 1 (PHLPP1) protects mice from lethal lipopolysaccharide (LPS) challenge and liveEscherichia coliinfection. Investigation of PHLPP1 function in macrophages reveals that it controls the magnitude and duration of inflammatory signaling by dephosphorylating the transcription factor STAT1 on Ser727 to inhibit its activity, reduce its promoter residency, and reduce the expression of target genes involved in innate immunity and cytokine signaling. This previously undescribed function of PHLPP1 depends on a bipartite nuclear localization signal in its unique N-terminal extension. Our data support a model in which nuclear PHLPP1 dephosphorylates STAT1 to control the magnitude and duration of inflammatory signaling in macrophages.HIGHLIGHTSPHLPP1 controls the transcription of genes involved in inflammatory signalingPHLPP1 dephosphorylates STAT1 on Ser727 to reduce its transcriptional activityPHLPP1 has a nuclear localization signal and a nuclear exclusion signalLoss of PHLPP1 protects mice from sepsis-induced death

Atypical protein kinase C (aPKC) isozymes are unique in the PKC superfamily in that they are not regulated by the lipid second messenger diacylglycerol, which has led to speculation about whether a different second messenger acutely controls their function. Here, using a genetically encoded reporter that we designed, aPKC-specific C kinase activity reporter (aCKAR), we found that the lipid mediator sphingosine 1-phosphate (S1P) promoted the cellular activity of aPKC. Intracellular S1P directly bound to the purified kinase domain of aPKC and relieved autoinhibitory constraints, thereby activating the kinase. In silico studies identified potential binding sites on the kinase domain, one of which was validated biochemically. In HeLa cells, S1P-dependent activation of aPKC suppressed apoptosis. Together, our findings identify a previously undescribed molecular mechanism of aPKC regulation, a molecular target for S1P in cell survival regulation, and a tool to further explore the biochemical and biological functions of aPKC.

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.

Emerging antibiotic resistance among pathogenic bacteria is an issue of great clinical importance, and new approaches to therapy are urgently needed. Anacardic acid, the primary active component of cashew nut shell extract, is a natural product used in the treatment of a variety of medical conditions, including infectious abscesses. Here, we investigate the effects of this natural product on the function of human neutrophils. We find that anacardic acid stimulates the production of reactive oxygen species and neutrophil extracellular traps, two mechanisms utilized by neutrophils to kill invading bacteria. Molecular modeling and pharmacological inhibitor studies suggest anacardic acid stimulation of neutrophils occurs in a PI3K-dependent manner through activation of surface-expressed G protein-coupled sphingosine-1-phosphate receptors. Neutrophil extracellular traps produced in response to anacardic acid are bactericidal and complement select direct antimicrobial activities of the compound.

Atypical protein kinase C (aPKC) isoenzymes are key modulators of insulin signalling, and their dysfunction correlates with insulin-resistant states in both mice and humans. Despite the engaged interest in the importance of aPKCs to type 2 diabetes, much less is known about the molecular mechanisms that govern their cellular functions than for the conventional and novel PKC isoenzymes and the functionally-related protein kinase B (Akt) family of kinases. Here we show that aPKC is constitutively phosphorylated and, using a genetically-encoded reporter for PKC activity, basally active in cells. Specifically, we show that phosphorylation at two key regulatory sites, the activation loop and turn motif, of the aPKC PKCζ in multiple cultured cell types is constitutive and independently regulated by separate kinases: ribosome-associated mammalian target of rapamycin complex 2 (mTORC2) mediates co-translational phosphorylation of the turn motif, followed by phosphorylation at the activation loop by phosphoinositide-dependent kinase-1 (PDK1). Live cell imaging reveals that global aPKC activity is constitutive and insulin unresponsive, in marked contrast to the insulin-dependent activation of Akt monitored by an Akt-specific reporter. Nor does forced recruitment to phosphoinositides by fusing the pleckstrin homology (PH) domain of Akt to the kinase domain of PKCζ alter either the phosphorylation or activity of PKCζ. Thus, insulin stimulation does not activate PKCζ through the canonical phosphatidylinositol-3,4,5-triphosphate-mediated pathway that activates Akt, contrasting with previous literature on PKCζ activation. These studies support a model wherein an alternative mechanism regulates PKCζ-mediated insulin signalling that does not utilize conventional activation via agonist-evoked phosphorylation at the activation loop. Rather, we propose that scaffolding near substrates drives the function of PKCζ.

The proto-oncogene Akt/protein kinase B (PKB) is a pivotal signal transducer for growth and survival. Growth factor stimulation leads to Akt phosphorylation at two regulatory sites (Thr-308 and Ser-473), acutely activating Akt signaling. Delineating the exact role of each regulatory site is, however, technically challenging and has remained elusive. Here, we used genetic code expansion to produce site-specifically phosphorylated Akt1 to dissect the contribution of each regulatory site to Akt1 activity. We achieved recombinant production of full-length Akt1 containing site-specific pThr and pSer residues for the first time. Our analysis of Akt1 site-specifically phosphorylated at either or both sites revealed that phosphorylation at both sites increases the apparent catalytic rate 1500-fold relative to unphosphorylated Akt1, an increase attributable primarily to phosphorylation at Thr-308. Live imaging of COS-7 cells confirmed that phosphorylation of Thr-308, but not Ser-473, is required for cellular activation of Akt. We found in vitro and in the cell that pThr-308 function cannot be mimicked with acidic residues, nor could unphosphorylated Thr-308 be mimicked by an Ala mutation. An Akt1 variant with pSer-308 achieved only partial enzymatic and cellular signaling activity, revealing a critical interaction between the γ-methyl group of pThr-308 and Cys-310 in the Akt1 active site. Thus, pThr-308 is necessary and sufficient to stimulate Akt signaling in cells, and the common use of phosphomimetics is not appropriate for studying the biology of Akt signaling. Our data also indicate that pThr-308 should be regarded as the primary diagnostic marker of Akt activity.

Conventional protein kinase C (PKC) family members are reversibly activated by binding to the second messengers Ca2+ and diacylglycerol, events that break autoinhibitory constraints to allow the enzyme to adopt an active, but degradation-sensitive, conformation. Perturbing these autoinhibitory constraints, resulting in protein destabilization, is one of many mechanisms by which PKC function is lost in cancer. Here, we address how a gain-of-function germline mutation in PKCα in Alzheimer’s disease (AD) enhances signaling without increasing vulnerability to down-regulation. Biochemical analyses of purified protein demonstrate that this mutation results in an ∼30% increase in the catalytic rate of the activated enzyme, with no changes in the concentrations of Ca2+ or lipid required for half-maximal activation. Molecular dynamics simulations reveal that this mutation has both localized and allosteric effects, most notably decreasing the dynamics of the C-helix, a key determinant in the catalytic turnover of kinases. Consistent with this mutation not altering autoinhibitory constraints, live-cell imaging studies reveal that the basal signaling output of PKCα-M489V is unchanged. However, the mutant enzyme in cells displays increased sensitivity to an inhibitor that is ineffective toward scaffolded PKC, suggesting the altered dynamics of the kinase domain may influence protein interactions. Finally, we show that phosphorylation of a key PKC substrate, myristoylated alanine-rich C-kinase substrate, is increased in brains of CRISPR-Cas9 genome-edited mice containing the PKCα-M489V mutation. Our results unveil how an AD-associated mutation in PKCα permits enhanced agonist-dependent signaling via a mechanism that evades the cell’s homeostatic down-regulation of constitutively active PKCα.

Many bioinformatics resources with unique perspectives on the protein landscape are currently available. However, generating new knowledge from these resources requires interoperable workflows that support cross-resource queries. In this study, we employ federated queries linking information from the Protein Kinase Ontology, iPTMnet, Protein Ontology, neXtProt, and the Mouse Genome Informatics to identify key knowledge gaps in the functional coverage of the human kinome and prioritize understudied kinases, cancer variants and post-translational modifications (PTMs) for functional studies. We identify 32 functional domains enriched in cancer variants and PTMs and generate mechanistic hypotheses on overlapping variant and PTM sites by aggregating information at the residue, protein, pathway and species level from these resources. We experimentally test the hypothesis that S768 phosphorylation in the C-helix of EGFR is inhibitory by showing that oncogenic variants altering S768 phosphorylation increase basal EGFR activity. In contrast, oncogenic variants altering conserved phosphorylation sites in the ‘hydrophobic motif’ of PKCβII (S660F and S660C) are loss-of-function in that they reduce kinase activity and enhance membrane translocation. Our studies provide a framework for integrative, consistent, and reproducible annotation of the cancer kinomes.