Publications by authors named "Alexandra C Newton"

116 Publications

mTORC2 controls the activity of PKC and Akt by phosphorylating a conserved TOR interaction motif.

Sci Signal 2021 Apr 13;14(678). Epub 2021 Apr 13.

Department of Pharmacology, University of California at San Diego, La Jolla, CA 92093, USA.

The complex mTORC2 is accepted to be the kinase that controls the phosphorylation of the hydrophobic motif, a key regulatory switch for AGC kinases, although whether mTOR directly phosphorylates this motif remains controversial. Here, we identified an mTOR-mediated phosphorylation site that we termed the TOR interaction motif (TIM; F-x-F-pT), which controls the phosphorylation of the hydrophobic motif of PKC and Akt and the activity of these kinases. The TIM is invariant in mTORC2-dependent AGC kinases, is evolutionarily conserved, and coevolved with mTORC2 components. Mutation of this motif in Akt1 and PKCβII abolished cellular kinase activity by impairing activation loop and hydrophobic motif phosphorylation. mTORC2 directly phosphorylated the PKC TIM in vitro, and this phosphorylation event was detected in mouse brain. Overexpression of PDK1 in mTORC2-deficient cells rescued hydrophobic motif phosphorylation of PKC and Akt by a mechanism dependent on their intrinsic catalytic activity, revealing that mTORC2 facilitates the PDK1 phosphorylation step, which, in turn, enables autophosphorylation. Structural analysis revealed that PKC homodimerization is driven by a TIM-containing helix, and biophysical proximity assays showed that newly synthesized, unphosphorylated PKC dimerizes in cells. Furthermore, disruption of the dimer interface by stapled peptides promoted 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.
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http://dx.doi.org/10.1126/scisignal.abe4509DOI Listing
April 2021

PKCα Is Recruited toContaining Phagosomes and Impairs Bacterial Replication by Inhibition of Autophagy.

Front Immunol 2021 18;12:662987. Epub 2021 Mar 18.

Laboratorio de Mecanismos Moleculares Implicados en el Tráfico Vesicular y la Autofagia-Instituto de Histología y Embriología (IHEM)- Universidad Nacional de Cuyo, CONICET- Facultad de Ciencias Médicas, Mendoza, Argentina.

Hijacking the autophagic machinery is a key mechanism through which invasive pathogens such as replicate in their host cells. We have previously demonstrated that the bacteria replicate in phagosomes labeled with the autophagic protein LC3, before escaping to the cytoplasm. Here, we show that the Ca-dependent PKCα binds to -containing phagosomes and that α-hemolysin, secreted by , promotes this recruitment of PKCα to phagosomal membranes. Interestingly, the presence of PKCα prevents the association of the autophagic protein LC3. Live cell imaging experiments using the PKC activity reporter CKAR reveal that treatment of cells with culture supernatants containing staphylococcal secreted factors transiently activates PKC. Functional studies reveal that overexpression of PKCα causes a marked inhibition of bacterial replication. Taken together, our data identify enhancing PKCα activity as a potential approach to inhibit replication in mammalian cells.
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http://dx.doi.org/10.3389/fimmu.2021.662987DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8013776PMC
March 2021

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

J Biol Chem 2021 Feb 19:100445. Epub 2021 Feb 19.

Department of Pharmacology, University of California at San Diego, La Jolla, CA 92093, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jbc.2021.100445DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8008189PMC
February 2021

PHLPPing the balance: restoration of protein kinase C in cancer.

Biochem J 2021 01;478(2):341-355

Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093, U.S.A.

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.
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http://dx.doi.org/10.1042/BCJ20190765DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8086891PMC
January 2021

The PHLPP1 N-Terminal Extension is a Mitotic Cdk1 Substrate and Controls an Interactome Switch.

Mol Cell Biol 2021 Jan 4. Epub 2021 Jan 4.

Department of Pharmacology, University of California, San Diego, California, USA.

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.
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http://dx.doi.org/10.1128/MCB.00333-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8088274PMC
January 2021

Location-specific inhibition of Akt reveals regulation of mTORC1 activity in the nucleus.

Nat Commun 2020 11 30;11(1):6088. Epub 2020 Nov 30.

Department of Pharmacology, University of California, San Diego, La Jolla, CA, USA.

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.
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http://dx.doi.org/10.1038/s41467-020-19937-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7705703PMC
November 2020

Hypothesis: Unifying model of domain architecture for conventional and novel protein kinase C isozymes.

IUBMB Life 2020 12 9;72(12):2584-2590. Epub 2020 Nov 9.

Department of Pharmacology, University of California, San Diego, La Jolla, California, USA.

Protein kinase C (PKC) family members are multi-domain proteins whose function is exquisitely tuned by interdomain interactions that control the spatiotemporal dynamics of their signaling. Despite extensive mechanistic studies on this family of enzymes, no structure of a full-length enzyme that includes all domains has been solved. Here, we take into account the biochemical mechanisms that control autoinhibition, the properties of each individual domain, and previous structural studies to propose a unifying model for the general architecture of PKC family members. This model shows how the C2 domains of conventional and novel PKC isozymes, which have different topologies and different positions in the primary structure, can occupy the same position in the tertiary structure of the kinase. This common architecture of conventional and novel PKC isozymes provides a framework for understanding how disease-associated mutations impair PKC function.
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http://dx.doi.org/10.1002/iub.2401DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8086893PMC
December 2020

How does the International Union of Biochemistry and Molecular Biology support education and training?

Biochem Mol Biol Educ 2021 Jan 6;49(1):7-8. Epub 2020 Nov 6.

Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.

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http://dx.doi.org/10.1002/bmb.21473DOI Listing
January 2021

PHLPPing the Script: Emerging Roles of PHLPP Phosphatases in Cell Signaling.

Annu Rev Pharmacol Toxicol 2021 01 30;61:723-743. Epub 2020 Sep 30.

Department of Pharmacology, University of California, San Diego, La Jolla, California 92093-0721, USA; email:

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.
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http://dx.doi.org/10.1146/annurev-pharmtox-031820-122108DOI Listing
January 2021

Pharmacology on Target.

Trends Pharmacol Sci 2020 04 28;41(4):227-230. Epub 2020 Feb 28.

Department of Pharmacology, University of California at San Diego, San Diego, CA 92093, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.tips.2020.02.002DOI Listing
April 2020

PHLPP1 counter-regulates STAT1-mediated inflammatory signaling.

Elife 2019 08 13;8. Epub 2019 Aug 13.

Department of Pharmacology, University of California, San Diego, San Diego, United States.

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 live infection. 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.
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http://dx.doi.org/10.7554/eLife.48609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6692130PMC
August 2019

The PHLPP2 phosphatase is a druggable driver of prostate cancer progression.

J Cell Biol 2019 06 15;218(6):1943-1957. Epub 2019 May 15.

Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

Metastatic prostate cancer commonly presents with targeted, bi-allelic mutations of the and tumor suppressor genes. In contrast, however, most candidate tumor suppressors are part of large recurrent hemizygous deletions, such as the common chromosome 16q deletion, which involves the AKT-suppressing phosphatase PHLPP2. Using RapidCaP, a genetically engineered mouse model of mutant metastatic prostate cancer, we found that complete loss of paradoxically blocks prostate tumor growth and disease progression. Surprisingly, we find that Phlpp2 is essential for supporting Myc, a key driver of lethal prostate cancer. Phlpp2 dephosphorylates threonine-58 of Myc, which renders it a limiting positive regulator of Myc stability. Furthermore, we show that small-molecule inhibitors of PHLPP2 can suppress MYC and kill mutant cells. Our findings reveal that the frequent hemizygous deletions on chromosome 16q present a druggable vulnerability for targeting MYC protein through PHLPP2 phosphatase inhibitors.
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http://dx.doi.org/10.1083/jcb.201902048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6548123PMC
June 2019

Protein kinases in tune.

IUBMB Life 2019 06 6;71(6):670-671. Epub 2019 May 6.

Department of Pharmacology, University of California San Diego, La Jolla, CA.

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http://dx.doi.org/10.1002/iub.2065DOI Listing
June 2019

Protein Kinase C Quality Control by Phosphatase PHLPP1 Unveils Loss-of-Function Mechanism in Cancer.

Mol Cell 2019 04 20;74(2):378-392.e5. Epub 2019 Mar 20.

Department of Pharmacology, University of California at San Diego, La Jolla, CA 92093, USA. Electronic address:

Protein kinase C (PKC) isozymes function as tumor suppressors in increasing contexts. In contrast to oncogenic kinases, whose function is acutely regulated by transient phosphorylation, PKC is constitutively phosphorylated following biosynthesis to yield a stable, autoinhibited enzyme that is reversibly activated by second messengers. Here, we report that the phosphatase PHLPP1 opposes PKC phosphorylation during maturation, leading to the degradation of aberrantly active species that do not become autoinhibited. Cancer-associated hotspot mutations in the pseudosubstrate of PKCβ that impair autoinhibition result in dephosphorylated and unstable enzymes. Protein-level analysis reveals that PKCα is fully phosphorylated at the PHLPP site in over 5,000 patient tumors, with higher PKC levels correlating (1) inversely with PHLPP1 levels and (2) positively with improved survival in pancreatic adenocarcinoma. Thus, PHLPP1 provides a proofreading step that maintains the fidelity of PKC autoinhibition and reveals a prominent loss-of-function mechanism in cancer by suppressing the steady-state levels of PKC.
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http://dx.doi.org/10.1016/j.molcel.2019.02.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6504549PMC
April 2019

Apical-basal polarity inhibits epithelial-mesenchymal transition and tumour metastasis by PAR-complex-mediated SNAI1 degradation.

Nat Cell Biol 2019 03 25;21(3):359-371. Epub 2019 Feb 25.

Department of Pharmacology, Moores Cancer Center, University of California, San Diego, La Jolla, CA, USA.

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.
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http://dx.doi.org/10.1038/s41556-019-0291-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6546105PMC
March 2019

Activation of atypical protein kinase C by sphingosine 1-phosphate revealed by an aPKC-specific activity reporter.

Sci Signal 2019 01 1;12(562). Epub 2019 Jan 1.

Department of Pharmacology, University of California at San Diego, La Jolla, CA 92037, USA.

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.
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http://dx.doi.org/10.1126/scisignal.aat6662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6657501PMC
January 2019

Protein kinase Cα gain-of-function variant in Alzheimer's disease displays enhanced catalysis by a mechanism that evades down-regulation.

Proc Natl Acad Sci U S A 2018 06 29;115(24):E5497-E5505. Epub 2018 May 29.

Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093;

Conventional protein kinase C (PKC) family members are reversibly activated by binding to the second messengers Ca 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 Ca 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.
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http://dx.doi.org/10.1073/pnas.1805046115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6004447PMC
June 2018

Genetic code expansion and live cell imaging reveal that Thr-308 phosphorylation is irreplaceable and sufficient for Akt1 activity.

J Biol Chem 2018 07 17;293(27):10744-10756. Epub 2018 May 17.

From the Departments of Biochemistry and

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 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.
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http://dx.doi.org/10.1074/jbc.RA118.002357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6036199PMC
July 2018

Integrative annotation and knowledge discovery of kinase post-translational modifications and cancer-associated mutations through federated protein ontologies and resources.

Sci Rep 2018 04 25;8(1):6518. Epub 2018 Apr 25.

Institute of Bioinformatics, University of Georgia, Athens, GA, 30602, USA.

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.
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http://dx.doi.org/10.1038/s41598-018-24457-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5916945PMC
April 2018

Protein kinase C: perfectly balanced.

Crit Rev Biochem Mol Biol 2018 04;53(2):208-230

a Department of Pharmacology , University of California at San Diego , La Jolla , CA , USA.

Protein kinase C (PKC) isozymes belong to a family of Ser/Thr kinases whose activity is governed by reversible release of an autoinhibitory pseudosubstrate. For conventional and novel isozymes, this is effected by binding the lipid second messenger, diacylglycerol, but for atypical PKC isozymes, this is effected by binding protein scaffolds. PKC shot into the limelight following the discovery in the 1980s that the diacylglycerol-sensitive isozymes are "receptors" for the potent tumor-promoting phorbol esters. This set in place a concept that PKC isozymes are oncoproteins. Yet three decades of cancer clinical trials targeting PKC with inhibitors failed and, in some cases, worsened patient outcome. Emerging evidence from cancer-associated mutations and protein expression levels provide a reason: PKC isozymes generally function as tumor suppressors and their activity should be restored, not inhibited, in cancer therapies. And whereas not enough activity is associated with cancer, variants with enhanced activity are associated with degenerative diseases such as Alzheimer's disease. This review describes the tightly controlled mechanisms that ensure PKC activity is perfectly balanced and what happens when these controls are deregulated. PKC isozymes serve as a paradigm for the wisdom of Confucius: "to go beyond is as wrong as to fall short."
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http://dx.doi.org/10.1080/10409238.2018.1442408DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5901981PMC
April 2018

Protein kinase C as a tumor suppressor.

Semin Cancer Biol 2018 02 2;48:18-26. Epub 2017 May 2.

Department of Pharmacology, University of California at San Diego, La Jolla, CA 92093-0721, USA. Electronic address:

Protein kinase C (PKC) has historically been considered an oncoprotein. This stems in large part from the discovery in the early 1980s that PKC is directly activated by tumor-promoting phorbol esters. Yet three decades of clinical trials using PKC inhibitors in cancer therapies not only failed, but in some cases worsened patient outcome. Why has targeting PKC in cancer eluded successful therapies? Recent studies looking at the disease for insight provide an explanation: cancer-associated mutations in PKC are generally loss-of-function (LOF), supporting an unexpected function as tumor suppressors. And, contrasting with LOF mutations in cancer, germline mutations that enhance the activity of some PKC isozymes are associated with degenerative diseases such as Alzheimer's disease. This review provides a background on the diverse mechanisms that ensure PKC is only active when, where, and for the appropriate duration needed and summarizes recent findings converging on a paradigm reversal: PKC family members generally function by suppressing, rather than promoting, survival signaling.
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http://dx.doi.org/10.1016/j.semcancer.2017.04.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5668200PMC
February 2018

Conventional protein kinase C in the brain: 40 years later.

Neuronal Signal 2017 Apr 10;1(2):NS20160005. Epub 2017 Apr 10.

Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093-0721, U.S.A.

Protein kinase C (PKC) is a family of enzymes whose members transduce a large variety of cellular signals instigated by the receptor-mediated hydrolysis of membrane phospholipids. While PKC has been widely implicated in the pathology of diseases affecting all areas of physiology including cancer, diabetes, and heart disease-it was discovered, and initially characterized, in the brain. PKC plays a key role in controlling the balance between cell survival and cell death. Its loss of function is generally associated with cancer, whereas its enhanced activity is associated with neurodegeneration. This review presents an overview of signaling by diacylglycerol (DG)-dependent PKC isozymes in the brain, and focuses on the role of the Ca-sensitive conventional PKC isozymes in neurodegeneration.
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http://dx.doi.org/10.1042/NS20160005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7373245PMC
April 2017

Reversing the Paradigm: Protein Kinase C as a Tumor Suppressor.

Trends Pharmacol Sci 2017 05 8;38(5):438-447. Epub 2017 Mar 8.

Laboratory of Cell and Developmental Signaling, National Cancer Institute at Frederick, Frederick, MD 21702, USA; Cancer Research UK Manchester Institute, Manchester, UK. Electronic address:

The discovery in the 1980s that protein kinase C (PKC) is a receptor for the tumor-promoting phorbol esters fueled the dogma that PKC is an oncoprotein. Yet 30+ years of clinical trials for cancer using PKC inhibitors not only failed, but in some instances worsened patient outcome. The recent analysis of cancer-associated mutations, from diverse cancers and throughout the PKC family, revealed that PKC isozymes are generally inactivated in cancer, supporting a tumor suppressive function. In keeping with a bona fide tumor suppressive role, germline causal loss-of-function (LOF) mutations in one isozyme have recently been identified in lymphoproliferative disorders. Thus, strategies in cancer treatment should focus on restoring rather than inhibiting PKC.
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http://dx.doi.org/10.1016/j.tips.2017.02.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5403564PMC
May 2017

PHLPPing through history: a decade in the life of PHLPP phosphatases.

Biochem Soc Trans 2016 12;44(6):1675-1682

Department of Pharmacology, University of California at San Diego, La Jolla, CA 92093-0721, U.S.A.

In the decade since their discovery, the PH domain leucine-rich repeat protein phosphatases (PHLPP) have emerged as critical regulators of cellular homeostasis, and their dysregulation is associated with various pathophysiologies, ranging from cancer to degenerative diseases, such as diabetes and heart disease. The two PHLPP isozymes, PHLPP1 and PHLPP2, were identified in a search for phosphatases that dephosphorylate Akt, and thus suppress growth factor signaling. However, given that there are over 200 000 phosphorylated residues in a single cell, and fewer than 50 Ser/Thr protein phosphatases, it is not surprising that PHLPP has many other cellular functions yet to be discovered, including a recently identified role in regulating the epigenome. Both PHLPP1 and PHLPP2 are commonly deleted in human cancers, supporting a tumor suppressive role. Conversely, the levels of one isozyme, PHLPP1, are elevated in diabetes. Thus, mechanisms to correctly control PHLPP activity in cells are critical for normal cellular homeostasis. This review summarizes the known functions of PHLPP and its role in disease.
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http://dx.doi.org/10.1042/BST20160170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5783572PMC
December 2016

KinView: a visual comparative sequence analysis tool for integrated kinome research.

Mol Biosyst 2016 11;12(12):3651-3665

Institute of Bioinformatics, University of Georgia, Athens, GA 30602, USA. and Department of Biochemistry & Molecular Biology, University of Georgia, Athens, GA 30602, USA.

Multiple sequence alignments (MSAs) are a fundamental analysis tool used throughout biology to investigate relationships between protein sequence, structure, function, evolutionary history, and patterns of disease-associated variants. However, their widespread application in systems biology research is currently hindered by the lack of user-friendly tools to simultaneously visualize, manipulate and query the information conceptualized in large sequence alignments, and the challenges in integrating MSAs with multiple orthogonal data such as cancer variants and post-translational modifications, which are often stored in heterogeneous data sources and formats. Here, we present the Multiple Sequence Alignment Ontology (MSAOnt), which represents a profile or consensus alignment in an ontological format. Subsets of the alignment are easily selected through the SPARQL Protocol and RDF Query Language for downstream statistical analysis or visualization. We have also created the Kinome Viewer (KinView), an interactive integrative visualization that places eukaryotic protein kinase cancer variants in the context of natural sequence variation and experimentally determined post-translational modifications, which play central roles in the regulation of cellular signaling pathways. Using KinView, we identified differential phosphorylation patterns between tyrosine and serine/threonine kinases in the activation segment, a major kinase regulatory region that is often mutated in proliferative diseases. We discuss cancer variants that disrupt phosphorylation sites in the activation segment, and show how KinView can be used as a comparative tool to identify differences and similarities in natural variation, cancer variants and post-translational modifications between kinase groups, families and subfamilies. Based on KinView comparisons, we identify and experimentally characterize a regulatory tyrosine (Y177) in the PLK4 C-terminal activation segment region termed the P+1 loop. To further demonstrate the application of KinView in hypothesis generation and testing, we formulate and validate a hypothesis explaining a novel predicted loss-of-function variant (D523N) in the regulatory spine of PKCβ, a recently identified tumor suppressor kinase. KinView provides a novel, extensible interface for performing comparative analyses between subsets of kinases and for integrating multiple types of residue specific annotations in user friendly formats.
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http://dx.doi.org/10.1039/c6mb00466kDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5508867PMC
November 2016

Second Messengers.

Cold Spring Harb Perspect Biol 2016 08 1;8(8). Epub 2016 Aug 1.

Department of Pharmacology, Howard Hughes Medical Institute, University of Washington School of Medicine, Seattle, Washington 98195.

Second messengers are small molecules and ions that relay signals received by cell-surface receptors to effector proteins. They include a wide variety of chemical species and have diverse properties that allow them to signal within membranes (e.g., hydrophobic molecules such as lipids and lipid derivatives), within the cytosol (e.g., polar molecules such as nucleotides and ions), or between the two (e.g., gases and free radicals). Second messengers are typically present at low concentrations in resting cells and can be rapidly produced or released when cells are stimulated. The levels of second messengers are exquisitely controlled temporally and spatially, and, during signaling, enzymatic reactions or opening of ion channels ensure that they are highly amplified. These messengers then diffuse rapidly from the source and bind to target proteins to alter their properties (activity, localization, stability, etc.) to propagate signaling.
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http://dx.doi.org/10.1101/cshperspect.a005926DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4968160PMC
August 2016

Protein kinase C mechanisms that contribute to cardiac remodelling.

Clin Sci (Lond) 2016 09;130(17):1499-510

Department of Pharmacology, Columbia University, New York, NY 10032, U.S.A.

Protein phosphorylation is a highly-regulated and reversible process that is precisely controlled by the actions of protein kinases and protein phosphatases. Factors that tip the balance of protein phosphorylation lead to changes in a wide range of cellular responses, including cell proliferation, differentiation and survival. The protein kinase C (PKC) family of serine/threonine kinases sits at nodal points in many signal transduction pathways; PKC enzymes have been the focus of considerable attention since they contribute to both normal physiological responses as well as maladaptive pathological responses that drive a wide range of clinical disorders. This review provides a background on the mechanisms that regulate individual PKC isoenzymes followed by a discussion of recent insights into their role in the pathogenesis of diseases such as cancer. We then provide an overview on the role of individual PKC isoenzymes in the regulation of cardiac contractility and pathophysiological growth responses, with a focus on the PKC-dependent mechanisms that regulate pump function and/or contribute to the pathogenesis of heart failure.
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http://dx.doi.org/10.1042/CS20160036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5024564PMC
September 2016

Bacterial spore coat protein kinases: A new twist to an old story.

Proc Natl Acad Sci U S A 2016 06 10;113(25):6811-2. Epub 2016 Jun 10.

Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093.

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http://dx.doi.org/10.1073/pnas.1607004113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4922189PMC
June 2016

Natural Product Anacardic Acid from Cashew Nut Shells Stimulates Neutrophil Extracellular Trap Production and Bactericidal Activity.

J Biol Chem 2016 Jul 13;291(27):13964-13973. Epub 2016 May 13.

Department of Pediatrics, University of California, San Diego, La Jolla, California 920934; Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California 92093,. Electronic address:

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.
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http://dx.doi.org/10.1074/jbc.M115.695866DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4933157PMC
July 2016

Gain-of-function mutations in protein kinase Cα (PKCα) may promote synaptic defects in Alzheimer's disease.

Sci Signal 2016 05 10;9(427):ra47. Epub 2016 May 10.

Department of Neurosciences and Division of Biology, Section of Neurobiology, University of California, San Diego, La Jolla, CA 92093, USA.

Alzheimer's disease (AD) is a progressive dementia disorder characterized by synaptic degeneration and amyloid-β (Aβ) accumulation in the brain. Through whole-genome sequencing of 1345 individuals from 410 families with late-onset AD (LOAD), we identified three highly penetrant variants in PRKCA, the gene that encodes protein kinase Cα (PKCα), in five of the families. All three variants linked with LOAD displayed increased catalytic activity relative to wild-type PKCα as assessed in live-cell imaging experiments using a genetically encoded PKC activity reporter. Deleting PRKCA in mice or adding PKC antagonists to mouse hippocampal slices infected with a virus expressing the Aβ precursor CT100 revealed that PKCα was required for the reduced synaptic activity caused by Aβ. In PRKCA(-/-) neurons expressing CT100, introduction of PKCα, but not PKCα lacking a PDZ interaction moiety, rescued synaptic depression, suggesting that a scaffolding interaction bringing PKCα to the synapse is required for its mediation of the effects of Aβ. Thus, enhanced PKCα activity may contribute to AD, possibly by mediating the actions of Aβ on synapses. In contrast, reduced PKCα activity is implicated in cancer. Hence, these findings reinforce the importance of maintaining a careful balance in the activity of this enzyme.
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http://dx.doi.org/10.1126/scisignal.aaf6209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5154619PMC
May 2016