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      Activation of Atypical Protein Kinase C by Sphingosine 1-Phosphate Revealed by Atypical Protein Kinase C-Specific Activity Reporter

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          Abstract

          Atypical protein kinase C (aPKC) isozymes are unique in the protein kinase C (PKC) superfamily in that they are not regulated by the lipid second messenger diacylglycerol. Whether a different second messenger acutely controls their function is unknown. Here we show that the lipid mediator, sphingosine 1-phosphate (S1P), controls the cellular activity of aPKC. Using a genetically-encoded reporter we designed, aPKC-specific C Kinase Activity Reporter (aCKAR), we demonstrate that intracellular S1P activates aPKC. Biochemical studies reveal that S1P directly binds to the kinase domain of aPKC to relieve autoinhibitory constraints. In silico studies identify potential binding sites on the kinase domain, one of which was validated biochemically. Lastly, functional studies reveal that S1P-dependent activation of aPKC suppresses apoptosis in HeLa cells. Taken together, our data reveal a previously undescribed molecular mechanism for controlling the cellular activity of atypical PKC and identify a new molecular target for S1P.

          One-sentence summary:

          The first genetically-encoded biosensor for atypical protein kinase C isozymes is used to identify intracellular sphingosine 1-phosphate as a novel activator of this class of protein kinase C isozymes, with computational, biochemical, and cellular studies identifying the mechanism and function of this activation.

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          Most cited references46

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          Sphingosine-1-phosphate signaling and its role in disease.

          The bioactive sphingolipid metabolite sphingosine-1-phosphate (S1P) is now recognized as a critical regulator of many physiological and pathophysiological processes, including cancer, atherosclerosis, diabetes and osteoporosis. S1P is produced in cells by two sphingosine kinase isoenzymes, SphK1 and SphK2. Many cells secrete S1P, which can then act in an autocrine or paracrine manner. Most of the known actions of S1P are mediated by a family of five specific G protein-coupled receptors. More recently, it was shown that S1P also has important intracellular targets involved in inflammation, cancer and Alzheimer's disease. This suggests that S1P actions are much more complex than previously thought, with important ramifications for development of therapeutics. This review highlights recent advances in our understanding of the mechanisms of action of S1P and its roles in disease. Copyright © 2011 Elsevier Ltd. All rights reserved.
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            PUMA induces the rapid apoptosis of colorectal cancer cells.

            Through global profiling of genes that were expressed soon after p53 expression, we identified a novel gene termed PUMA (p53 upregulated modulator of apoptosis). The protein encoded by PUMA was found to be exclusively mitochondrial and to bind to Bcl-2 and Bcl-X(L) through a BH3 domain. Exogenous expression of PUMA resulted in an extremely rapid and profound apoptosis that occurred much earlier than that resulting from exogenous expression of p53. Based on its unique expression patterns, p53 dependence, and biochemical properties, PUMA may be a direct mediator of p53-associated apoptosis.
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              Emerging biology of sphingosine-1-phosphate: its role in pathogenesis and therapy.

              Membrane sphingolipids are metabolized to sphingosine-1-phosphate (S1P), a bioactive lipid mediator that regulates many processes in vertebrate development, physiology, and pathology. Once exported out of cells by cell-specific transporters, chaperone-bound S1P is spatially compartmentalized in the circulatory system. Extracellular S1P interacts with five GPCRs that are widely expressed and transduce intracellular signals to regulate cellular behavior, such as migration, adhesion, survival, and proliferation. While many organ systems are affected, S1P signaling is essential for vascular development, neurogenesis, and lymphocyte trafficking. Recently, a pharmacological S1P receptor antagonist has won approval to control autoimmune neuroinflammation in multiple sclerosis. The availability of pharmacological tools as well as mouse genetic models has revealed several physiological actions of S1P and begun to shed light on its pathological roles. The unique mode of signaling of this lysophospholipid mediator is providing novel opportunities for therapeutic intervention, with possibilities to target not only GPCRs but also transporters, metabolic enzymes, and chaperones.
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                Author and article information

                Journal
                101465400
                34171
                Sci Signal
                Sci Signal
                Science signaling
                1945-0877
                1937-9145
                5 July 2019
                01 January 2019
                01 January 2019
                25 July 2019
                : 12
                : 562
                : eaat6662
                Affiliations
                [1 ]Department of Pharmacology, University of California at San Diego, La Jolla, CA 92037, USA
                [2 ]Division of Biochemistry, Department of Biochemistry and Molecular Biology, Graduate School of Medicine, Kobe University, Kobe 650-0017, Japan
                Author notes

                Author contributions: T.K., I.T., T.O., C.A.P. and A.N.V. performed the experiments. T.K. and A.C.N wrote the manuscript. A.D.C. performed the modeling and docking under the mentorship of J.A.M. T.K., S.N., and A.C.N. conceived the project and designed the experiments.

                [* ]Corresponding author. anewton@ 123456ucsd.edu (A.C.N.); tkajimot@ 123456med.kobe-u.ac.jp (T.K.)
                Article
                PMC6657501 PMC6657501 6657501 nihpa1039689
                10.1126/scisignal.aat6662
                6657501
                30600259
                e8627c67-edfc-4a04-908c-bac13c48be3e
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