title: Autocrine Signaling Underlies Fast Repetitive Plasma Membrane Translocation of Conventional and Novel Protein Kinase C Isoforms in β Cells.
abstract: PKC signaling has been implicated in the regulation of many cell functions, including metabolism, cell death, proliferation, and secretion. Activation of conventional and novel PKC isoforms is associated with their Ca(2+)- and/or diacylglycerol (DAG)-dependent translocation to the plasma membrane. In β cells, exocytosis of insulin granules evokes brief (<10 s) local DAG elevations ("spiking") at the plasma membrane because of autocrine activation of P2Y1 purinoceptors by ATP co-released with insulin. Using total internal reflection microscopy, fluorescent protein-tagged PKCs, and signaling biosensors, we investigated whether DAG spiking causes membrane recruitment of PKCs and whether different classes of PKCs show characteristic responses. Glucose stimulation of MIN6 cells triggered DAG spiking with concomitant repetitive translocation of the novel isoforms PKCδ, PKCϵ, and PKCη. The conventional PKCα, PKCβI, and PKCβII isoforms showed a more complex pattern with both rapid and slow translocation. K(+) depolarization-induced PKCϵ translocation entirely mirrored DAG spiking, whereas PKCβI translocation showed a sustained component, reflecting the subplasma membrane Ca(2+) concentration ([Ca(2+)]pm), with additional effect during DAG spikes. Interference with DAG spiking by purinoceptor inhibition prevented intermittent translocation of PKCs and reduced insulin secretion but did not affect [Ca(2+)]pm elevation or sustained PKCβI translocation. The muscarinic agonist carbachol induced pronounced transient PKCβI translocation and sustained recruitment of PKCϵ. When rise of [Ca(2+)]pm was prevented, the carbachol-induced DAG and PKCϵ responses were somewhat reduced, but PKCβI translocation was completely abolished. We conclude that exocytosis-induced DAG spikes efficiently recruit both conventional and novel PKCs to the β cell plasma membrane. PKC signaling is thus implicated in autocrine regulation of β cell function.
authors.affiliation: From the Department of Medical Cell Biology, Uppsala University, Biomedical Centre, Box 571, 75123 Uppsala, Sweden.
authors.forename: Anne
authors.initials: A
authors.lastname: Wuttke
authors.affiliation: From the Department of Medical Cell Biology, Uppsala University, Biomedical Centre, Box 571, 75123 Uppsala, Sweden.
authors.forename: Qian
authors.initials: Q
authors.lastname: Yu
authors.affiliation: From the Department of Medical Cell Biology, Uppsala University, Biomedical Centre, Box 571, 75123 Uppsala, Sweden anders.tengholm@mcb.uu.se.
authors.forename: Anders
authors.initials: A
authors.lastname: Tengholm
mesh_terms: D000818:Animals; D019898:Autocrine Communication / Q000187:drug effects; D002217:Carbachol / Q000494:pharmacology; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D004075:Diglycerides / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology; D050417:Insulin-Secreting Cells / Q000201:enzymology; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D011976:Receptors, Muscarinic / Q000378:metabolism; D058472:Receptors, Purinergic P2Y1 / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects
chemical_list: C011439:1,2-diacylglycerol; D004075:Diglycerides; D007328:Insulin; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D008565:Membrane Proteins; D011976:Receptors, Muscarinic; D058472:Receptors, Purinergic P2Y1; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D002217:Carbachol; D011493:Protein Kinase C; D005947:Glucose
keywords: PKC; calcium; diacylglycerol; exocytosis; oscillations; purinergic receptor; β cell