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1.
Mol Cell Endocrinol ; 177(1-2): 107-15, 2001 May 25.
Article in English | MEDLINE | ID: mdl-11377826

ABSTRACT

The mechanism by which glucose and other fuels stimulate phosphoinositide-specific phospholipase C (PLC) in pancreatic islet beta cells is not known. Previous studies have suggested that glucose may couple to PLC beta 1 and PLC delta 1. To determine directly if fuels activate these PLC isozymes, clones stably overexpressing PLC beta 1 or PLC delta 1 were generated in the fuel-sensitive beta cell line RINm5F, and secretagogue regulation of these PLC isoforms was determined. Overexpression of PLC beta 1 or PLC delta 1 significantly increased PLC activity in isolated cell fractions, consistent with overexpression of active PLC isoforms in these clones. In paired experiments, stimulation of inositol phosphate (IP) accumulation by the fuel glyceraldehyde was enhanced in clones overexpressing PLC beta 1, in parallel with the G-protein alpha subunit activator, AlF(4)(-), suggesting a coupling between glyceraldehyde and this PLC isoform. In contrast, overexpression of PLC delta 1 had no effect on glyceraldehyde- or AlF(4)(-)-stimulated IP accumulation. Similarly, IP accumulation stimulated by ionomycin was enhanced in PLC beta 1, but not PLC delta 1 clones, indicating that increases in intracellular free calcium [Ca(2+)](i) can regulate PLC beta 1 but not PLC delta 1 overexpressed in this cell line. Interestingly, [Arg(8)] vasopressin-stimulated, but not carbachol-stimulated, IP accumulation was significantly increased in clones overexpressing either PLC beta 1 or PLC delta 1. These studies illustrate unique pathways coupling diverse secretagogues to specific PLC isoforms in islet beta cells, and demonstrate that glyceraldehyde can activate PLC beta 1 but not PLC delta 1; whereas, vasopressin, but not carbachol, can stimulate either isoform.


Subject(s)
Glyceraldehyde/pharmacology , Islets of Langerhans/enzymology , Isoenzymes/metabolism , Type C Phospholipases/metabolism , Vasopressins/pharmacology , Animals , Carbachol/pharmacology , Cell Line , Enzyme Activation/drug effects , Inositol Phosphates/metabolism , Insulin/metabolism , Insulin Secretion , Isoenzymes/drug effects , Phospholipase C beta , Phospholipase C delta , Rats , Transfection , Type C Phospholipases/drug effects
2.
EMBO J ; 20(4): 743-54, 2001 Feb 15.
Article in English | MEDLINE | ID: mdl-11179219

ABSTRACT

Three classes of mammalian phosphoinositide-specific phospholipase C (PLC) have been characterized, PLCbeta, PLCgamma and PLCdelta, that are differentially regulated by heterotrimeric G-proteins, tyrosine kinases and calcium. Here we describe a fourth class, PLCepsilon, that in addition to conserved PLC domains, contains a GTP exchange factor (GRF CDC25) domain and two C-terminal Ras-binding (RA) domains, RA1 and RA2. The RA2 domain binds H-Ras in a GTP-dependent manner, comparable with the Ras-binding domain of Raf-1; however, the RA1 domain binds H-Ras with a low affinity in a GTP-independent manner. While G(alpha)q, Gbetagamma or, surprisingly, H-Ras do not activate recombinant purified protein in vitro, constitutively active Q61L H-Ras stimulates PLC(epsilon) co-expressed in COS-7 cells in parallel with Ras binding. Deletion of either the RA1 or RA2 domain inhibits this activation. Site-directed mutagenesis of the RA2 domain or Ras demonstrates a conserved Ras-effector interaction and a unique profile of activation by Ras effector domain mutants. These studies identify a novel fourth class of mammalian PLC that is directly regulated by Ras and links two critical signaling pathways.


Subject(s)
Type C Phospholipases/metabolism , ras Proteins/metabolism , Amino Acid Sequence , Animals , COS Cells , Cloning, Molecular , Molecular Sequence Data , Mutagenesis , Phosphoinositide Phospholipase C , Protein Binding , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Deletion , Sequence Homology, Amino Acid , Spodoptera , Type C Phospholipases/chemistry , Type C Phospholipases/genetics
3.
Endocrine ; 8(2): 161-7, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9704573

ABSTRACT

The aim of this study was to examine the effects of insulin and phorbol 12-myristate 13-acetate (PMA), an activator of classic and novel PKCs, on the translocation of PKC from cytosol to membrane in H4IIE (H4) rat hepatoma cells. Six PKC isoforms were expressed, including PKC-mu and PKC-lambda, identified for the first time in this hepatoma-cell line. Insulin induced translocation of PKC-delta from the cytosol to the membrane fraction as early as 15 min and maximally at 60 min with levels returning to that of controls by 180 min. Insulin also decreased levels of PKC-zeta in membranes at 5, 10, 15, and 30 min, but had no effect on cytosol levels. Ten minutes of PMA treatment translocated PKC-delta completely, and 24 h of PMA treatment downregulated PKC-delta. Neither acute nor chronic PMA had any effect on PKC-zeta. These studies establish the ability of both insulin and PMA to activate PKC-delta in H4 cells, and coupled with our previous work demonstrating a diminution of the effect of insulin on gene transcription in PKC downregulated cells, suggest that insulin may exert specific effects, in part, through a PKC-dependent pathway.


Subject(s)
Insulin/pharmacology , Isoenzymes/metabolism , Liver Neoplasms, Experimental/enzymology , Protein Kinase C/metabolism , Animals , Biological Transport/drug effects , Blotting, Western , Cell Fractionation , Cell Membrane/enzymology , Cytosol/enzymology , Kinetics , Liver Neoplasms, Experimental/ultrastructure , Rats , Tumor Cells, Cultured
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