Polycystin-1 activates the calcineurin/NFAT (nuclear factor of activated T-cells) signaling pathway.
Regulation of intracellular Ca(2+) mobilization has been associated with the functions of polycystin-1 (PC1) and polycystin-2 (PC2), the protein products of the PKD1 and PKD2 genes. We have now demonstrated that PC1 can activate the calcineurin/NFAT (nuclear factor of activated T-cells) signaling pathway through Galpha(q) -mediated activation of phospholipase C (PLC). Transient transfection of HEK293T cells with an NFAT promoter-luciferase reporter demonstrated that membrane-targeted PC1 constructs containing the membrane proximal region of the C-terminal tail, which includes the heterotrimeric G protein binding and activation domain, can stimulate NFAT luciferase activity. Inhibition of glycogen synthase kinase-3beta by LiCl treatment further increased PC1-mediated NFAT activity. PC1-mediated activation of NFAT was completely inhibited by the calcineurin inhibitor, cyclosporin A. Cotransfection of a construct expressing the Galpha(q) subunit augmented PC1-mediated NFAT activity, whereas the inhibitors of PLC (U73122) and the inositol trisphosphate and ryanodine receptors (xestospongin and 2-aminophenylborate) and a nonspecific Ca(2+) channel blocker (gadolinium) diminished PC1-mediated NFAT activity. PC2 was not able to activate NFAT. An NFAT-green fluorescent protein nuclear localization assay demonstrated that PC1 constructs containing the C-tail only or the entire 11-transmembrane spanning region plus C-tail induced NFAT-green fluorescent protein nuclear translocation. NFAT expression was demonstrated in the M-1 mouse cortical collecting duct cell line and in embryonic and adult mouse kidneys by reverse transcriptase-PCR and immunolocalization. These data suggest a model in which PC1 signaling leads to a sustained elevation of intracellular Ca(2+) mediated by PC1 activation of Galpha(q) followed by PLC activation, release of Ca(2+) from intracellular stores, and activation of store-operated Ca(2+) entry, thus activating calcineurin and NFAT.
Puri
Sanjeev
S
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160, USA.
Magenheimer
Brenda S
BS
Maser
Robin L
RL
Ryan
Erin M
EM
Zien
Christopher A
CA
Walker
Danielle D
DD
Wallace
Darren P
DP
Hempson
Scott J
SJ
Calvet
James P
JP
D021581:Active Transport, Cell Nucleus; D000818:Animals; D015153:Blotting, Western; D001897:Boronic Acids / Q000494:pharmacology; D019703:Calcineurin / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D005682:Gadolinium / Q000494:pharmacology; D017930:Genes, Reporter; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007668:Kidney / Q000196:embryology / Q000378:metabolism; D018021:Lithium Chloride / Q000494:pharmacology; D008156:Luciferases / Q000378:metabolism; D047028:Macrocyclic Compounds; D051379:Mice; D008807:Mice, Inbred BALB C; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D050778:NFATC Transcription Factors; D010080:Oxazoles / Q000494:pharmacology; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000502:physiology*; D011760:Pyrrolidinones / Q000494:pharmacology; D018160:Receptors, Cytoplasmic and Nuclear / Q000037:antagonists & inhibitors; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism; D015398:Signal Transduction; D050396:TRPP Cation Channels; D013997:Time Factors; D014018:Tissue Distribution; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism
D001897:Boronic Acids; D019703:Calcineurin; D002118:Calcium; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D004791:Enzyme Inhibitors; D004963:Estrenes; D005682:Gadolinium; D038362:Glycogen Synthase Kinase 3; D000071679:Glycogen Synthase Kinase 3 beta; D049452:Green Fluorescent Proteins; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018021:Lithium Chloride; D008156:Luciferases; D047028:Macrocyclic Compounds; D050778:NFATC Transcription Factors; D010080:Oxazoles; D011506:Proteins; D011760:Pyrrolidinones; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D050396:TRPP Cation Channels; D010738:Type C Phospholipases; C506926:ITPR1 protein, human; C494005:Nfatc1 protein, mouse; C108644:xestospongin A; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C028592:3-aminobenzeneboronic acid; C000605506:GSK3B protein, human; C000605507:Gsk3b protein, mouse