Proteolytic cleavage and nuclear translocation of fibrocystin is regulated by intracellular Ca2+ and activation of protein kinase C
ABSTRACT Fibrocystin, a type I membrane protein of unknown function, is the protein affected in the autosomal recessive form of polycystic kidney disease. Here we show that fibrocystin undergoes regulated proteolysis. Several proteolytic cleavages occur within the predicted ectodomain, whereas at least one cleavage occurs within the cytoplasmic portion. The latter generates a C-terminal intracellular fragment that harbors the nuclear localization signal KRKVSRLAVTGERTATPAPKIPRIT and translocates to the nucleus. Proteolytic cleavage of fibrocystin occurs constitutively in long term cultures of polarized inner medullary collecting duct cells (mIMCD-3). Activation of protein kinase C and release of intracellular Ca2+ are required for proteolysis under these conditions. In short term cultures of human embryonic kidney 293 cells (HEK-293), proteolytic cleavage of fibrocystin can be elicited by stimulation of intracellular Ca2+ release or activation of protein kinase C. These results identify a novel Ca2+-dependent pathway that signals from fibrocystin located in the cell membrane to the nucleus.
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ABSTRACT: FPC (fibrocystin or polyductin) is a single transmembrane receptor-like protein, responsible for the human autosomal recessive polycystic kidney disease (ARPKD). It was recently proposed that FPC undergoes a Notch-like cleavage and subsequently the cleaved carboxy(C)-terminal fragment translocates to the nucleus. To study the functions of the isolated C-tail, we expressed the intracellular domain of human FPC (hICD) in renal epithelial cells. By 3-dimensional (3D) tubulogenesis assay, we found that in contrast to tubule-like structures formed from control cells, hICD-expressing cells exclusively formed cyst-like structures. By western blotting, we showed that the Akt/mTOR pathway, indicated by increased phosphorylation of Akt at serine 473 and S6 kinase 1 at threonine 389, was constitutively activated in hICD-expressing cells, similar to that in FPC knockdown cells and ARPKD kidneys. Moreover, application of mTOR inhibitor rapamycin reduced the size of the cyst-like structures formed by hICD-expressing cells. Application of either LY294002 or wortmannin inhibited the activation of both S6K1 and Akt. Expression of full-length FPC inhibited the activation of S6 and S6 kinase whereas co-expression of hICD with full-length FPC antagonized the inhibitory effect of full-length FPC on mTOR. Taken together, we propose that FPC modulates the PI3K/Akt/mTOR pathway and the cleaved C-tail regulates the function of the full-length protein.PLoS ONE 05/2014; 9(5):e95630. DOI:10.1371/journal.pone.0095630 · 3.53 Impact Factor
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ABSTRACT: The portable synchrotron MIRRORCLE-20 was converted to IR and FIR beam source for the life science research. To collect the whole radiation, we installed the magic mirror and the circular mirror in the MIRRORCLE-20. The repetition of 40 Hz and the vacuum level of 10-5 [Pa] were improved to 100 Hz and 10-7 [Pa], respectively. We observed 0.5 mW peak power and 0.35 mW average power of IR beam. The lifetime of electron beam was enhanced about two times.
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ABSTRACT: Autosomal recessive polycystic kidney disease (ARPKD), an inherited disorder characterized by the formation of cysts in renal collecting ducts and biliary dysgenesis, is caused by mutations of PKHD1. The expression of PKHD1 is tissue-specific and developmentally regulated. Here, we show that a 2.0-kb genomic fragment containing the proximal promoter of the mouse Pkhd1 gene directs tissue-specific expression of a lacZ reporter gene in transgenic mice. LacZ is expressed in renal collecting ducts beginning during embryonic development but is not expressed in extrarenal tissues. The Pkhd1 promoter contains a binding site for the transcription factor hepatocyte nuclear factor-1β (HNF-1β) that is required for activity in transfected cells. Mutation of the HNF-1β binding site abolishes the expression of the lacZ reporter gene in renal collecting ducts. Transgenes containing the 2.0-kb promoter and 2.7-kb of additional genomic sequence extending downstream to the second exon are expressed in the kidney, intrahepatic bile ducts, and male reproductive tract. This pattern overlaps with the endogenous expression of Pkhd1 and coincides with sites of expression of HNF-1β. We conclude that the proximal 2.0 kb promoter is sufficient for tissue-specific expression of Pkhd1 in renal collecting ducts in vivo, and HNF-1β is required for Pkhd1 promoter activity in collecting ducts. Additional genomic sequences located in exons 1-2 or elsewhere in the gene locus are required for expression in extrarenal tissues.American journal of physiology. Renal physiology 06/2014; 307(3). DOI:10.1152/ajprenal.00422.2013 · 3.30 Impact Factor