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. 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. Autosomal recessive polycystic kidney disease (ARPKD) 2The abbreviations used are: PKD, polycystic kidney disease; ARPKD, autosomal recessive PKD; PKC, protein kinase C; RIP, regulated intramembrane proteolysis; PMA, phorbol 12-myristate 13-acetate; EGFP, enhanced green fluorescent protein; DsRed, red fluorescent protein; MDCK, Madin-Darby canine kidney cells; NLS, nuclear localization sequence; APP, amyloid precursor protein; STAT, signal transducers and activators of transcription; LDLR, low density lipoprotein receptor; RyR, ryanodine receptor; FC, fibrocystin; DAPI, 4′,6-diamidino-2-phenylindole. 2The abbreviations used are: PKD, polycystic kidney disease; ARPKD, autosomal recessive PKD; PKC, protein kinase C; RIP, regulated intramembrane proteolysis; PMA, phorbol 12-myristate 13-acetate; EGFP, enhanced green fluorescent protein; DsRed, red fluorescent protein; MDCK, Madin-Darby canine kidney cells; NLS, nuclear localization sequence; APP, amyloid precursor protein; STAT, signal transducers and activators of transcription; LDLR, low density lipoprotein receptor; RyR, ryanodine receptor; FC, fibrocystin; DAPI, 4′,6-diamidino-2-phenylindole. is a hereditary cause of kidney failure in infants and children. ARPKD affects 1 in 20,000 individuals and is characterized by aberrant epithelial cell proliferation, which causes cystic dilation of the renal collecting ducts, and abnormal development of intrahepatic bile ducts (1Lieberman E. Salinas-Madrigal L. Gwinn J.L. Brennan L.P. Fine R.N. Landing B.H. Medicine (Baltimore). 1971; 50: 277-318Crossref PubMed Scopus (139) Google Scholar). Affected individuals present with bilateral kidney enlargement, intrauterine kidney failure, and oligohydramnios; the latter causes pulmonary hypoplasia and limb and facial abnormalities. Children who survive the perinatal period or develop ARPKD later in life develop chronic kidney disease and portal hypertension due to congenital hepatic fibrosis. ARPKD is caused by mutations of the polycystic kidney and hepatic disease gene 1 (PKHD1) located on chromosome 6 (2Zerres K. Mucher G. Bachner L. Deschennes G. Eggermann T. Kaariainen H. Knapp M. Lennert T. Misselwitz J. von Muhlendahl K.E. Neumann H.P.H. Pirson Y. Rudnik-Schoneborn S. Steinbicker V. Wirth B. Scharer K. Nat. Genet. 1994; 7: 429-432Crossref PubMed Scopus (228) Google Scholar). The protein encoded by PKHD1 is termed fibrocystin (also called polyductin, or tigmin (3Xiong H. Chen Y. Yi Y. Tsuchiya K. Moeckel G. Cheung J. Liang D. Tham K. Xu X. Chen X.Z. Pei Y. Zhao Z.J. Wu G. Genomics. 2002; 80: 96-104Crossref PubMed Scopus (61) Google Scholar, 4Onuchic L.F. Furu L. Nagasawa Y. Hou X. Eggermann T. Ren Z. Bergmann C. Senderek J. Esquivel E. Zeltner R. Rudnik-Schoneborn S. Mrug M. Sweeney W. Avner E.D. Zerres K. Guay-Woodford L.M. Somlo S. Germino G.G. Am. J. Hum. Genet. 2002; 70: 1305-1317Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar, 5Ward C.J. Hogan M.C. Rossetti S. Walker D. Sneddon T. Wang X. Kubly V. Cunningham J.M. Bacallao R. Ishibashi M. Milliner D.S. Torres V.E. Harris P.C. Nat. Genet. 2002; 30: 259-269Crossref PubMed Scopus (583) Google Scholar)). Fibrocystin is an ∼500,000 dalton type I membrane protein comprised of a large N-terminal ectodomain, a single transmembrane segment, and a short C-terminal cytoplasmic domain. The ectodomain contains arrays of IPT (Ig-like, plexins, transcription factors) domains and PbH1 (parallel β-helix repeats) domains. The structure of fibrocystin suggests that it may function as a receptor for an as yet unidentified ligand. However, the authentic function of fibrocystin and the mechanism of signal transduction remain unknown. Recent studies suggest that the molecular pathogenesis of PKD may involve primary cilia and associated Ca2+-dependent signaling (6Nauli S.M. Alenghat F.J. Luo Y. Williams E. Vassilev P. Li X. Elia A.E. Lu W. Brown E.M. Quinn S.J. Ingber D.E. Zhou J. Nat. Genet. 2003; 33: 129-137Crossref PubMed Scopus (1609) Google Scholar). Primary cilia are present on the apical membrane of renal tubular epithelial cells, and bending of the cilia in response to fluid flow shear stress elicits an increase in cytosolic Ca2+ concentration ([Ca2+]). Polycystin-1 and polycystin-2, which are affected in the autosomal dominant form of PKD, are located in primary cilia and are required for an initial Ca2+ influx that is triggered by fluid flow shear stress (6Nauli S.M. Alenghat F.J. Luo Y. Williams E. Vassilev P. Li X. Elia A.E. Lu W. Brown E.M. Quinn S.J. Ingber D.E. Zhou J. Nat. Genet. 2003; 33: 129-137Crossref PubMed Scopus (1609) Google Scholar). Subsequently, the increase in [Ca2+]i is amplified by Ca2+-induced Ca2+ release. Inhibitors of intracellular Ca2+ release abolish the primary cilia-dependent increase in [Ca2+]i, which suggests that Ca2+-induced Ca2+ release is an obligatory step in the primary cilia signaling cascade (6Nauli S.M. Alenghat F.J. Luo Y. Williams E. Vassilev P. Li X. Elia A.E. Lu W. Brown E.M. Quinn S.J. Ingber D.E. Zhou J. Nat. Genet. 2003; 33: 129-137Crossref PubMed Scopus (1609) Google Scholar). Fibrocystin is located in the primary cilium as well as the basal body, which anchors the primary cilium in the cell body (7Masyuk T.V. Huang B.Q. Ward C.J. Masyuk A.I. Yuan D. Splinter P.L. Punyashthiti R. Ritman E.L. Torres V.E. Harris P.C. LaRusso N.F. Gastroenterology. 2003; 125: 1303-1310Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 8Menezes L.F. Cai Y. Nagasawa Y. Silva A.M. Watkins M.L. Da Silva A.M. Somlo S. Guay-Woodford L.M. Germino G.G. Onuchic L.F. Kidney Int. 2004; 66: 1345-1355Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 9Wang S. Luo Y. Wilson P.D. Witman G.B. Zhou J. J. Am. Soc. Nephrol. 2004; 15: 592-602Crossref PubMed Scopus (124) Google Scholar, 10Ward C.J. Yuan D. Masyuk T.V. Wang X. Punyashthiti R. Whelan S. Bacallao R. Torra R. LaRusso N.F. Torres V.E. Harris P.C. Hum. Mol. Genet. 2003; 12: 2703-2710Crossref PubMed Scopus (260) Google Scholar). Together with some similarities in disease manifestations, the overlapping subcellular localization of fibrocystin and polycystins suggests that they may be involved in a common pathway. However, so far, no such pathway has been described. Recent studies suggest that ciliary signaling may involve the regulated proteolysis of polycystin-1. Polycystin-1 undergoes regulated intramembrane proteolysis (RIP), which releases a C-terminal fragment that translocates to the nucleus. Disruption of primary cilia formation causes the accumulation of the fragment in the nucleus, where it affects AP-1 activity. The C-terminal fragment of polycystin-1 has also been shown to interact with STAT6 and the coactivator p100 in the nucleus. Here we show that fibrocystin undergoes regulated proteolytic cleavage releasing a cytoplasmic fragment that translocates to the nucleus. We identify factors that are necessary and sufficient to trigger proteolytic cleavage, and we define a nuclear translocation signal located within the cytoplasmic portion of fibrocystin. Reagents and Antibodies—Dantrolene, caffeine, ruthenium red, calphostin, thapsigargin, carbachol, and phorbol 12-myristate 13-acetate (PMA) were from Sigma. Mifepristone and anti-V5 antibody were from Invitrogen. Monoclonal mouse anti-fibrocystin antibody was described previously (10Ward C.J. Yuan D. Masyuk T.V. Wang X. Punyashthiti R. Whelan S. Bacallao R. Torra R. LaRusso N.F. Torres V.E. Harris P.C. Hum. Mol. Genet. 2003; 12: 2703-2710Crossref PubMed Scopus (260) Google Scholar). To generate rabbit anti-fibrocystin serum (IgG8739), rabbits were immunized with a polyacrylamide gel-purified glutathione S-transferase fusion protein containing the cytoplasmic domain of mouse fibrocystin (amino acids 3869-4060). Plasmids—cDNA encoding full-length (12,225 bp) human fibrocystin was assembled from 12 PCR fragments that were synthesized using human kidney cDNA as template and verified by sequencing. A V5 epitope tag was attached in-frame to the C terminus by removing the stop codon and inserting the DNA fragment into pcDNA3.1-V5/His (Invitrogen). The resultant plasmid was termed pFC-V5. The plasmid pGeneFC was created by inserting the fibrocystin coding region into pGene/V5 (Invitrogen). Inserting a cDNA encoding EGFP into pGene generated the plasmid pGeneEGFP. The plasmid pSwitch was from Invitrogen. To construct the plasmid pFCA-EGFP, a PCR fragment encoding the C terminus of mouse fibrocystin (amino acids 3876-4059) was inserted 5′ to the EGFP coding region of pEGFP. Subsequently, the DNA fragment encoding FCA-EGFP was excised and inserted into pcDNA3.1. To construct the plasmid pFCA-DsRed, the EGFP coding region in pFCA-EGFP was replaced with a DNA fragment encoding DsRed2 (Clontech). The plasmids pEGFP-FC(3876-4059), pEGFP-FC(3876-3970), pEGFP-FC(3973-4031), pEGFP-FC(4043-4059), pEGFP-FC(3876-3940) pEGFP-FC3941-3970), pEGFP-FC(3946-3951), pEGFP-FC(3946-3954), pEGFP-FC(3941-3951), pEGFP-FC(3952-3970), pEGFP-FC(3949-3970), pEGFP-FC(3946-3970), and pEGFP-FC(3941-3963) were generated by inserting DNA encoding the corresponding regions of mouse fibrocystin into pEGFP-C3. To construct the plasmids pFC(3973-4031)-V5 and pFC(3875-3970)-V5, PCR fragments encoding the corresponding regions of mouse fibrocystin were cloned into pcDNA3.1-V5/His. The plasmid pLDLR-GV encoding the human low density lipoprotein receptor (LDLR) fused at its C terminus to Gal4-VP16 was a generous gift from Dr. Thomas Südhof (University of Texas Southwestern Medical School, Dallas, TX). The plasmid pFC-GV was generated by inserting the Gal4-VP16 coding sequence into the ApaI site of human fibrocystin (at codon 3918). The pG5-luc reporter plasmid was a generous gift from Richard Baer (Columbia University, New York, NY) (11Yu X. Wu L.C. Bowcock A.M. Aronheim A. Baer R. J. Biol. Chem. 1998; 273: 25388-25392Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). The plasmids pEF-neo and pEF-PKCαA/E were generous gifts from Gottfried Baier (Medical University of Innsbruck, Innsbruck, Austria). Cell Culture, Transfection, and Generation of Stable Cell Lines—mIMCD-3 cells were plated at a density of 500,000 cells/100-mm dish, and 24 and 48 h later, the cells were transfected with 2 μg of pFC-V5 using Effectene (Qiagen, Valencia, CA). Cells were incubated for an additional 72 h and lysed in 100 μl of buffer containing Tris-buffered saline, 0.5% Triton X-100, protease inhibitor mixture (Hoffmann-La Roche Inc.). Twenty μl of the lysates were analyzed by SDS-PAGE, and immunoblot analysis was performed using horseradish peroxidase-conjugated anti-V5 as described previously (12Hiesberger T. Shao X. Gourley E. Reimann A. Pontoglio M. Igarashi P. J. Biol. Chem. 2005; 280: 10578-10586Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). mIMCD3/FC and mIMCD3/EGFP cell lines with of fibrocystin and EGFP, were by cells with μg of pSwitch and μg of pGeneFC or μg of pGeneEGFP. Stable were of in containing and To for cells from were with for 48 h or and were analyzed by using horseradish peroxidase-conjugated cells transfected with the receptor were a generous gift from (University of Medical J. Williams Mol. Google Scholar). cells, cells, or cells were plated in and with μg of encoding μg of reporter and μg of were performed as described previously T. Y. Shao X. A.M. X. Somlo S. Igarashi P. J. 2004; PubMed Scopus Google Scholar). was as the of and containing nuclear cell membrane or were generated as described previously Brown Li A. J.L. Full Text Full Text PDF PubMed Scopus Google Scholar). was by the of the membrane protein and the nuclear protein cell nuclear To nuclear nuclear was with and cells were in and transfected with plasmids using or 48 h later, cells were with and to kidney were and was performed using anti-fibrocystin and were by of Fibrocystin a C-terminal DNA fragment encoding human fibrocystin was assembled from 12 PCR cloned into the plasmid and verified by sequencing. To of the a C-terminal V5 epitope tag was and the resultant plasmid was termed pFC-V5. inner medullary collecting duct cells which fibrocystin T. Y. Shao X. A.M. X. Somlo S. Igarashi P. J. 2004; PubMed Scopus Google were transfected with pFC-V5 or and were analyzed an antibody the C-terminal V5 epitope tag a molecular corresponding to full-length fibrocystin as well as a of proteolytic fragments To the proteolytic cleavage of we generated mIMCD3/FC cells in which the of human fibrocystin can be by with analysis of cells fibrocystin the of proteolytic fragments to in The of proteolytic cleavage of fibrocystin were from the molecular of the proteolytic the cytoplasmic domain of human fibrocystin the epitope tag has a molecular of the fragment is by cleavage in the cytoplasmic domain to the transmembrane Proteolytic fragments of fibrocystin that the as well as the C-terminal V5 epitope tag a molecular of that fragments to were generated by proteolytic cleavage in the ectodomain of fibrocystin. The site of cleavage the fragment may be within the transmembrane To the subcellular localization of the proteolytic we and nuclear and analyzed the by that were to the transmembrane domain to were in the membrane as In the fragment containing the C terminus of fibrocystin was in the nuclear suggests that the cytoplasmic domain of fibrocystin undergoes proteolytic cleavage, releasing a fragment that translocates to the nucleus. In some as well as that it may additional cleavage or and The fragment was in the nuclear and the membrane long of the that is or is subcellular To fibrocystin undergoes proteolytic a nuclear nuclear of cells were analyzed by A antibody the C-terminal domain of fibrocystin a protein corresponding in to The fragment was using the C-terminal domain of fibrocystin were also the that is to of Fibrocystin a the localization of the cytoplasmic domain of fibrocystin in kidney of were with an antibody for the cytoplasmic region of the that the cytoplasmic domain of fibrocystin was located in the of renal tubular epithelial cells The cytoplasmic domain was also in the as well as the primary cilia the cells were in a the fibrocystin in the a To define the mechanism of nuclear the cytoplasmic domain of fibrocystin was to EGFP or and in The subcellular localization of the fusion was by A protein containing the cytoplasmic domain of fibrocystin (amino acids 3876-4059) and EGFP was located in the nucleus, whereas EGFP by was in the a fusion protein containing the cytoplasmic domain of fibrocystin and was located in the nucleus. the fusion were in the were in that a To the nuclear cells were transfected with plasmids encoding the cytoplasmic domain fused to DsRed, and the were with an that the fusion containing the cytoplasmic domain were located in To identify the region within the cytoplasmic domain of fibrocystin that is required for nuclear plasmids encoding EGFP fused to the N-terminal portion (amino acids the portion (amino acids and the C-terminal portion (amino acids of the cytoplasmic domain were transfected into an EGFP fusion protein containing the N-terminal portion was located in the nucleus, whereas fusion containing the or C-terminal in the To these using a cell type and plasmids encoding the V5 N-terminal portion and portion of the cytoplasmic domain were transfected into analysis of nuclear and cytosolic that the N-terminal portion the portion was present in the nuclear To define the sequence nuclear plasmids encoding regions of the cytoplasmic domain of fibrocystin fused to EGFP were and the subcellular of the fusion were These the nuclear localization signal to be KRKVSRLAVTGERTATPAPKIPRIT within sequence nuclear Ca2+ and to Fibrocystin the proteolytic cleavage of fibrocystin is affected by in intracellular Ca2+ concentration and Ca2+-induced Ca2+ mIMCD3/FC cells were with to of fibrocystin with to intracellular Ca2+ J. Biol. 2004; PubMed Scopus Google the of the fragment of Ca2+ by with also the of with ruthenium red, a Ca2+ or with which with ryanodine receptor intracellular Ca2+ the formation of To fibrocystin cleavage, we a to the to the proteolytic cleavage of amyloid precursor protein and low density lipoprotein X. PubMed Scopus Google Scholar, P. J. J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). A plasmid encoding the transcription Gal4-VP16 fused to the C terminus of fibrocystin was Proteolytic cleavage of the fibrocystin fusion protein releases which translocates to the and a reporter gene with fibrocystin of cells with a plasmid encoding with cells density lipoprotein receptor fused to which is to proteolysis. The increase in was in the of that was required for fibrocystin cleavage To intracellular Ca2+ release is required is also sufficient to fibrocystin we a short term cell of human embryonic kidney cells These cells and an increase of intracellular Ca2+ with D.E. J. 1998; PubMed Scopus Google Scholar). basal no proteolytic fragments of transfected fibrocystin were However, the of proteolysis of fibrocystin fibrocystin proteolysis was by with the protein kinase C inhibitor C. latter a of in the Ca2+-induced cleavage of fibrocystin. To fibrocystin proteolysis in response to intracellular Ca2+ the reporter was cells were with the plasmids pFC-GV and pG5-luc and incubated with or a with that stimulation of intracellular Ca2+ release was sufficient to fibrocystin cleavage we fibrocystin cleavage be by activation of a cell receptor that intracellular Ca2+ release. These cells the receptor which to with the with activation of C and Ca2+ release M. S.M. B. D. J. J. 1994; PubMed Scopus Google Scholar). to triggered fibrocystin proteolysis in cells and cells, whereas with 100 fibrocystin cleavage in cells these results that stimulation of intracellular Ca2+ release is sufficient to trigger fibrocystin Activation of and for the Generation of of fibrocystin proteolysis in the of the inhibitor C an of of the of or To activation of is sufficient to fibrocystin cleavage, cells were transfected with pFC-V5 and to activators of protein with PMA, an of PKC, a increase of proteolytic cleavage, which was by with C In no cleavage was cells were or with the protein kinase A However, also the of by the which it to activation with fibrocystin To we used additional of the and activation of mIMCD3/FC cells, in which fibrocystin is under the of a were with C 24 h and cleavage was analyzed h with C of fibrocystin a of fibrocystin proteolysis we a constitutively of G. B. H. H. G. A. Baier G. Mol. Biol. PubMed Google Scholar). of a plasmid encoding in fibrocystin proteolysis and of whereas of plasmid The or of serum To the cleavage of fibrocystin by reporter were cells were with pFC-GV and and was by with or of the Fibrocystin proteolysis was by activity. with whereas the of activity. of no on cells the these results that proteolysis is The that fibrocystin is to proteolytic least fragments that the C terminus of fibrocystin are the of the proteolytic fragments under a fragment was The fragment contains of the cytoplasmic domain of fibrocystin and was in the nucleus. In full-length fibrocystin has been in the primary basal body and apical and to a in the These suggest that fibrocystin undergoes regulated proteolysis releasing a cytoplasmic C-terminal fragment that translocates from the apical or to the nucleus. The protease that fibrocystin cleavage inhibitor studies suggest that it is or to that are to regulated a of fibrocystin to be In the of proteolytic cleavage to the transmembrane nuclear translocation of the cytoplasmic of type I membrane signaling of cytoplasmic fragments generated by is described for also in the of APP, and lipoprotein protein 2004; 15: PubMed Scopus Google Scholar). signaling that are for and disease the cleavage releasing the nuclear fragment is by an obligatory proteolytic in the ectodomain of the is that the proteolytic cleavage in the ectodomain of fibrocystin is also required for a step that releases the cytoplasmic a protein affected in the autosomal dominant form of PKD, was to be to proteolysis in a with of to proteolysis of generates a C-terminal cytoplasmic fragment that translocates to the release. The C-terminal cytoplasmic fragment of polycystin-1 has been shown to AP-1 and the of the latter with In the polycystin-1 C-terminal cytoplasmic fragment with STAT6 and the coactivator p100 and However, of the C-terminal cytoplasmic domain of fibrocystin no on or reporter which suggests that at least some of the of the cytoplasmic domains of polycystin-1 and fibrocystin are We the nuclear localization sequence within the fragment of fibrocystin to be sequence a NLS, which is characterized by of acids that are by a PubMed Scopus Google Scholar). to be in the in the are the site of of and of and cell and transcription by factors A.I. 2003; PubMed Scopus Google Scholar). The localization of suggests that fibrocystin may an in these Several lines of that the release of intracellular Ca2+ is a for fibrocystin proteolysis. cleavage of fibrocystin in a long term cell be by of Ca2+-induced Ca2+ release. cleavage of fibrocystin was under basal in a short term cell cleavage and nuclear translocation be by or stimulation of intracellular Ca2+ release. These suggest that signaling that [Ca2+]i as or of fibrocystin Ca2+-dependent signaling cascade that is to be in the development of polycystic kidney disease primary Primary cilia located on the apical of renal tubular epithelial cells are involved in fluid caused by the flow of fluid the epithelial cell the cilium and an increase in Proteolytic cleavage of polycystin-1 to involve primary The of primary cilia or of fluid flow results in the accumulation of the polycystin-1 C-terminal cytoplasmic fragment in the nucleus. is that primary cilia are also involved in the of fibrocystin proteolysis. Fibrocystin proteolysis was in long term cultures of cells, which form primary and no cleavage was in short term cell which primary However, no of fluid flow on fibrocystin cleavage has been be required to the of primary cilia and the increase in [Ca2+]i on fibrocystin of fibrocystin is by activation of using the inhibitor and a constitutively that activation of is necessary and sufficient to cause fibrocystin proteolysis. Activation of can the for release of intracellular that activation is to be to Ca2+ release. are by intracellular Ca2+ release and of Ca2+-induced Ca2+ release. it is activation of fibrocystin cleavage, one is that the of intracellular fibrocystin to interact with the activation is also involved in the of of been shown to and the of in R. M. C. S. L. B. S. A. 2004; PubMed Scopus Google Scholar). of fibrocystin proteolysis by of activation or intracellular Ca2+ release a to In we a in which cytosolic Ca2+ concentration and activation of a proteolytic that to the release of the cytoplasmic C-terminal fragment of fibrocystin. of the of a NLS, the C-terminal fragment is to the nucleus. A of fibrocystin in a signaling cascade is by similarities with to We are to and Thomas for and and for