Calcium Dependence of Polycystin-2 Channel Activity Is Modulated by Phosphorylation at Ser812

Polycystin-2 (PC-2) is a non-selective cation channel that, when mutated, results in autosomal dominant polycystic kidney disease. In an effort to understand the regulation of this channel, we investigated the role of protein phosphorylation in PC-2 function. We demonstrated the direct incorporation of phosphate into PC-2 in cells and tissues and found that this constitutive phosphorylation occurs at Ser812, a putative casein kinase II (CK2) substrate domain. Ser812 can be phosphorylated by CK2 in vitro and substitution S812A results in failure to incorporate phosphate in cultured epithelial cells. Non-phosphorylated forms of PC-2 traffic normally in the endoplasmic reticulum and cilial compartments and retain homo- and hetero-multimerization interactions with PC-2 and polycystin-1, respectively. Single-channel studies of PC-2, S812A, and a substitution mutant, T721A, not related to phosphorylation show that PC-2 and S812A function as divalent cation channels with similar current amplitudes across a range of holding potentials; the T721A channel is not functional. Channel open probabilities for PC-2 and S812A show a bell-shaped dependence on cytoplasmic Ca2+ but there is a shift in this Ca2+ dependence such that S812A is 10-fold less sensitive to Ca2+ activation/inactivation than the wild type PC-2 channel. In vivo analysis of PC-2-dependent enhanced intracellular Ca2+ transients found that S812A resulted in enhanced transient duration and relative amplitude intermediate between control cells and those overexpressing wild type PC-2. Phosphorylation at Ser812 modulates PC-2 channel activity and factors regulating this phosphorylation are likely to play a role in the pathogenesis of polycystic kidney disease. Polycystin-2 (PC-2) is a non-selective cation channel that, when mutated, results in autosomal dominant polycystic kidney disease. In an effort to understand the regulation of this channel, we investigated the role of protein phosphorylation in PC-2 function. We demonstrated the direct incorporation of phosphate into PC-2 in cells and tissues and found that this constitutive phosphorylation occurs at Ser812, a putative casein kinase II (CK2) substrate domain. Ser812 can be phosphorylated by CK2 in vitro and substitution S812A results in failure to incorporate phosphate in cultured epithelial cells. Non-phosphorylated forms of PC-2 traffic normally in the endoplasmic reticulum and cilial compartments and retain homo- and hetero-multimerization interactions with PC-2 and polycystin-1, respectively. Single-channel studies of PC-2, S812A, and a substitution mutant, T721A, not related to phosphorylation show that PC-2 and S812A function as divalent cation channels with similar current amplitudes across a range of holding potentials; the T721A channel is not functional. Channel open probabilities for PC-2 and S812A show a bell-shaped dependence on cytoplasmic Ca2+ but there is a shift in this Ca2+ dependence such that S812A is 10-fold less sensitive to Ca2+ activation/inactivation than the wild type PC-2 channel. In vivo analysis of PC-2-dependent enhanced intracellular Ca2+ transients found that S812A resulted in enhanced transient duration and relative amplitude intermediate between control cells and those overexpressing wild type PC-2. Phosphorylation at Ser812 modulates PC-2 channel activity and factors regulating this phosphorylation are likely to play a role in the pathogenesis of polycystic kidney disease. Autosomal dominant polycystic kidney disease (ADPKD) 1The abbreviations used are: ADPKD, autosomal dominant polycystic kidney disease; GST, glutathione S-transferase; HA, hemagglutinin; MDCK, Madin-Darby canine kidney cells; DMEM, Dulbecco's modified Eagle's medium; H-DMEM, Ham's F-12:DMEM; IF, immunofluorescent cell staining; IP, immunoprecipitation; Endo H, endoglycosidase H; ER, endoplasmic reticulum; ERD, ER retention domain; CK2, casein kinase II; MES, 4-morpholineethanesulfonic acid; CIP, calf intestinal phosphatase. consists of at least two genetically distinct disorders characterized by bilateral renal cyst formation and progressive renal enlargement. ADPKD affects more than 1 in 1000 live births and is the most common monogenic cause of kidney failure in humans. PKD1 and PKD2, the genes mutated in almost all ADPKD patients, encode polycystin-1 (Pc-1) and polycystin-2 (PC-2), respectively (1Igarashi P. Somlo S. J. Am. Soc. 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Nature. 2000; 408: 990-994Crossref PubMed Scopus (669) Google Scholar, 5Koulen P. Cai Y. Geng L. Maeda Y. Nishimura S. Witzgall R. Ehrlich B.E. Somlo S. Nat. Cell Biol. 2002; 4: 191-197Crossref PubMed Scopus (560) Google Scholar). In mammalian species, PC-2 and Pc-1 have been localized to the apical primary cilia of renal tubule cells and epithelial cells in culture (6Pazour G.J. San Agustin J.T. Follit J.A. Rosenbaum J.L. Witman G.B. Curr. Biol. 2002; 12: R378-R380Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar, 7Yoder B.K. Hou X. Guay-Woodford L.M. J. Am. Soc. Nephrol. 2002; 13: 2508-2516Crossref PubMed Scopus (731) Google Scholar, 8Nauli 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 (1629) Google Scholar) as well as other cellular locations. PC-2 has also been found in monocilia at the embryonic node that play a critical role in left-right axis determination (9McGrath J. Somlo S. Makova S. Tian X. Brueckner M. Cell. 2003; 114: 61-73Abstract Full Text Full Text PDF PubMed Scopus (618) Google Scholar). The PC-2 Ca2+ channel is thought to be part of flow sensor complexes in both renal tubular and node cilia (8Nauli 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 (1629) Google Scholar, 9McGrath J. Somlo S. Makova S. Tian X. Brueckner M. Cell. 2003; 114: 61-73Abstract Full Text Full Text PDF PubMed Scopus (618) Google Scholar). Protein phosphorylation is a post-translational modification that often plays a role in regulation of channel function (10Catterall W.A. Annu. Rev. Cell Dev. Biol. 2000; 16: 521-555Crossref PubMed Scopus (1952) Google Scholar). 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Commun. 1999; 259: 539-543Crossref PubMed Scopus (36) Google Scholar). Protein kinase X, a novel cAMP-dependent serine/threonine kinase that regulates epithelial migration and morphogenesis is aberrantly expressed in kidney tissues from ADPKD patients (15Li X. Li H.P. Amsler K. Hyink D. Wilson P.D. Burrow C.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9260-9265Crossref PubMed Scopus (47) Google Scholar). Mutations in Nek-family kinases Nek1 and Nek8 underlie polycystic kidney phenotypes in a pair of naturally occurring mouse models (16Upadhya P. Birkenmeier E.H. Birkenmeier C.S. Barker J.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 217-221Crossref PubMed Scopus (131) Google Scholar, 17Liu S. Lu W. Obara T. Kuida S. Lehoczky J. Dewar K. Drummond I.A. 2002; PubMed Scopus Google Scholar). kinases by regulating in epithelial cells S. Lu W. Obara T. Kuida S. Lehoczky J. Dewar K. Drummond I.A. 2002; PubMed Scopus Google Scholar). PC-2 is to have at least serine/threonine phosphorylation in cytoplasmic (2Mochizuki T. Wu G. Hayashi T. Xenophontos S.L. Veldhuisen B. Saris J.J. Reynolds D.M. Cai Y. Gabow P.A. Pierides A. Kimberling W.J. Breuning M.H. Deltas C.C. Peters D.J. Somlo S. Science. 1996; 272: 1339-1342Crossref PubMed Scopus (1181) Google Scholar). In the current we to PC-2 is phosphorylated and the functional role of such phosphorylation We found that PC-2 is phosphorylated at Ser812 in Phosphorylation at this not the the with channel studies show that of phosphorylation at Ser812 results in a in the of the PC-2 channel to Phosphorylation of PC-2 is an factor in regulating the activity of the channel in for human by with the of of the at of protein a of the of human PC-2 used to cells from an mouse to cells and by protein and and that results characterized by and and found to be The used in this from cultured of the other used as PC-2 and have been Y. Maeda Y. A. Wu G. Hayashi T. T. Witzgall R. Somlo S. 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PC-2 is phosphorylated in the mouse kidney cells with and by PC-2 from mouse kidney phosphorylated the that PC-2 is phosphorylated in the kidney in The of Phosphorylation of PC-2 of the phosphorylation in PC-2 are in the the phosphorylation of PC-2 occurs in the we phosphorylation of a of PC-2, Y. Maeda Y. A. Wu G. Hayashi T. T. Witzgall R. Somlo S. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). of PC-2 is and to both the ER and the cell Y. Maeda Y. A. Wu G. Hayashi T. T. Witzgall R. Somlo S. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). In to PC-2, not phosphorylated in vivo that the for constitutive phosphorylation for PC-2 is the the for we at of phosphorylation of the Ser812, and We with all by into and in vivo phosphorylation not phosphorylated that the more substrate for phosphorylation are the in the Phosphorylation analysis and the T721A and S812A substitution Ser812 as the of constitutive phosphorylation of PC-2 Ser812 by is a substrate for casein kinase II (CK2) (2Mochizuki T. Wu G. Hayashi T. Xenophontos S.L. Veldhuisen B. Saris J.J. Reynolds D.M. Cai Y. Gabow P.A. Pierides A. Kimberling W.J. Breuning M.H. Deltas C.C. Peters D.J. Somlo S. Science. 1996; 272: 1339-1342Crossref PubMed Scopus (1181) Google a protein that CK2 is expressed in renal epithelial cells the polycystic kidney disease mouse kidney as well as epithelial cells by CK2 is expressed in mouse kidney and cells We PC-2 is phosphorylated by CK2 in the of PC-2 with the phosphorylation for phosphorylation by of the ER retention that both and Ser812 resulted in of phosphorylation by CK2 at and Ser812 phosphorylated whereas not are with Ser812 a of phosphorylation by Phosphorylation at Ser812 for Protein there in the of PC-2, there is that PC-2 is expressed in the ER and primary cilia of epithelial cells (6Pazour G.J. San Agustin J.T. Follit J.A. Rosenbaum J.L. Witman G.B. Curr. Biol. 2002; 12: R378-R380Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar, 7Yoder B.K. Hou X. Guay-Woodford L.M. J. Am. Soc. Nephrol. 2002; 13: 2508-2516Crossref PubMed Scopus (731) Google Scholar, Y. Maeda Y. A. Wu G. Hayashi T. T. Witzgall R. Somlo S. J. 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We used to the role of phosphorylation at Ser812 in PC-2 and hetero-multimerization PC-2, and the forms and S812A by when with a We to with the We have cell overexpressing Pc-1 with a and an and S812A with Pc-1 in a similar to wild type and PC-2 Phosphorylation at Ser812 not of PC-2 with Phosphorylation of the PC-2 Channel to by we the of phosphorylation on PC-2 channel channel of wild T721A, and S812A forms of the PC-2 channel in P. Cai Y. Geng L. Maeda Y. Nishimura S. Witzgall R. Ehrlich B.E. Somlo S. Nat. Cell Biol. 2002; 4: 191-197Crossref PubMed Scopus (560) Google Scholar). of in from cell wild type and PC-2 demonstrated by with not all on the of the channel used as the current not channel activity when on either of the channel P. Cai Y. Geng L. Maeda Y. Nishimura S. Witzgall R. Ehrlich B.E. Somlo S. Nat. 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Cell Biol. 2002; 4: 191-197Crossref PubMed Scopus (560) Google and we the of phosphorylation on this of the channel C and We found that at on the PC-2 a channel open than S812A at S812A a open than PC-2 C and the channel open the range of Ca2+ from to 1 both and S812A forms of the PC-2 channel a bell-shaped dependence on cytoplasmic Ca2+ there a shift in the Ca2+ dependence such that PC-2 a open of at whereas S812A a open of at 10-fold Ca2+ The S812A substitution results in of phosphorylation of PC-2 but also in an in protein the in Ca2+ dependence the of the of phosphorylation of other we channel studies in the of calf intestinal either on the of the on the with on the resulted in Ca2+ dependence to that in the S812A substitution that the to Ca2+ is the of of phosphorylation on the of the channel not The open of wild type PC-2 channel occurs at a Ca2+ at the Ca2+ plays an role in PC-2 channel function in phosphorylation is an in the normal function of PC-2. We have an in vivo for PC-2 channel activity P. Cai Y. Geng L. Maeda Y. Nishimura S. Witzgall R. Ehrlich B.E. Somlo S. Nat. Cell Biol. 2002; 4: 191-197Crossref PubMed Scopus (560) Google Scholar). of wild type PC-2 in cells results in enhanced amplitude and duration of Ca2+ from intracellular with PC-2 this is on Ca2+ also in cells. We used this to Ca2+ by the S812A of PC-2. of resulted in transient in intracellular Ca2+ in control cells as well as cells overexpressing either PC-2 S812A both the relative amplitude and for the Ca2+ transients the of wild type PC-2 resulted in the relative amplitude and duration of the the relative amplitude and duration of Ca2+ the S812A Ca2+ transients with amplitude and duration intermediate between PC-2 overexpressing and cells The are with the that phosphorylation at Ser812 is for the normal by PC-2 to in intracellular Ca2+ that phosphorylation at Ser812 modulates the Ca2+ dependence of polycystin-2 channel function in Mutations in the PC-2 channel underlie ADPKD. of in of the G. Somlo S. Mol. Genet. 2000; PubMed Scopus Google with the of PC-2 with and with PC-1 F. Germino F.J. Cai Y. X. Somlo S. Germino G.G. Nat. Genet. 16: PubMed Scopus Google Scholar). at least D.M. Hayashi T. Cai Y. Veldhuisen B. T. Qian F. Maeda Y. Li R. E. Wu Breuning M.H. Germino G.G. Peters Somlo S. J. 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Genet. 2003; 33: 129-137Crossref PubMed Scopus (1629) Google and we have that Ca2+ modulates PC-2 channel but other factors for the regulation of the channel activity have not been to this we investigated the role of protein phosphorylation in the regulation of PC-2. We found the PC-2 is phosphorylated in vivo at a putative CK2 is the phosphorylation by at in the PC-2 CK2 can this in and of this phosphorylation of PC-2 in vivo and phosphorylation by CK2 in phosphorylation can channels by and is that Ser812 occurs in a as for retention of PC-2 in the ER Y. Maeda Y. A. Wu G. Hayashi T. T. Witzgall R. Somlo S. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). we found that interactions with PC-2 and PC-1 are by phosphorylation at the and of the channel by phosphorylation at this intracellular Ca2+ is in the range of D.E. Cell. Full Text PDF PubMed Scopus Google Scholar). of in this range in of the wild type PC-2 channel but not the S812A channel. 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