Inositol Diphosphate Signaling Regulates Telomere Length

Abstract

Activation of phospholipase C-dependent inositol polyphosphate signaling pathways generates distinct messengers derived from inositol 1,4,5-trisphosphate that control gene expression and mRNA export. Here we report the regulation of telomere length by production of a diphosphorylinositol tetrakisphosphate, PP-IP4, synthesized by the KCS1 gene product. Loss of PP-IP4 production results in lengthening of telomeres, whereas overproduction leads to their shortening. This effect requires the presence of Tel1, the yeast homologue of ATM, the protein mutated in the human disease ataxia telangiectasia. Our data provide in vivo evidence of a regulatory link between inositol polyphosphate signaling and the checkpoint kinase family and describe a third nuclear process modulated by phospholipase C activation. Activation of phospholipase C-dependent inositol polyphosphate signaling pathways generates distinct messengers derived from inositol 1,4,5-trisphosphate that control gene expression and mRNA export. Here we report the regulation of telomere length by production of a diphosphorylinositol tetrakisphosphate, PP-IP4, synthesized by the KCS1 gene product. Loss of PP-IP4 production results in lengthening of telomeres, whereas overproduction leads to their shortening. This effect requires the presence of Tel1, the yeast homologue of ATM, the protein mutated in the human disease ataxia telangiectasia. Our data provide in vivo evidence of a regulatory link between inositol polyphosphate signaling and the checkpoint kinase family and describe a third nuclear process modulated by phospholipase C activation. Appropriate cellular responses to environmental changes involve intracellular second messenger systems that transduce the signals from cytoplasm to nucleus, thereby initiating adaptive genetic programs. One well described intracellular messenger system works through receptor-coupled activation of phospholipase C (PLC), 1The abbreviations used are: PLC, phospholipase C; IP, inositol phosphate; IP6, inositol hexakisphosphate; IP3, inositol 1,4,5-trisphosphate; IP4, inositol tetrakisphosphate; IP5, inositol pentakisphosphate; PP-IP4, diphosphorylinositol tetrakisphosphate; PP2-IP3, (bis)diphosphorylinositol trisphosphate; PP-IP5, diphosphorylinositol pentakisphosphate; HPLC, high performance liquid chromatography; kin–, catalytically inactive kinase; PIKK, phosphatidylinositol 3-kinase-related kinase. which hydrolyzes phosphatidylinositol 4,5-bisphosphate to yield inositol 1,4,5-trisphosphate (IP3) (reviewed in Refs. 1Berridge M.J. Irvine R.F. Nature. 1989; 341: 197-205Crossref PubMed Scopus (3313) Google Scholar and 2Majerus P.W. Annu. Rev. Biochem. 1992; 61: 225-250Crossref PubMed Scopus (349) Google Scholar). In metazoans, cellular production of IP3 functions as a signal for calcium release from intracellular stores through allosteric activation of an IP3 receptor channel. Recent studies indicate that IP3 also plays an important role as precursor to multiple inositol polyphosphates (IPs), each with potentially unique signaling capability (reviewed in Ref. 3Irvine R.F. Schell M.J. Nat. Rev. Mol. Cell. Biol. 2001; 2: 327-338Crossref PubMed Scopus (542) Google Scholar). This signaling potential is not restricted to the cytoplasm, because IPs are known to function in nuclear processes in budding yeast (reviewed in Ref. 4York J.D. Bradshaw R.A. Dennis E. Handbook of Cell Signaling. 2. Academic Press, Orlando, FL2003: 229-232Google Scholar). Activation of yeast phospholipase C (Plc1) produces IP3, which is rapidly phosphorylated by the dual specificity 6-/3-kinase Ipk2 (initially cloned as ArgRIII/Arg82 (5Dubois E. Messenguy F. Mol. Gen. Genet. 1994; 243: 315-324Crossref PubMed Scopus (41) Google Scholar)) to yield IP4 and IP5 (6Flick J.S. Thorner J. Mol. Cell. Biol. 1993; 13: 5861-5876Crossref PubMed Scopus (171) Google Scholar, 7Odom A.R. Stahlberg A. Wente S.R. York J.D. Science. 2000; 287: 2026-2029Crossref PubMed Scopus (346) Google Scholar, 8Saiardi A. Nagata E. Luo H.R. Sawa A. Luo X. Snowman A.M. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2306-2311Crossref PubMed Scopus (95) Google Scholar). This phosphorylation step is coupled to transcriptional regulation, possibly through chromatin remodeling (7Odom A.R. Stahlberg A. Wente S.R. York J.D. Science. 2000; 287: 2026-2029Crossref PubMed Scopus (346) Google Scholar, 9Steger D.J. Haswell E.S. Miller A.L. Wente S.R. O'Shea E.K. Science. 2003; 299: 114-116Crossref PubMed Scopus (315) Google Scholar, 10Shen X. Xiao H. Ranallo R. Wu W. Wu C. Science. 2003; 299: 112-114Crossref PubMed Scopus (292) Google Scholar, 11El Alami M. Messenguy F. Scherens B. Dubois E. Mol. Microbiol. 2003; 49: 457-468Crossref PubMed Scopus (60) Google Scholar). IP5 is then phosphorylated by the 2-kinase Ipk1, generating IP6, which is required for efficient mRNA export from the nucleus (12York J.D. Odom A.R. Murphy R. Ives E.B. Wente S.R. Science. 1999; 285: 96-100Crossref PubMed Scopus (448) Google Scholar, 13Ives E.B. Nichols J. Wente S.R. York J.D. J. Biol. Chem. 2000; 275: 36575-36583Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 14Feng Y. Wente S.R. Majerus P.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 875-879Crossref PubMed Scopus (80) Google Scholar). Both IP5 and IP6 can be converted to the diphosphoryl inositols PP-IP4 and PP-IP5 through the action of Kcs1, a kinase required for normal vacuolar morphology (15Huang C.F. Voglmaier S.M. Bembenek M.E. Saiardi A. Snyder S.H. Biochemistry. 1998; 37: 14998-15004Crossref PubMed Scopus (43) Google Scholar, 16Dubois E. Scherens B. Vierendeels F. Ho M.M. Messenguy F. Shears S.B. J. Biol. Chem. 2002; 277: 23755-23763Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 17Saiardi A. Caffrey J.J. Snyder S.H. Shears S.B. J. Biol. Chem. 2000; 275: 24686-24692Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 18Saiardi A. Erdjument-Bromage H. Snowman A.M. Tempst P. Snyder S.H. Curr. Biol. 1999; 9: 1323-1326Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). PP-IP5 has recently been suggested to play a role in chemotaxis in Dictyostelium (19Luo H.R. Huang Y.E. Chen J.C. Saiardi A. Ijima IM. Ye K. Huang Y. Nagata E. Devreotes P. Snyder S.H. Cell. 2003; 114: 559-572Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). A nuclear role for Kcs1 is implied by its initial cloning as a regulator of mitotic DNA recombination, and inositol kinase activity is required for this regulation, but further understanding of its nuclear activity is lacking (20Huang K.N. Symington L.S. Genetics. 1995; 141: 1275-1285Crossref PubMed Google Scholar, 21Luo H.R. Saiardi A. Yu H. Nagata E. Ye K. Snyder S.H. Biochemistry. 2002; 41: 2509-2515Crossref PubMed Scopus (67) Google Scholar). Recent work has provided further linking of IP production and nuclear function through an important family of protein serine/threonine kinases known as phosphatidylinositol 3-kinase related kinases (PIKKs). IP6 stimulates DNA repair by non-homologous end joining with mammalian proteins in vitro (22Hanakahi L.A. Bartlet-Jones M. Chappell C. Pappin D. West S.C. Cell. 2000; 102: 721-729Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Non-homologous end joining can be reconstituted in vitro with a limited number of purified proteins, including the Ku heterodimer, which binds DNA ends and IP6 (23Ma Y. Lieber M.R. J. Biol. Chem. 2002; 277: 10756-10759Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 24Hanakahi L.A. West S.C. EMBO J. 2002; 21: 2038-2044Crossref PubMed Scopus (87) Google Scholar), and the PIKK, DNA-dependent protein kinase (DNA-PKcs) (reviewed in Refs. 25Lieber M.R. Ma Y. Pannicke U. Schwarz K. Nat Rev Mol. Cell. Biol. 2003; 4: 712-720Crossref PubMed Scopus (793) Google Scholar and 26Smith G.C.M. Jackson S.P. Genes Dev. 1999; 13: 916-934Crossref PubMed Scopus (769) Google Scholar). Other PIKKs, namely ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) gene products in mammalian cells and Tel1 and Mec1 in yeast, have been shown to have important activities in DNA damage checkpoint and telomere maintenance (27Abraham R.T. Genes Dev. 2001; 15: 2177-2196Crossref PubMed Scopus (1676) Google Scholar, 28Keith C.T. Schreiber S.L. Science. 1995; 270: 50-51Crossref PubMed Scopus (451) Google Scholar, 29Zhou B.S. Elledge S.J. Nature. 2000; 408: 433-439Crossref PubMed Scopus (2645) Google Scholar). Telomeres are specialized protein-nucleic acid structures at the ends of linear, eukaryotic chromosomes that preserve genetic information during DNA replication, promote genomic stability, and are important in both cellular senescence and oncogenic transformation (reviewed in Refs. 30Blackburn E.H. Cell. 2001; 108: 661-673Abstract Full Text Full Text PDF Scopus (1772) Google Scholar, 31Vega L.R. Mateyak M.K. Zakian V.A. Nat. Rev. Mol. Cell. Biol. 2003; 4: 948-959Crossref PubMed Scopus (100) Google Scholar, 32Smogorzewska A. De Lange T. Annu. Rev. Biochem. 2004; 73: 177-208Crossref PubMed Scopus (667) Google Scholar). Telomeric DNA is maintained through the action of the ribonucleoprotein telomerase, which acts in late S-phase to add template-independent, species-specific TG-rich repeats to the lagging strand, ensuring that chromosome length and coding sequences are maintained. Telomere length is heterogeneous among chromosomes and cells, with the average length established by a dynamic equilibrium between forces that shorten (exonuclease access, low telomerase activity) and those that lengthen telomeric sequences (capping proteins, high telomerase activity). Here we describe a role for the phospholipase C-dependent IP signaling pathway in the regulation of telomere length through production of an inositol diphosphate, PP-IP4, synthesized by Kcs1. This modulation is dependent on Tel1 providing a functional connection between PIKKs and inositol phosphates in vivo. Strains, Media, and Genetic Methods—Yeast strains used in this study are isogenic with W303α (leu2–3,112 his3–11,15 ura3–1 ade2–1 trp1–1 rad5–535 can1–100) except for changes introduced by transformation as indicated. Yeast were grown either in standard rich (yeast-peptone-dextrose) medium or in complete minimal medium containing 2% glucose or galactose and lacking appropriate nutrients to verify and maintain plasmids and markers. Yeast genetic manipulations were performed using standard procedures. Deletion strains plc1::KANMX, ipk2::KANMX, ipk1::KANMX, tel1::ura3, and yku70::ura3 ura3::HIS3 were generated as described previously (7Odom A.R. Stahlberg A. Wente S.R. York J.D. Science. 2000; 287: 2026-2029Crossref PubMed Scopus (346) Google Scholar, 12York J.D. Odom A.R. Murphy R. Ives E.B. Wente S.R. Science. 1999; 285: 96-100Crossref PubMed Scopus (448) Google Scholar, 33Greenwell P.W. Kronmal S.L. Porter S.E. Gassenhuber J. Obermaier B. Petes T.D. Cell. 1995; 82: 823-829Abstract Full Text PDF PubMed Scopus (349) Google Scholar, 34Porter S.E. Greenwell P.W. Ritchie K.B. Petes T.D. Nucleic Acids Res. 1996; 24: 582-585Crossref PubMed Scopus (203) Google Scholar). Double and triple deletion strains were constructed by mating to isogenic strains of the opposite mating type followed by sporulation of the resulting diploid and dissection of tetrads. Nutrient requirements, inositol labeling, and PCR were used to screen spores for double mutations. The entire KCS1 and DDP1 open reading frames were deleted from the diploid Saccharomyces cerevisiae strain W303 and replaced with the selectable marker HIS3 as described (35Stolz L.E. Huynh C.V. Thorner J. York J.D. Genetics. 1998; 148: 1715-1729Crossref PubMed Google Scholar). Tetrads were dissected using a Zeiss micromanipulator following sporulation of the heterozygous diploids on 0.3% potassium acetate plates. The kcs1::HIS3 and ddp1::HIS3 spores both segregated 2:2 (not shown) and were identified by growth patterns on medium lacking histidine as well as by PCR analysis. Gene Identification and Plasmid Construction—To identify candidate mammalian inositol diphosphoryl synthases, human expressed sequence tag data bases were searched with BLASTP, gapped BLASTP, or TBLASTN with appropriate query sequences. The full-length human cDNA 646420 (designated as an of yeast from as an in The entire open reading the for the the and of derived from and by in the of of and to The entire open reading of the KCS1 gene by from S. cerevisiae genomic DNA and a to an were to with the and to the inactive Kcs1 generated by a PCR both and to The presence of the by the resulting in strains were performed by type and catalytically inactive of and using the containing a In each the from the with and and the of were and The for on by of the The entire coding sequence in a from the described previously and provided by Thorner (6Flick J.S. Thorner J. Mol. Cell. Biol. 1993; 13: 5861-5876Crossref PubMed Scopus (171) Google Scholar). This the multiple cloning of the the with the resulting of cells were of complete minimal medium containing and were for with at inositols were as described (35Stolz L.E. Huynh C.V. Thorner J. York J.D. Genetics. 1998; 148: 1715-1729Crossref PubMed Google Scholar). inositol were in a and to analysis. inositol were to and on a were with the following to followed by an at for using a IP were in the as described (12York J.D. Odom A.R. Murphy R. Ives E.B. Wente S.R. Science. 1999; 285: 96-100Crossref PubMed Scopus (448) Google Scholar). PP-IP4 and PP-IP5 were on and to by mammalian diphosphoryl inositol Shears S.B. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). of Telomere of this are as previously K.B. J.C. Petes T.D. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar). DNA from grown to and with on a The were to and at to a derived from containing telomeric DNA and a of the The with using that the are the in the the effect because the used for is from the chromosome of the is of telomeric sequences. The the resulting from at the of the on the The the of the to generated by of in in as a of a high of we the that is a of this type of in or of the of has the in yeast strains lengthening of telomere recombination, the in Ritchie K.B. J.C. Petes T.D. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar). of in S. of the of inositol phosphates in budding yeast is in A phospholipase C (Plc1) and inositol polyphosphate Ipk1, and Kcs1, function to of IP hydrolyzes phosphatidylinositol 4,5-bisphosphate to release IP3 from the (6Flick J.S. Thorner J. Mol. Cell. Biol. 1993; 13: 5861-5876Crossref PubMed Scopus (171) Google Scholar). IP3 as for the nuclear inositol kinase Ipk2 known as which both the and to IP5 (7Odom A.R. Stahlberg A. Wente S.R. York J.D. Science. 2000; 287: 2026-2029Crossref PubMed Scopus (346) Google Scholar, A. Caffrey J.J. Snyder S.H. Shears S.B. 2000; PubMed Scopus Google Scholar). IP5 is phosphorylated at the by to IP6 (12York J.D. Odom A.R. Murphy R. Ives E.B. Wente S.R. Science. 1999; 285: 96-100Crossref PubMed Scopus (448) Google Scholar). Kcs1 has been to function as a inositol diphosphoryl to PP-IP4 and PP-IP5 from IP5 and IP6 A. Caffrey J.J. Snyder S.H. Shears S.B. J. Biol. Chem. 2000; 275: 24686-24692Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). of inositol through a Shears S.B. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). further study the role of kinases and in inositol we a of yeast strains using equilibrium of yeast cells with that the IP is IP6, of the from cellular Other including PP-IP5, are at low but are not of A in the IP pathway is in cells deletion of the IP5 2-kinase cells, which IP5 and a diphosphoryl PP-IP4 (12York J.D. Odom A.R. Murphy R. Ives E.B. Wente S.R. Science. 1999; 285: 96-100Crossref PubMed Scopus (448) Google Scholar). Deletion of KCS1 in the strain the of PP-IP4, which can be with production of PP-IP4 is dependent on Kcs1 in vivo A. Caffrey J.J. Snyder S.H. Shears S.B. J. Biol. Chem. 2000; 275: 24686-24692Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). of KCS1 not IP6 that PP-IP4 is not required for of IP6 of the DDP1 gene a of PP-IP5, which is not at in cells Shears S.B. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). of KCS1 in the strain leads to an an of PP-IP5 production the of a second inositol diphosphoryl (designated which IP6 to a PP-IP5 This is in of the work of that Kcs1 is the IP6 kinase activity in yeast A. Caffrey J.J. Snyder S.H. Shears S.B. J. Biol. Chem. 2000; 275: 24686-24692Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). from have IP6 kinase further the presence of a second kinase not not that is by as from cells IP6 kinase and we were not to this activity using Ipk2 a of not is that the of the PP-IP5 generated by Kcs1 and are not is that kinases thereby generating distinct PP-IP5 products that are not by analysis. The of PP-IP5 in the strain either activity is by Kcs1 of its or Kcs1 the of to Kcs1 is PP-IP5 activity also function as a kinase to Kcs1 in the of PP-IP4 from as of cells a but of PP-IP4 (not are DDP1 is in is not known Kcs1 or activity is for generating this results with previously data to the pathway in and Telomere in IPs in the regulation of nuclear function and DNA repair processes in mammalian and yeast systems provided the to pathways in budding of non-homologous end joining repair in or yeast that of inositol signaling pathways not in of non-homologous end joining in yeast not is of that yeast PIKKs, Tel1 and which are of and that play an important role in the regulation of telomere of function by the length of in yeast in inositol that strains with the or have telomeres, as with the average of in cells In cells have type The in IP among the strains is the to the diphosphoryl inositols PP-IP4 phosphorylated as and PP-IP5 The cells have a telomere in the opposite to IP6 and PP-IP5, and have PP-IP4 This that of PP-IP5 production is not for the data PP-IP4 production or a as a regulator of telomere cells not the in gene expression or mRNA export in cells, that the telomere is distinct from the previously nuclear activities of the which of PP-IP5, has to (not telomere length is in and double and triple for and are with of Kcs1 a results phospholipase C-dependent PP-IP4 production through Kcs1 activity as a regulator of telomere in PP-IP4 production to further telomere we in which on IP production in cells (12York J.D. Odom A.R. Murphy R. Ives E.B. Wente S.R. Science. 1999; 285: 96-100Crossref PubMed Scopus (448) Google Scholar). strains have IP6 in PP-IP4 and normal telomere length In cells have of PP-IP4 (not shown) and further of to an average length of from the in type Kcs1 is from this the cells have a telomere data are with a of function effect by the telomere lengthening in the of inositol and the with in and production of PP-IP4, we a of modulation of telomere length of for of the telomere length that inositol kinase activity is required for normal telomere we in strains with either Kcs1, Kcs1 or a human inositol diphosphoryl Both Kcs1 and expression telomere length in the whereas Kcs1 expression not that kinase activity is The of Kcs1 to in that the not and be in performed in the which and of the Kcs1 strains of PP-IP4 in the growth of cells in which is a of PP-IP4 as with of strains that expression of KCS1 telomere length to the strain expression of inactive kinase in this strain not to telomere length but also further telomeres, of a protein at the telomere with this telomere length to the length in cells in the cells, PP-IP4 to Kcs1 data not results indicate a for kinase activity and PP-IP4 as well as a role for a protein as a that or Kcs1 of Telomeres this evidence for PP-IP4 as a regulator of telomere we used to identify of the PP-IP4 of the initial report that IP6 with DNA-dependent protein we Tel1 and to be because both have important in maintenance of the yeast telomere S.E. Greenwell P.W. Ritchie K.B. Petes T.D. Nucleic Acids Res. 1996; 24: 582-585Crossref PubMed Scopus (203) Google Scholar). that the effect of PP-IP4 be in cells a of the IP of cells an average telomere length of with Double and and triple strains were constructed and for telomere length In a the of Kcs1 function length of with Ku and Kcs1 in the of the PP-IP4 effect on in the of we telomere length in both and (not in further the we not evidence of or senescence to have a either the of function effect be in the of or is dependent on the Ku In of PP-IP4 at the telomere are in the of Both the double and and triple strains have from cells This that the IPs function in the pathway of telomere regulation as this of Tel1 function leads to telomeres, the data are with Kcs1 production of PP-IP4 as a regulator of Tel1 Here we report a role of a phospholipase C-dependent signaling pathway in the control of telomere deletion of each of the in the pathway to PP-IP4 an lengthening of The telomere is in yeast lacking inositol phosphates as well as in those lacking Kcs1 kinase which PP-IP5 by of cells with PP-IP5 have the opposite of PP-IP5 the the kinase activity of Kcs1 be to IP6 on the IP which is for normal telomere IP5 is a known for Kcs1, the is that PP-IP4 leads to and of PP-IP4 or Kcs1 phosphorylation of inositol as IP4 as the at the we also report that further in PP-IP4 to further of The of and of PP-IP4 that this signaling the equilibrium of telomere dual also Kcs1 and its products from a number of in the maintenance of in yeast, of which have been to either lengthen or shorten but not regulation by a intracellular messenger pathway telomere maintenance the potential control of providing a to the average telomere length on growth or environmental the of PP-IP4 has on telomere length in the of Tel1, work also a functional between IPs and the activity of a family linking important nuclear signaling that of Tel1 and in PP-IP4 both to telomeres, the is that PP-IP4 Tel1 kinase either through or kinase with Tel1 a of this IP Kcs1 a role as a regulator of Tel1 activity is with of the initial cloning of KCS1 as a second of a mitotic (20Huang K.N. Symington L.S. Genetics. 1995; 141: 1275-1285Crossref PubMed Google because of Tel1 function P.W. Kronmal S.L. Porter S.E. Gassenhuber J. Obermaier B. Petes T.D. Cell. 1995; 82: 823-829Abstract Full Text PDF PubMed Scopus (349) Google Scholar). Tel1 is not the potential of IP regulation as candidate in this pathway of telomere regulation the protein but their at the telomere the as well as Tel1 lengthening telomeres, telomerase to the telomere and is that the activity of Kcs1 that access, the equilibrium to average of telomere the for Kcs1 and inositol in cellular signaling we at this that processes are Other important be from studies a screen for deletion with a telomere that with length to the and S.H. T. S. R. J. C. A. M. M.J. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar). The of the PP-IP4 pathway were not identified in this the to identify of the described previously deletion strains with telomere The of mammalian for both the inositol diphosphoryl and Tel1 that an have important for human Our data the role of IPs in nuclear function and provide the of the described pathways of IPs in Activation of phospholipase C produces unique IP which have been to nuclear processes including transcriptional chromatin and mRNA export. that the of activity a in the IP pathway its low at plays a regulatory role in the Our evidence for a regulatory role of the diphosphoryl inositol PP-IP4 in the telomere length the that a role of phospholipase C activation is to an of IP messengers to control nuclear of the York and Petes for and Odom and for strain