Inositol diphosphate signaling regulates telomere length.

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), 1 which hydrolyzes phosphatidylinositol 4,5-bisphosphate to yield inositol 1,4,5-trisphosphate (IP 3 ) (reviewed in Refs. 1 and 2). In metazoans, cellular production of IP 3 functions as a signal for calcium release from intracellular stores through allosteric activation of an IP 3

receptor channel.
Recent studies indicate that IP 3 also plays an important role as precursor to multiple inositol polyphosphates (IPs), each with potentially unique signaling capability (reviewed in Ref. 3). This signaling potential is not restricted to the cytoplasm, because IPs are known to function in nuclear processes in budding yeast (reviewed in Ref. 4). Activation of yeast phospholipase C (Plc1) produces IP 3 , which is rapidly phosphorylated by the dual specificity 6-/3-kinase Ipk2 (initially cloned as ArgRIII/Arg82 (5)) to yield IP 4 and IP 5 (6 -8). This phosphorylation step is coupled to transcriptional regulation, possibly through chromatin remodeling (7, 9 -11). IP 5 is then phosphorylated by the 2-kinase Ipk1, generating IP 6 , which is required for efficient mRNA export from the nucleus (12)(13)(14). Both IP 5 and IP 6 can be converted to the diphosphoryl inositols PP-IP 4 and PP-IP 5 through the action of Kcs1, a kinase required for normal vacuolar morphology (15)(16)(17)(18). PP-IP 5 has recently been suggested to play a role in chemotaxis in Dictyostelium (19). 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 (20,21).
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). IP 6 stimulates DNA repair by non-homologous end joining with mammalian proteins in vitro (22). 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 IP 6 (23,24), and the PIKK, DNA-dependent protein kinase (DNA-PK cs ) (reviewed in Refs. 25 and 26). 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 (27)(28)(29).
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. 30 -32). 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-IP 4 , synthesized by Kcs1. This modulation is dependent on Tel1 providing a functional connection between PIKKs and inositol phosphates in vivo.
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 (35). 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 hIP6K, an orthologue of yeast Kcs1) was obtained from Research Genetics, Inc. as an insert in pBluescript SK(Ϫ). The entire open reading frame minus the codons for the first six aminoterminal amino acids was inserted in-frame into the NcoI and XhoI sites of pGEX-KG, derived from pGEX-2T (Amersham Biosciences) and kindly donated by Dr. John Moskow in the laboratory of Dr. Daniel Lew (Duke University, Department of Pharmacology and Cancer Biology), to generate pGEX-hIP6K. The entire open reading frame of the KCS1 gene was amplified by polymerase chain reaction (PCR) from S. cerevisiae genomic DNA and inserted into a pGEX vector to create an amino-terminal glutathione S-transferase fusion. Primers were designed to incorporate EcoRI sites in-frame with the initiation sites and to place XhoI sites after the stop codons. Catalytically inactive Kcs1 was generated by a PCR strategy converting both Asp 791 and Lys 793 to alanine. The presence of the desired mutations was confirmed by sequencing the resulting construct termed pGEX-kcs1kin Ϫ . Complementation analyses in haploid kcs1⌬ strains were performed by expressing wild type and catalytically inactive forms of Ksc1 and hIP6K using the episomal plasmid pRS426 containing a galactose-inducible promoter. In each case, the insert was removed from the pGEX vector with EcoRI and XhoI and ligated into the EcoRI-XhoI sites of pRS426GAL. These constructs were termed pRS-KCS1, pRS-kcs1kin Ϫ , and pRS-hIP6K.
The construct for PLC1 overexpression was based on pYEX4T, modified by removal of the LEU2 sequence. The entire PLC1 coding sequence was removed in a BamHI-NotI fragment from the pGAL1-10-PLC1 vector described previously and provided by Dr. Jeremy Thorner (6). This fragment was inserted into the modified multiple cloning site of the pYEX4T vector behind the CUP1 promoter with the resulting plasmid pYEX-PLC1.
Inositol Radiolabeling of Yeast-Approximately 10 5 cells were inoculated into 1 ml of appropriately modified complete minimal medium containing myo-[ 3 H]inositol (American Radiolabel Corporation, St. Louis, MO) and were labeled for 24 h with shaking at 30°C. Soluble inositols were harvested as described (35). Water-soluble inositol isomers were frozen quickly in a dry ice-isopropanol bath and stored at Ϫ80°C prior to HPLC analysis. To analyze inositol isomers, samples were thawed, equilibrated to 10 mM ammonium phosphate, pH 3.5, and resolved on a 4.6 ϫ 125-mm Partisphere SAX-HPLC strong anion exchange column (Whatman). Samples were eluted with the following gradient: 10 mM ammonium phosphate increased linearly to 1.7 M ammonium phosphate over 25 min followed by an isocratic flow at 1.7 M ammonium phosphate for 50 min. Radioactivity was measured using a BetaRAM TM in-line detector (INUS Systems, Tampa, FL). Individual IP isomers were assigned in the chromatograms as described (12). PP-IP 4 and PP-IP 5 were assigned based on published elution profiles and sensitivity to hydrolysis by mammalian diphosphoryl inositol pyrophosphatase (36).
Southern Analysis of Telomere Length-Details of this analysis are as published previously (37). Genomic DNA was prepared from 5-ml cultures grown to saturation and digested with PstI before electro-phoresis on a 1% Tris borate-EDTA-agarose gel. The fragments were transferred to Hybond Nϩ nylon membranes (Amersham Biosciences) and hybridized at 65°C to a probe derived from pYT14 containing telomeric DNA and a portion of the YЈ subtelomeric repeat. The probe was labeled with [␣-32 P]dCTP using Ready-To-Go labeling beads (Amersham Biosciences). Note that the Southern blot strategies are the accepted methods in the field; however, the effect appears small because the PstI site used for cleaving is about 800 bp from the chromosome end. Thus less than half of the fragment is composed of telomeric sequences. The bottom smear represents the terminal fragment resulting from cutting at the last YЈ elements (one of the subtelomeric repeats) on the chromosome. The bands near the top of the gel reflect hybridization to fragments generated by cutting tandem YЈ elements of two different sizes. These bands vary in intensity, even in wild-type strains, probably as a consequence of a high rate of unequal recombination. Although we cannot exclude the possibility that there is a somewhat higher rate of this type of recombination in one or more of the mutants, none of them has the dramatic phenotype seen in yeast strains undergoing alternative lengthening of telomere recombination, where the upper bands become extremely prominent. For example, see Fig. 3 in Ritchie et al. (37). Fig. 1. A single phospholipase C (Plc1) and three inositol polyphosphate kinases, Ipk2, Ipk1, and Kcs1, function to generate over seven species of water-soluble IP messengers. Plc1 hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to release IP 3 from the membrane (6). IP 3 serves as substrate for the nuclear inositol kinase Ipk2 (also known as Arg82), which phosphorylates both the D-6 and D-3 position hydroxyl groups to produce IP 5 (7,38). IP 5 is phosphorylated at the 2-hydroxyl by Ipk1 to generate IP 6 (12). Kcs1 has been reported to function as a inositol diphosphoryl synthase to produce PP-IP 4 and PP-IP 5 from IP 5 and IP 6 substrates, respectively (17). Dephosphorylation of inositol diphosphates occurs through a phosphatase Ddp1 (36).

Genetics of Inositol Polyphosphate Synthesis in S. cerevisiae-A graphic summary of the metabolism of soluble inositol phosphates in budding yeast is depicted in
To further study the role of these kinases and phosphatases in inositol metabolism we analyzed a series of yeast mutant strains using isotopic equilibrium radiolabeling. Steady state metabolic radiolabeling of wild-type yeast cells with [ 3 H]inositol indicates that the major soluble IP is IP 6 , representing ϳ2% of the recovered radioactivity from soluble cellular extracts (Fig. 2). Other IPs, including PP-IP 5 , are present at low levels FIG. 1. Schema of phospholipase C-dependent inositol polyphosphate metabolism in S. cerevisiae. A single phospholipase C (Plc1) hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to release IP 3 from the membrane. IP 3 serves as substrate for the inositol kinase Ipk2, which phosphorylates both the D-6 and D-3 position hydroxyl groups to produce Ins(1,3,4,5,6)P 5 . IP 5 is then phosphorylated by Ipk1 at the 2-hydroxyl to generate IP 6 . IP 6 can be further phosphorylated to PP-IP 5 by one of two inositol diphosphoryl synthases in this strain, Kcs1, or a second kinase, Ids1. Kcs1 also phosphorylates IP 5 to make PP-IP 4 . The kinase(s) responsible for synthesis of PP 2 -IP 3 is currently not defined, although it is likely that Kcs1 or Ids1 functions at this step. The inositol diphosphates PP-IP 4 , PP 2 -IP 3 , and PP-IP 5 are dephosphorylated to IP 5 and IP 6 by an inositol diphosphoryl phosphatase Ddp1. Note that the ring position phosphates that Kcs1 and Ids1 phosphorylate are undetermined; thus it is possible that the PP-IP 5 products of each kinase are distinct isomers (which are omitted from the diagram only for the sake of simplicity).
but are not observed under these labeling conditions (therefore HPLC traces only show relevant times of elution). A significant branch point in the IP pathway is exposed in metabolically labeled cells upon deletion of the IP 5 2-kinase (ipk1⌬) cells, which accumulate IP 5 and a diphosphoryl inositol, PP-IP 4 ( Fig.  2A) (12). Deletion of KCS1 in the ipk1⌬ strain abrogates the peak of PP-IP 4 , which can be complemented with active Kcs1; thus production of PP-IP 4 is primarily dependent on Kcs1 in vivo ( Fig. 2A) (17). Disruption of KCS1 alone does not alter IP 6 levels, demonstrating that PP-IP 4 is not required for synthesis of IP 6 ( Fig. 2A). Disruption of the DDP1 gene exposes a peak of PP-IP 5 , which is not seen at appreciable levels in wild-type cells (Fig. 2B) (36). Elimination of KCS1 in the ddp1⌬ strain (kcs1⌬ ddp1⌬) leads to an increase rather than an ablation of PP-IP 5 production (Fig. 2B), demonstrating the existence of a second inositol diphosphoryl synthase (designated Ids1), which phosphorylates IP 6 to a PP-IP 5 isomer (see Fig. 1 schematic). This is surprising in light of the published work of others that Kcs1 is the major IP 6 kinase activity in yeast (17). Cellular extracts prepared from kcs1⌬ ddp1⌬ mutants have significant IP 6 kinase activity, further indicating the presence of a second kinase (data not shown). Additionally, it does not appear that Ids1 is encoded by IPK2, as extracts from ipk2⌬ kcs1⌬ mutant cells harbor IP 6 kinase activity, and we were not able to detect this activity using recombinant Ipk2 under a range of conditions (data not shown).
It is noteworthy that the chemical nature of the PP-IP 5 isomers generated by Kcs1 and Ids1 are not known; thus it is plausible that these kinases phosphorylate different positions, thereby generating distinct PP-IP 5 products that are not resolved by our HPLC analysis. The observed elevation of PP-IP 5 in the kcs1⌬ ddp1⌬ strain suggests either that: 1) Ids1 activity is negatively regulated by Kcs1 and/or one of its products; or 2) Kcs1 converts the product of Ids1 to PP 2 -IP 4 ; thus when Kcs1 is deleted, PP-IP 5 accumulates. Ids1 activity may also function as a minor kinase relative to Kcs1 in the synthesis of PP-IP 4 from Ins(1,3,4,5,6)P 5 as analysis of ipk1⌬ kcs1⌬ddp1⌬ mutant cells reveals a small but significant peak of PP-IP 4 (not shown). Furthermore, PP 2 -IP 3 derivatives are observed when DDP1 is eliminated in ipk1⌬ strains; however, it is not known whether Kcs1 or Ids1 activity is responsible for generating this product(s). Collectively, our new results along with previously published data lead to the schematic pathway depicted in Fig. 1.
Inositol Phosphates and Telomere Length in Vivo-Evidence implicating IPs in the regulation of nuclear function and DNA repair processes in mammalian and yeast systems provided the impetus to examine these pathways in budding yeast. Examination of non-homologous end joining repair in plc1, ipk2, ipk1, or kcs1 mutant yeast revealed that loss of inositol signaling pathways does not result in detectable disruption of non-homologous end joining in living yeast (data not shown). It is of interest that yeast possess two PIKKs, Tel1 and Mec1, which are orthologues of ATM and ATR that play an important evolutionarily conserved role in the regulation of telomere length. We therefore continued our examination of PIKK-related function by determining the length of telomeres in yeast defective in inositol metabolism. We find that strains with the genotypes plc1⌬, ipk2⌬, or kcs1⌬ have similarly elongated telomeres, averaging 375 bp as compared with the average of 325 bp observed in wild-type cells (Fig. 3A). In contrast, ipk1⌬ cells have telomeres slightly shorter than wild type (Fig. 3A). The change in IP profile shared among the first three mutant strains is the inability to synthesize the diphosphoryl inositols PP-IP 4 (and downstream higher phosphorylated forms such as PP 2 -IP 3 ) and PP-IP 5 (Fig. 1). The ipk1⌬ cells have a telomere shift in the opposite direction, fail to synthesize IP 6 and PP-IP 5 , and have increased PP-IP 4 levels (Fig. 2). This finding clearly demonstrates that loss of PP-IP 5 production is not responsible for the lengthened telomeres. Instead, these data implicate PP-IP 4 production or a downstream metabolite as a negative regulator of telomere length. Importantly, kcs1⌬ cells do not universally display the defects in gene expression or mRNA export observed in plc1⌬, ipk2⌬, or ipk1⌬ cells, suggesting that the observed telomere phenotype is distinct from the previously reported nuclear activities of IP 4 , IP 5 , and/or IP 6 . Furthermore, the kcs1⌬ ddp1⌬ strain, which accumulates large amounts of PP-IP 5 , has long telomeres identical to kcs1⌬ alone (not shown). Wild-type telomere length is observed in ddp1⌬ strains, and double and triple mutants for ddp1⌬, ipk1⌬, and kcs1⌬ are consistent with loss of Kcs1 being a dominant phe- notype. Collectively, these results implicate phospholipase Cdependent PP-IP 4 production through Kcs1 activity as a negative regulator of telomere length.
To test whether increases in PP-IP 4 production would lead to further telomere shortening, we overexpressed PLC1 in several mutant strains, which (based on our previous studies) significantly elevates IP production in cells (12). Wild-type strains overexpressing PLC1 have increased IP 6 levels (20-fold), no detectable increase in PP-IP 4 levels, and normal telomere length (Fig. 3B). In contrast, ipk1⌬ cells overexpressing PLC1 have significantly elevated levels of PP-IP 4 (not shown) and undergo further shortening of telomeres to an average length of 265 bp from the 325 bp observed in wild type (Fig. 3B). When Kcs1 is eliminated from this strain, the cells again have a long telomere phenotype (Fig. 3B). These data are consistent with a gain of function effect mediated by PP-IP 4 . Comparing the telomere lengthening in the absence of inositol diphosphates and the shortening seen with increases in Plc1-and Kcs1mediated production of PP-IP 4 , we see a range of modulation of telomere length of over 120 bp, accounting for roughly onethird of the total wild-type telomere length (Fig. 3B).
To confirm that inositol kinase activity is required for normal telomere length, we examined telomeres in kcs1⌬ strains complemented with either wild-type Kcs1, kinase-inactivated Kcs1 (kcs1kin Ϫ ), or a human inositol diphosphoryl synthase (hIP6K) (Fig. 4). Both Kcs1 and hIP6K expression complement telomere length in the kcs1⌬ strain, whereas Kcs1 kinase-inactive ex-pression does not (Fig. 4), demonstrating that kinase activity is required. The ability of Kcs1 to complement in dextrose indicated that the GAL promoter was not "off" and may be leaky in these strains. We therefore performed complementation analysis in the ipk1⌬ kcs1⌬ strains, which enable detection and quantification of PP-IP 4 . Indeed, the Kcs1 strains produced significant levels of PP-IP 4 even in dextrose, confirming the leaky expression. Furthermore, growth of cells in galactose, which is a strong inducer of GAL promoters, only modestly elevated PP-IP 4 levels as compared with dextrose (Fig. 4). Analysis of ipk1⌬ kcs1⌬ complemented strains confirm that expression of KCS1 restores telomere length equivalent to the ipk1⌬ strain alone (Fig. 4). Intriguingly, expression of inactive kinase in this strain not only fails to complement telomere length but also further lengthens telomeres, suggestive of a direct kinase-effector protein interaction at the telomere (Fig.  4). Consistent with this idea, hIP6K only partially restores telomere length to the length observed in ipk1⌬ cells in the ipk1⌬ kcs1⌬ mutant cells, despite PP-IP 4 synthesis equivalent to exogenous Kcs1 (HPLC data not shown). These results indicate a requirement for kinase activity and PP-IP 4 production, as well as a possible role for a non-catalytic protein component, such as a determinant that mediates proper localization or regulation.
Kcs1 Regulation of Telomeres Requires Tel1-With this evidence for PP-IP 4 as a regulator of telomere length, we used genetics to identify other components of the novel PP-IP 4 pathway. Because of the initial report that IP 6 interacts with DNAdependent protein kinase, we considered Tel1 and Ku70/80 to be reasonable effector candidates, because both have important yet independent roles in maintenance of the yeast telomere (34). We predicted that the effect of PP-IP 4 would be absent in cells missing a critical component of the IP effector pathway.
Analysis of yku70 mutant cells indicates an average telomere length of ϳ150 bp (Fig. 5A), consistent with published reports. Double (ipk1⌬ yku70⌬ and kcs1⌬ yku70⌬) and triple (ipk1⌬ kcs1⌬ yku70⌬) mutant strains were constructed and tested for telomere length (Fig. 5A). In a yku70⌬ background, the loss of Kcs1 function again lengthens telomeres about 50 bp (average length of 200 bp) consistent with Ku and Kcs1 functioning in independent pathways. To probe the gain of the PP-IP 4 effect on shortening telomeres in the absence of Ku, we examined telomere length in both yku70⌬ ipk1⌬ (Fig. 5A) and yku70⌬ ipk1⌬ overexpressing Plc1 (not shown). Neither condition resulted in further shortening beyond the 150-bp length. Furthermore, we did not find evidence of recombination or senescence to suggest telomeres have reached a critical length, indicating either the gain of function effect cannot be reproduced in the setting of short telomeres or is dependent on the Ku pathway.
In contrast, all effects of PP-IP 4 at the telomere are abolished in the absence of Tel1. Both the double (ipk1⌬ tel1⌬ and kcs1⌬ tel1⌬) and triple (ipk1⌬ kcs1⌬ tel1⌬) mutant strains have stable, shortened telomeres indistinguishable from tel1⌬ cells (Fig. 5B). This result indicates that the IPs function in the same pathway of telomere regulation as this ATM homologue. Because loss of Tel1 function leads to short telomeres, the data are consistent with Kcs1 production of PP-IP 4 acting as a negative regulator of Tel1 function. DISCUSSION Here we report a novel role of a phospholipase C-dependent signaling pathway in the control of telomere length. Individual deletion of each of the three enzymes in the synthetic pathway to PP-IP 4 causes an identical lengthening of telomeres. The long telomere phenotype is seen in yeast lacking all soluble inositol phosphates (plc1-null) as well as in those lacking only Kcs1 kinase activity, which do synthesize PP-IP 5 by way of Ids1 activity. Because ipk1-null cells with no PP-IP 5 have the opposite phenotype, loss of PP-IP 5 cannot explain the longer telomeres observed. Therefore the kinase activity of Kcs1 must be producing something else, proximal to IP 6 on the IP pathway, which is needed for normal telomere length. Because IP 5 is a known substrate for Kcs1, the simplest interpretation is that PP-IP 4 loss leads to longer telomeres. We cannot formally exclude PP 2 -IP 3 and other molecules downstream of PP-IP 4 or Kcs1 phosphorylation of another inositol substrate such as IP 4 as the molecules acting at the telomere. Importantly, we also report that further increases in PP-IP 4 synthesis lead to further shortening of telomeres. The opposing effects of gain and loss of PP-IP 4 demonstrate that this signaling molecule regulates the equilibrium of telomere length. These dual criteria also distinguish Kcs1 and its products from a number of reported genes involved in the maintenance of telomeres in yeast, most of which have only been reported to either lengthen or shorten telomeres but not both. Such regulation by a signalingactivated intracellular messenger pathway puts telomere maintenance under the potential control of extracellular stimuli, providing a mechanism to alter the average telomere length based on growth conditions or other environmental inputs.
Because neither the loss nor gain of PP-IP 4 has any impact on telomere length in the absence of Tel1, our work also provides a novel functional interaction between IPs and the activity of a PIKK family member, linking two important nuclear signaling pathways. Given that loss of Tel1 and increase in PP-IP 4 both lead to shortened telomeres, the simplest explanation is that PP-IP 4 4. Complementation of telomere phenotype requires Kcs1 kinase activity. ipk1⌬ or ipk1⌬ kcs1⌬ cells were transformed with plasmids encoding various inositol diphosphoryl synthases behind a GAL10 promoter. Cultures were grown in complete minimal medium-Ura with either dextrose (D) or galactose (G) as carbon source, and telomeric sequences were probed as described under "Experimental Procedures." The plasmids, from left to right, are pKCS1 (encoding wildtype Kcs1), pVEC (empty vector control), pkcs1kin Ϫ (encoding an active site point mutant of Kcs1), and pIP6K (encoding a human inositol diphosphoryl synthase). The length of wild type (wt) telomeres is indicated by the oval and arrow on right. nd, not detected. role as a negative regulator of Tel1 activity is consistent with earlier reports of the initial cloning of KCS1 as a second site suppressor of a mitotic recombination phenotype (20) because loss of Tel1 function increases recombination rates (33). Tel1 is not the only potential target of IP regulation as other candidate effectors in this pathway of telomere regulation include the Mre11⅐Rad50⅐Xrs2 protein complex components, but their precise biochemical roles at the telomere remain elusive. Because the Mre11⅐Rad50⅐Xrs2 complex as well as Tel1 favors lengthening telomeres, they must somehow favor telomerase access to the telomere end, and it is likely that the activity of Kcs1 opposes that access, thus changing the equilibrium to favor average loss of telomere sequence. Given the reported roles for Kcs1 and inositol diphosphates in cellular signaling events, we cannot rule out at this point that other processes are involved. Other important regulators may be gleaned from recent studies describing a genome-wide screen for deletion mutants with a telomere phenotype that yielded many mutants with length alterations similar to the plc1 and kcs1 phenotypes (39). The components of the PP-IP 4 pathway were not identified in this laborious screen; however, the authors concede they failed to identify one-third of the described previously deletion strains with telomere phenotypes. The existence of mammalian homologues for both the inositol diphosphoryl synthases and Tel1 indicates that such an interaction may have important implications for human disease.
Our data expand the role of IPs in nuclear function and provide new insights into the complexity of the described metabolic pathways of soluble IPs in yeast. Activation of phospholipase C produces several unique IP messengers, which have been found to modulate nuclear processes including transcriptional control, chromatin remodeling, and mRNA export. We see that the loss of Ipk1 activity uncovers a branch point in the IP synthetic pathway that, despite its low abundance at steady state, plays a significant regulatory role in the cell. Our evidence for a regulatory role of the diphosphoryl inositol PP-IP 4 in determining the telomere length set point reinforces the hypothesis that a primordial role of phospholipase C activation is to generate an ensemble of IP messengers to control several nuclear events.