JBC Transcription and Nuclear Factor Monoclonals

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Originally published In Press as doi:10.1074/jbc.M505089200 on June 8, 2005

J. Biol. Chem., Vol. 280, Issue 30, 27654-27661, July 29, 2005
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Molecular Definition of a Novel Inositol Polyphosphate Metabolic Pathway Initiated by Inositol 1,4,5-Trisphosphate 3-Kinase Activity in Saccharomyces cerevisiae*

Andrew M. Seeds{ddagger}, Robert J. Bastidas{ddagger}, and John D. York{ddagger}§

From the Departments of {ddagger}Pharmacology and Cancer Biology and of §Biochemistry, Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710

Received for publication, May 9, 2005 , and in revised form, June 7, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The production of inositol polyphosphate (IPs) and pyrophosphates (PP-IPs) from inositol 1,4,5-trisphosphate (I(1,4,5)P3) requires the 6-/3-/5-kinase activity of Ipk2 (also known as Arg82 and inositol polyphosphate multikinase). Here, we probed the distinct roles for I(1,4,5)P3 6- versus 3-kinase activities in IP metabolism and cellular functions reported for Ipk2. Expression of either I(1,4,5)P3 6- or 3-kinase activity rescued growth of ipk2-deficient yeast at high temperatures, whereas only 6-kinase activity enabled growth on ornithine as the sole nitrogen source. Analysis of IP metabolism revealed that the 3-kinase initiated the synthesis of novel pathway consisting of over eleven IPs and PP-IPs. This pathway was present in wild-type and ipk2 null cells, albeit at low levels as compared with inositol hexakisphosphate synthesis. The primary route of synthesis was: I(1,4,5)P3 -> I(1,3,4,5)P4 -> I(1,2,3,4,5)P5 -> PP-IP4 -> PP2-IP3 and required Kcs1 (or possibly Ipk2), Ipk1, a novel inositol pyrophosphate synthase, and then Kcs1 again, respectively. Mutation of kcs1 ablated this pathway in ipk2 null cells and overexpression of Kcs1 in ipk2 mutant cells phenocopied IP3K expression, confirming it harbors a novel 3-kinase activity. Our work provides a revised genetic map of IP metabolism in yeast and evidence for dosage compensation between IPs and PP-IPs downstream of I(1,4,5)P3 in the regulation of nucleocytoplasmic processes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cells respond to diverse extracellular stimuli by activating phospholipase C (PLC), which hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate inositol 1,4,5-trisphosphate (I(1,4,5)P3)1 and diacylglycerol. I(1,4,5)P3 acts through allosteric activation of the IP3 receptor that releases calcium from intracellular stores (1, 2). I(1,4,5)P3 has also been shown to be a precursor for the production of other inositol polyphosphates (IPs), including inositol tetrakisphosphate (IP4), inositol pentakisphosphate (IP5), inositol hexakisphosphate (IP6) and inositol pyrophosphates (PP-IPs) (3, 4). These second messengers have been implicated in the regulation of cellular functions such as mRNA export, transcription, chromatin remodeling, DNA metabolism, vesicular trafficking, chemotaxis, and environmental stress responses (5–14).

Cloning and characterization of several evolutionarily conserved inositol polyphosphate kinases (IPKs) has reinvigorated interest in "orphan" IP messengers (15–17). In yeast, activation of phospholipase C results in production of I(1,4,5)P3, and its sequential phosphorylation to IP6 via the activities of two kinases, Ipk2 and Ipk1 (5, 8, 18). Ipk2 has been found to sequentially phosphorylate I(1,4,5)P3 on its D-6 and D-3 positions to generate I(1,3,4,5,6)P5. Ipk1 functions as a 2-kinase to convert I(1,3,4,5,6)P5 to IP6. Additionally, both Ipk2 and Ipk1 have been shown to have promiscuous activities suggesting that each may facilitate generation of complex branches of IP metabolites. Subsequent metabolism of IP5 and IP6 by inositol pyrophosphate synthases, such as Kcs1 and a novel activity designated Ids1, generate PP-IP4 and PP-IP5 (10, 13, 19). It appears that the metabolic functions of IPKs have been conserved across eukaryotes as they are required for IP6 synthesis (18, 20–27).

The enzymatic promiscuity Ipk2 and its action early in complex IP metabolic pathways may account for the pleiotropic biological defects observed in ipk2 mutant yeast. To further dissect the roles of the kinase activity of Ipk2 we have utilized heterologous complementation analysis in ipk2 mutant yeast cells (20, 21, 23). Here, in order to specifically assess the role of 3-kinase activity, we studied the effects of expression of a Drosophila I(1,4,5)P3 3-kinase {beta} isoform (dmIP3K), whose only reported enzymatic function was to generate inositol 1,3,4,5-tetrakisphosphate (I(1,3,4,5)P4) (21, 28). This work has led to an unexpected finding that expression of dmIP3K initiates a novel IP pathway, whose molecular basis we describe. Additionally, we provide evidence for dosage compensation among IP species in the regulation of Ipk2-mediated nuclear and cytoplasmic processes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Strains and Media—Yeast were grown in either rich medium (yeastpeptone-dextrose), or complete minimal (CM) medium lacking the appropriate nutrients for maintenance of plasmids containing markers. Yeast strains used in this study were from previous studies or generated by mating strains from previous studies (5, 8, 10). Ornithine plates were made as previously described (5, 29).

High Performance Liquid Chromatography Columns and Gradients—Two different Partisphere SAX columns and elution gradients were used in this study. Method 1 utilized a custom-made narrow-bore column and was obtained from Capital HPLC Ltd. (12.5 cm x 2.1 mm). IPs were eluted with a linear gradient of ammonium phosphate (pH 3.5) (AP) from 10 mM to 1.7 M over the course of 12 min and isocratic elution at 1.7 M AP for 23 min (flow rate of 0.4 ml/min). Method 2 achieved higher resolution IPs by using a wider-bore Whatman column (12.5 cm x 4.6 mm) and a longer elution gradient as follows: a linear gradient from 10 to 85 mM AP over 5 min, then 85 mM to 1.7 M AP over 65 min, and then isocratic elution at 1.7 M AP for 30 min all at a flow rate of 1 ml/min.



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  		FIG. 1.
Comparison of I(1,4,5)P3 6- versus 3-kinase function in the rescue of ipk2{Delta}-specific growth defects. Strains of ipk2{Delta}, ipk2{Delta}ipk1{Delta}, or ipk2{Delta}kcs1{Delta} (B only) were transformed with empty pRS314 vector (Vec) or pRS314 containing dmIpk2 or dmIP3K. dmIpk2 has I(1,4,5)P3 6-kinase activity, whereas dmIP3K has 3-kinase activity. The strains were then serially diluted (1/10) and spotted onto plates containing complete minimal (CM), ornithine as the sole nitrogen source (ORN) (A) or YPD medium (B) and grown for 2 days at the indicated temperatures.

 
Plasmid Construction—Construction of plasmids pRS314-dmIpk2 and pRS314-dmIP3K{beta} (which we will refer to as "dmIP3K" throughout the remainder of this report) and Kcs1 expression constructs (pRS-Kcs1 and pRS-kcs1kin-) was described previously (10, 21). The plasmids were then transformed into different yeast strains using standard yeast transformation techniques. The pCR®2.1 vector (Invitrogen) containing the entire scIpk1 coding region with a PCR-generated SalI site at the 5' end was provided by Dr. Makoto Fujii (York laboratory, Duke University). The scIpk1 coding region was subcloned from pCR®2.1 using SalI and EcoRI and ligated into the pGEX-2T vector. The vector was transformed into Escherichia coli (DH5{alpha}) for expression of recombinant protein.

In Vivo Labeling of Yeast Cultures—Yeast cultures were incubated at 30 °C in minimal medium lacking the appropriate amino acids and 150 µM CuSO4 to late logarithmic phase. [3H]Inositol (American Radiolabeled Chemicals) was added to a final concentration of 80 µCi/ml. For pulse-chase analysis: yeast strains were grown to late logarithmic phase in 50 ml of unlabeled CM medium. The cells were washed and resuspended in 500 µl of inositol-free CM medium supplemented with 1 mCi/ml [3H]inositol. After labeling for 10 min the cells were washed, resuspended in 50 ml of inositol-replete CM medium without label, and incubated at 30 °C. Aliquots were taken at various time points, and the cells were frozen on dry ice until they could be harvested and analyzed by Partisphere strong-anion exchange HPLC. Soluble IPs were harvested and analyzed by HPLC using a strong-anion exchange column as described previously (8). Alternatively, the IPs were harvested for enzyme treatment as described below.

Enzyme Analysis of [3H]Inositol-labeled Yeast Extracts—Labeled yeast strains were resuspended in 50 mM Tris, pH 7.5, and lysed for 30 s with a bead beater (Biospec Products) using glass beads (B. Braun Biotech International). The lysate was immediately boiled for 5 min, and then the entire procedure was repeated a second time. Extracts containing soluble IPs were recovered by centrifugation. Reactions were carried out by incubating the extracts for 1 h at 37 °C in a buffer containing 10 mM Tris, pH 7.5, and 10 mM NaCl with 500 ng of human Type I 5-phosphatase and/or human diphosphoryl inositol polyphosphate phosphohydrolase (DIPP). The reactions were stopped by addition of 200 µl of 10 mM ammonium phosphate, pH 3.5, and analyzed by Partisphere strong-anion exchange HPLC.

Bacterial Expression of dmIP3 and scIpk1—Transformed E. coli (DH5{alpha}) were grown at 37 °C to an A600 of 0.6 and induced with 0.1 mM isopropyl-1-thio-{beta}-D -galactopyranoside for 4 h at 30 °C.Thecells were recovered by centrifugation at 4 °C, resuspended in ice-cold 50 mM Tris-HCl, pH 7.5, 50 mM KCl, 5 mM dithiothreitol, Complete Mini protease inhibitor mixture (Roche Applied Science) and lysed with four passes through a cell cracker (a high shear fluid processing system for cell rupture, Microfluidics Corp.). The lysates were cleared by centrifugation at 14,000 x g. The glutathione S-transferase fusion proteins were purified over glutathione-Sepharose (Amersham Biosciences) according to the manufacturer's instructions. A buffer containing 50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 5 mM dithiothreitol, and 20 mM glutathione was used to elute the proteins from the glutathione Sepharose. Proteins were quantified by modified Bradford method and SDS-PAGE analysis.

Inositol Phosphate Kinase Assay—All unlabeled IPs were purchased from Cell Signals, Inc., and [3H]I(1,3,4,5)P4 was purchased from PerkinElmer Life Sciences. [32P]I(1,3,4,5)P4 was synthesized in buffer containing 50 mM Tris, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 5 µM I(1,4,5)P3, trace amounts of [32P]ATP, and 7.5 µg of dmIP3K. [3H]I(1,3,4,5,6)P5 standard was synthesized by incubating [3H]I(1,3,4,5)P4 with recombinant dmIpk2 and ATP as described (21). Kinase assay conditions were carried out essentially as described by Stevenson-Paulik et al. (20).

Kinetic Assays—The Km and Vmax values of the enzymatic interaction between scIpk1 and I(1,3,4,5)P4 and various substrates were determined. The following reaction mixture was prepared: 10 mM Tris, pH 7.5, 10 mM NaCl, 4 mM ATP, 20 mM MgCl2, 10,000 cpm/µl [32P]I(1,3,4,5)P4, 500 ng of scIpk1, and various concentrations of unlabeled I(1,3,4,5)P4 in a 20-µl reaction volume. The reaction was stopped by the addition of 4 µl of 1 M KH2PO4. The amount of product formed was quantified by thin layer chromatography. The Km and Vmax values were obtained from a nonlinear curve fit to the Michaelis-Menten equation using GraphPad Prism version 4.01.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Comparative roles of I(1,4,5)P3 6- and 3-Kinase Activities in Yeast—Studies of yeast Ipk2 have shown that its predominant in vivo catalytic function is to convert I(1,4,5)P3 -> I(1,4,5,6)P4 -> I(1,3,4,5,6)P5 via 6- and 3-kinase activities. Recombinant yeast Ipk2 has been shown biochemically, but not in vivo, to possess a less efficient I(1,4,5)P3 3-kinase activity. Of interest, both kinase-dependent and independent roles have been described for Ipk2 in regulating biological functions (5–8, 18, 20, 30, 31).

To further probe the kinase-dependent roles for Ipk2, we compared I(1,4,5)P3 6- versus 3-kinase activities for functional complementation. To accomplish this, we genetically added back I(1,4,5,6)P4 or I(1,3,4,5)P4 production by heterologously expressing Drosophila IPKs, either dmIpk2 or dmIP3K, in ipk2-deficient yeast. Both kinases have been shown to be members of a family of IP kinases bearing a signature PXXXDXKXG motif that includes the following: Ipk2 6-/3-/5-kinases; IP6 kinases, which function as inositol pyrophosphate synthases and utilize IP5 and IP6 substrates; and IP3Ks, which were not found in budding yeast or plants, but were present in fly, mouse, and human genomes (4, 17). We recently reported the cloning and biochemical characterization of dmIP3K and dmIpk2 and showed they possess I(1,4,5)P3 3-kinase and I(1,4,5)P3 6-kinase activities, respectively (21). Functional analysis of ipk2-deficient cells that expressed either dmIpk2 or dmIP3K revealed that 6-kinase, but not 3-kinase activity could rescue growth on ornithine as a sole nitrogen source (Fig. 1A). This data, coupled with our previous analysis of the ipk2–3 mutant (5), indicated that production of I(1,4,5,6)P4, but not I(1,3,4,5)P4, was necessary for activation of the ArgR-Mcm1 transcriptional complex. When we analyzed the transgenic strains for growth at high temperatures, we found that either 6- or 3-kinase activity was able to complement the temperature-sensitive phenotype of ipk2 null yeast (Fig. 1B). To rule out that complementation analysis may be related to downstream metabolites, such as those required for mRNA export pathways as described for Ipk1 (8), we tested the expression in ipk2 ipk1 double mutant cells. Rescue of growth on ornithine as the sole nitrogen source or temperature sensitivity by dmIpk2 did not require the presence of Ipk1; however, the rescue of temperature sensitivity by dmIP3K did require Ipk1 (Fig. 1, A and B). Additionally, to determine if 6- or 3-kinase complementation of temperature sensitivity required pyrophosphate synthesis, we analyzed ipk2 kcs1 double mutants. We report that dmIpk2 and IP3K were able to partially (not fully) rescue under these conditions (Fig. 1B). Our data indicate that I(1,2,3,4,5)P5 production and a downstream PP-IP were required for sustaining temperature-sensitive complementation. Of interest, the ipk1-dependent temperature-sensitive phenotype may be of use for forward genetic strategies aimed at identification of regulators and receptors of Ipk1 pathways. Of note, we have previously published that ipk1 mutation is not temperature-sensitive by plating assays (only after 20 generations; see York et al. (8)).

Discovery of a Novel IP Metabolic Pathway Initiated by I(1,4,5)P3 3-Kinase Activity—We next analyzed the effect of heterologous expression of dmIpk2 and dmIP3K on IP metabolism in ipk2 null yeast. We previously reported that dmIpk2 expression was able to fully complement Ipk2 enzyme function in cells by converting I(1,4,5)P3 to I(1,3,4,5,6)P5 (21). Remarkably, analysis of ipk2-deficient cells expressing dmIP3K revealed the synthesis of several new IP metabolites, including novel IP3, IP4, IP5, and PP-IP species (Fig. 2).

To determine the genes required for synthesis of the new species downstream of I(1,3,4,5)P4, we examined the role of inositol pyrophosphate synthase and 2-kinase activities. Kcs1 has been shown to synthesize PP-IP4{alpha} and PP-IP5{alpha} from I(1,3,4,5,6)P5 and IP6 precursors (14, 19). Of note, we have implemented a symbol-based nomenclature ({alpha}, {beta}, and {gamma}) to enable the distinction of the growing list of PP-IP isomers and species. As we describe below, the basis for assigning distinct isomers was that they had unique HPLC elution profiles and unique genetic routes of synthesis. At this time we do not have chemical structures that enable definition of the ring positions harboring pyrophosphates. Loss of Kcs1 in ipk2{Delta} cells expressing dmIP3K resulted in the obvious elimination of the most polar PP-IP species detected, PP2-IP3{beta} (Fig. 2). We used the "{beta}" designation due to its distinct elution profile from PP2-IP3{alpha} (which is synthesized downstream of I(1,3,4,5,6)P5 -> PP-IP4{alpha}) and the fact that it is synthesized through I(1,3,4,5)P4 -> I(1,2,3,4,5)P5 -> PP-IP4{beta} (also see below). These data indicate the existence of a second pyrophosphate synthase gene product, designated here as Ips1, required for the synthesis of PP-IP4{beta}. It is unclear at this point whether or not Ips1 is similar or identical to the activity identified previously as Ids1, which is required for the synthesis of PP-IP5{beta} (10). We next tested whether yeast Ipk1 was required for the pathway. When dmIP3K was expressed in ipk2{Delta}ipk1{Delta} cells, there was a loss of IP5 and all PP-IPs, along with the accumulation of unique IP3 and IP4 species (Fig. 2). We therefore conclude that phosphorylation of I(1,4,5)P3 on the D-3 position initiates a novel IP synthesis pathway that requires Ipk1, a novel inositol pyrophosphate synthase, and Kcs1.

Does this novel pathway exist in wild-type or ipk2 mutant cells? Earlier work by our laboratory and the Shears laboratory indicated that IPs with similar HPLC elution profiles to those we observed in Fig. 2 exist at low levels in ipk2-deficient and wild-type cells (8, 18). We therefore re-examined several combinations of kinase mutants using a high resolution and high sensitivity radiolabeling system (Fig. 3). Using this method, examination of ipk2 null cells revealed a similar pattern of IPs to those observed in the dmIP3K-expressing cells (compare Fig. 3, top and second traces). Of note, this method also exposed that the number of unique IP and PP-IP species in ipk2-deficient cells expressing dmIP3K was substantially greater than we previously thought (compare Fig. 3, top trace to Fig. 2, second trace). The presence of these IPs in ipk2-deficient cells led us to speculate that another yeast gene product harbors I(1,4,5)P3 3-kinase activity. We therefore analyzed ipk2 ipk1 and ipk2 kcs1 double mutant cells (Fig. 3, bottom two traces). Similar to results shown above, loss of Ipk1 in ipk2 null cells resulted in the disappearance of IP5 and PP-IP species, and a corresponding accumulation of IP3 and IP4. Loss of Kcs1 in ipk2 null cells ablated the synthesis of nearly all IP and PP-IPs detectable using this high sensitivity method. This indicates that Kcs1 may regulate or act as a I(1,4,5)P3 3-kinase and that Ipk1 functions in the conversion of IP4 to the higher IPs of this pathway. High sensitivity labeling of wild-type cells indicated similar species were present (data not shown) consistent with results of others (18).



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  		FIG. 2.
Molecular dissection of a new IP pathway initiated by I(1,4,5)P3 3-kinase activity. Kinase mutant yeast strains: ipk2{Delta}, ipk2{Delta}kcs1{Delta}, or ipk2{Delta}ipk1{Delta} were transformed with either empty pRS314 vector (Vec) or pRS314 vector containing dmIP3K (dmIP3K)as indicated. Strains were radiolabeled to isotopic equilibrium with metabolic precursor [3H]inositol and harvested at late logarithmic phase, and soluble extracts containing inositol polyphosphates were separated by strong-anion exchange HPLC. Elution positions of various IP and PP-IP species are indicated. The PP-IPs were identified as PP-IP4 (pyrophosphate species generated through phosphorylation of IP5) and PP2-IP3 (pyrophosphate species generated through phosphorylation of PP-IP4). Symbols are assigned to the PP-IPs to distinguish between the different isomers identified in this study and in previous studies. Black bars indicate multiple species.

 
Ipk1 Harbors I(1,3,4,5)P4 2-Kinase Activity—Our data indicated that the dmIP3K-dependent IP synthesis pathway was initiated by D-3 phosphorylation of I(1,4,5)P3 to I(1,3,4,5)P4. Because deletion of Ipk1 blocked the pathway and caused a buildup of I(1,3,4,5)P4, we hypothesized that the second step was carried out by Ipk1 phosphorylation of I(1,3,4,5)P4 to generate I(1,2,3,4,5)P5. To test this, we carried out in vitro kinase assays using both recombinant dmIP3K and scIpk1 (Fig. 4). Incubation of I(1,4,5)P3 with ATP and dmIP3K resulted in the generation of IP4 that we previously demonstrated to be I(1,3,4,5)P4 (Fig. 4B) (21). When I(1,4,5)P3 was incubated with ATP, dmIP3K, and scIpk1 we observed the formation of an IP5 product that did not co-elute with I(1,3,4,5,6)P5 standard (Fig. 4C). This product was not a PP-IP based on its insensitivity to DIPP. Additionally, we observed that this IP5 was completely hydrolyzed to IP4 when treated with recombinant type I 5-phosphatase (Fig. 4D), an enzyme previously found to utilize only I(1,4,5)P3 and I(1,3,4,5)P4 (3). We therefore concluded that the structure had to be authentic I(1,2,3,4,5)P5 based on its elution after the I(1,3,4,5,6)P5 standard, the requirement of 2-kinase activity, sensitivity to 5-phosphatase, and the fact that the only other mono-phosphorylated isomer capable of being synthesized from I(1,3,4,5)P4 was I(1,2,3,4,5)P5. We examined the kinetic properties of this reaction and found that scIpk1 phosphorylated I(1,3,4,5)P4 with a Km of 148 µM and Vmax of 60 nmol/min/mg (Fig. 4E).



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  		FIG. 3.
Kcs1 and Ipk1 are required for ipk2{Delta}-independent IP synthesis. High resolution and sensitivity isotopic equilibrium radio-labeling was performed on ipk2{Delta} plus dmIP3K (upper trace), ipk2{Delta} (second trace), ipk2{Delta}ipk1{Delta} (third trace), or ipk2{Delta}kcs1{Delta} (bottom trace) strains as described under "Materials and Methods." Soluble extracts containing [3H]IP and PP-IP molecules were separated by HPLC using higher resolution column and protracted elution gradient as compared with Fig. 2. Elution positions of various IP and PP-IP species are indicated.

 
To understand whether this novel activity is conserved in eukaryotes, we tested whether Ipk1 2-kinase orthologs were able to phosphorylate I(1,3,4,5)P4. Recombinant rat Ipk1 was not able to phosphorylate I(1,3,4,5)P4 in vitro, and co-expression of rat Ipk1 and dmIP3K in ipk2{Delta}ipk1{Delta} cells did not restore I(1,2,3,4,5)P5, PP-IP4{beta}, or PP2-IP3{beta} synthesis (data not shown). Conversely, Arabidopsis thaliana Ipk1 was able to phosphorylate I(1,3,4,5)P4.2 This suggests that the ability of the 2-kinase to use I(1,3,4,5)P4 as a substrate was species-specific in eukaryotes.



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  		FIG. 4.
Budding yeast Ipk1 functioned as an I(1,3,4,5)P 2-kinase. 100 nM I(1,4,5)P3 was incubated at 37 °C with no kinase (A 4), 100 ng of recombinant dmIP3K (B), or both dmIP3K and scIpk1 (C) in a buffer containing 50 mM HEPES (pH 7.5), 50 mM NaCl, 20 mM MgCl2, and 4 mM ATP. A duplicate reaction to C was treated with human Type I 5-phosphatase at 37 °C (D). In panels A–D, reaction products were resolved by Partisphere strong anion exchange HPLC, and elution profile of a I(1,3,4,5,6)P5 standard was superimposed in C (gray line). E, the kinetic parameters of scIpk1 phosphorylation of I(1,3,4,5)P4 were determined using the above reaction conditions while the I(1,3,4,5)P4 concentration was varied. Km and Vmax values were obtained from a nonlinear curve fit to the Michaelis-Menten equation using GraphPad Prism version 4.01. The R2 value was 0.9697.

 
Characterization of the IP Species Produced by the I(1,4,5,)P3 3-Kinase Pathway—To understand the molecular structure of the species produced by this novel pathway, we used enzymatic digestion of the novel IPs derived from mutant cells. Our typical method for preparing IPs from radiolabeled extracts utilized 0.5 M HCl, which impaired enzymatic analysis. To circumvent this problem, we rapidly boiled radiolabeled extracts isolated under neutral pH conditions to ensure the inactivation of endogenous enzymes. Extracts prepared from ipk2 null cells expressing dmIP3K in this manner appeared similar to those prepared by the acid-chloroform extraction method used in previous analysis (compare Fig. 2, second trace, with Fig. 5, top trace). To determine if any of the species were I(1,4,5)P3, I(1,3,4,5)P4, or I(1,2,3,4,5)P5 we treated extracts with recombinant human Type I 5-phosphatase, which dephosphorylates the D-5 these substrates. Surprisingly, none of the IPs or PP-IPs in this extract were hydrolyzed by the 5-phosphatase (Fig. 5, second trace). To confirm that the 5-phosphatase was active under these conditions, we spiked these extracts with [3H]I(1,3,4,5)P4 standard and observed that it was completely hydrolyzed to I(1,3,4)P3 by the 5-phosphatase (not shown). We next treated extracts prepared from ipk2 ipk1 double mutant cells expressing dmIP3K and found that, although the IP4 species was sensitive to 5-phosphatase, consistent with it being I(1,3,4,5)P4, the IP3 species was not (data not shown). Thus, the IP3 species found in either extract expressing dmIP3K was likely I(3,4,5)P3 based on its elution profile. Additionally, we have partially purified an IP 1-phosphatase activity from yeast, which we have designated Inp1, encoded by an unknown gene.3 Given that the IP4 species in ipk2 null cells expressing dmIP3K was not 5-phosphatase-sensitive, the elution position relative to other IP4 standards, and the inability to be phosphorylated by either recombinant Ipk1 or Ipk2 (not shown) suggested that it was likely I(2,3,4,5)P4. This species may arise from either Inp1 cleavage of I(1,2,3,4,5)P5 or by phosphorylation of I(3,4,5)P3 by a 2-kinase. Of note, we were unsuccessful in our attempts to phosphorylate I(3,4,5)P3 with recombinant yeast Ipk1 (not shown); however, it was possible that the specific activity of the kinase toward this substrate was low or that we did not have proper conditions that mimicked those in cells.



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  		FIG. 5.
Enzymatic identification of IP and PP-IP species present in the I(1,4,5)P3 3-kinase pathway. DIPP and Type I 5-phosphatase treatment of labeled yeast extracts. Radiolabeled IP extracts were prepared from ipk2-deficient cells expressing dmIP3K and digested with control (No enz), 500 ng of recombinant human type I inositol polyphosphate 5-phosphatase (5Ptase), 500 ng of recombinant human diphosphoryl inositol polyphosphate phosphatase (DIPP), or both (DIPP/5Ptase). The resulting reactants were then separated by HPLC as described in Fig. 2. Elution positions of various IP and PP-IP species are indicated.

 
We next examined the identities of PP-IP species in ipk2{Delta} cells expressing dmIP3K. DIPP specifically removes the {beta}-phosphate from PP-IPs and has no reported activity against the IP monophosphates (34). Recombinant human DIPP completely dephosphorylated the two major species that eluted at 20 and 30 min with a concomitant increase in the levels of I(1,2,3,4,5)P5 (Fig. 5, third trace). The observed increase in IP4 levels in DIPP-treated extracts confirmed that PP-IP3{alpha} and PP2-IP2{alpha} were present as shown in Fig. 3 (top trace) (note that under low resolution HPLC this species co-eluted with IP5). We did not observe evidence of IP6 after treatment. Additionally, we simultaneously treated extracts with both DIPP and 5-phosphatase. We observed a disappearance of PP-IP4{beta} and PP2-IP3{beta}, the reduction of a major peak of I(1,2,3,4,5)P5, and the formation of I(1,3,4)P3 and I(1,2,3,4)P4 (Fig. 5, bottom trace). We conclude that the major IP5 released by DIPP treatment was I(1,2,3,4,5)P5 based on: 1) this IP5 species co-eluted exactly with authentic I(1,2,3,4,5)P5; 2) it was fully susceptible to 5-phosphatase treatment generating a product that coeluted with the I(1,2,3,4)P4 standard; and 3) that its synthesis in cells required Ipk1. Furthermore, recombinant type I 5-phosphatase was unable to hydrolyze any additional IP5 species tested, and we are not aware of any literature demonstrating that 5-phosphatases have ring-position promiscuity (i.e. 4- or 3-phosphatase activities). Collectively, these data corroborated our genetic evidence that PP-IP4{beta} and PP2-IP3{beta} arose through sequential phosphorylation of I(1,2,3,4,5)P5, via the action of two pyrophosphate synthase steps, likely through Ips1 or Ids1 and then Kcs1. Additionally they suggested that I(1,3,4,5)P4 was the likely precursor for PP-IP3{alpha} and PP2-IP2{alpha}. A second IP5 (IP5x) species was identified through this experiment, based on the observation of an IP5 species that was not susceptible to 5-phosphatase treatment (compare Fig. 5, second and bottom traces).

Pulse-chase Analysis of the IP3K-dependent Synthesis Pathway—To further examine the order of cellular IP synthesis we used pulse-chase analysis of ipk2 null cells expressing dmIP3K. Overnight cultures were grown to late logarithmic phase, pulse labeled with medium labeled with [3H]inositol for 10 min, washed, and then chased with medium supplemented with excess cold inositol. Examination of IP profiles revealed that IP3 and IP4 pools accumulated first, and were followed by the synthesis of IP5, PP-IP4{beta}, and PP2-IP3{beta} (Fig. 6A). These data were consistent with the kinetics we observed for scIpk1 phosphorylation of I(1,3,4,5)P4. The relatively high Km that scIpk1 exhibits for I(1,3,4,5)P4 may help explain our observation that the substrate of the 2-kinase (I(1,3,4,5)P4) accumulated before a significant amount of I(1,2,3,4,5)P5 was synthesized.

Treatment of the pulse-labeled extracts with 5-phosphatase revealed that at early time points, I(1,4,5)P3 and I(1,3,4,5)P4 were the predominant species (Table I). At progressively later time points I(3,4,5)P3 (90 min or more) and I(2,3,4,5)P5 (after 240 min) became the predominant species, because the majority of IP3 and IP4 were no longer 5-phosphatase-susceptible. Presumably, I(3,4,5)P3 accumulates early from the high levels of I(1,3,4,5)P4, whereas I(2,3,4,5)P4 was not synthesized until later, because it was generated from I(1,2,3,4,5)P5. I(1,2,3,4,5)P5 was the only IP5 species detected at any of the tested time points, further supporting its role as the substrate for PP-IP4 and PP2-IP3 synthesis (Table I).


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  		TABLE I
5-Phosphatase susceptibility of extracts from pulse labeled strains

5-Phosphatase susceptibility was determined by comparing HPLC traces of extracts incubated with or without 5-phosphatase.

 
In ipk2{Delta}ipk1{Delta} cells expressing dmIP3K I(1,3,4,5)P4 was the only IP4 species detected at any of the tested time points. At later time points the IP3 species was not susceptible to 5-phosphatase hydrolysis, indicating it was I(3,4,5)P3. Based on these findings, together with the genetic and pulse labeling data presented above, we propose the primary synthesis pathway to be I(1,4,5)P3 -> I(1,3,4,5)P4 -> I(1,2,3,4,5)P5 -> PP-IP4{beta} -> PP2-IP3{beta}. Given the complexity of additional species, it also appears that there were several branches to the pathway, which may arise from phosphatase and promiscuity of IPK kinase activities.

Kcs1 Functions as an I(1,4,5)P3 3-Kinase in ipk2{Delta} Cells— Two lines of evidence support that Kcs1 may function as an I(1,4,5,)P3 3-kinase: the work of Dubois and colleagues that reported that Kcs1 had an undetermined I(1,4,5)P3 kinase activity in vitro (13); and our observation that deletion of KCS1 ablated synthesis of higher IPs in an ipk2 null. Dubois and co-workers also reported that Kcs1 overexpression in ipk2{Delta} cells resulted in the production of several higher phosphorylated IPs, which resembled profiles we reported here for ipk2 null cells expressing dmIP3K. We directly compared the IP profiles generated by either expression of dmIP3K or Kcs1 in ipk2{Delta} cells. This analysis revealed that I(1,3,4,5)P4, I(2,3,4,5)P4, I(1,2,3,4,5)P5, and PP-IP4{beta} were synthesized in both extracts; however, there were significant differences in the relative levels of the different PP-IP4 species (Fig. 7A, top and middle traces). When Kcs1 was expressed in ipk2{Delta}ipk1{Delta} cells, the synthesis of I(1,2,3,4,5)P5, PP-IP4{beta}, and PP2-IP3{beta} was abolished; however we observed the production of PP-IP3{alpha} and PP2-IP2{alpha} species (Fig. 7A, bottom trace). Treatment of extracts from ipk2{Delta}ipk1{Delta} cells overexpressing Kcs1 with 5-phosphatase and/or DIPP confirmed that I(1,3,4,5)P4 was a precursor to PP-IP synthesis demonstrating that Kcs1 functioned in cells as an I(1,4,5)P3 3-kinase (Fig. 7B). Of note, treatment with 5-phosphatase caused almost complete hydrolysis of IP3 to I(1,4)P2 indicating that it was I(1,4,5)P3 and not I(3,4,5)P3 (Fig. 7B).



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  		FIG. 6.
Pulse-chase analysis of IP metabolism in yeast mutants expressing dmIP3K. ipk2{Delta} (A) or ipk2{Delta}ipk1{Delta} (B) expressing pRS314 vector containing dmIP3K{beta} were grown overnight to logarithmic phase, pulse labeled with 1 mCi/ml [3H]inositol for 10 min, washed, and chased for indicated times with medium containing excess cold-inositol. Radiolabeled extracts were harvested and analyzed by Partisphere strong-anion exchange HPLC as described for Fig. 2. Elution positions of various IP and PP-IP species are indicated.

 
Our results indicate that Kcs1 is both a pyrophosphate synthase and a I(1,4,5)P3 3-kinase. Previous experiments examining the pyrophosphate synthase activity of Kcs1 demonstrated that its activity requires a highly conserved PXXXDXKXG motif that is required for ATP binding (10, 35). To determine if this site is also important for the 3-kinase activity of Kcs1, we generated a double point mutant (D786A,K788A) that lacks its diphosphoryl synthase activity. When this mutant was expressed in ipk2{Delta} cells, we did not observe the Kcs1-dependent synthesis pathway, indicating that the 3-kinase activity uses the same catalytic domain as the pyrophosphate synthase activity (data not shown).



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  		FIG. 7.
Identification of a novel I(1,4,5)P3 3-kinase activity for Kcs1. A, ipk2{Delta} was transformed with vector containing pRS314-dmIP3K (top trace) or pRS426-Kcs1 under control of the Gal4 promoter (middle trace). Additionally, ipk2{Delta}ipk1{Delta} was transformed with pRS426-Kcs1 (bottom trace). Strains were radiolabeled to isotopic equilibrium, and soluble extracts were harvested and separated by HPLC as described for Fig. 2. B, enzymatic treatment of radiolabeled prepared from ipk2 ipk1 double mutant cells overexpressing Kcs1. The extracts were then incubated at 37 °C with no enzyme (top trace), 500 ng of human Type I 5-phosphatase (second trace), or 500 ng of human DIPP (third trace), or both (bottom trace). The products were then analyzed as described for Fig. 2. Elution positions of various IP and PP-IP species are indicated.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our results may be summarized into four main findings: 1) we distinguish between the roles of 6- versus 3-kinase activities associated with Ipk2 function in cells; 2) we serendipitously have discovered and defined a molecular basis for a new IP/PP-IP pathway in yeast that is initiated by I(1,4,5)P3 3-kinase activity; 3) we demonstrate novel in vivo activities for Kcs1 and Ipk1 that have ramifications for re-interpreting previously published work; and 4) we provide evidence for new phosphatase and inositol pyrophosphate synthase activities in yeast: designated Inp1 and Ips1, respectively.

Our initial hypothesis for the heterologous expression of Drosophila IPKs in ipk2 mutant yeast was that such experiments would allow us to distinguish between 6- and 3-kinase activities. These experiments also provide a means to "add-back" I(1,4,5,6)P4 and I(1,3,4,5)P4 production in cells without complications due to protein components, because the Drosophila enzymes have less than 50 residues, out of over 350, in common with yeast Ipk2 (note: the concept of "add-back" was first proposed in the York laboratory by Drs. Audrey Odom and Jill Stevenson-Paulik while studying the A. thaliana Ipk2). Although dmIpk2, and inferred 6-kinase activity, was able to complement growth on ornithine as a sole nitrogen source and growth at high temperatures, dmIP3K I(1,4,5)P3 3-kinase activity was only able to complement temperature growth. Thus, this provided the first evidence that synthesis of I(1,3,4,5)P4 was unable to restore regulation of gene expression as judged by growth on ornithine. These data further support our initial evidence that I(1,4,5,6)P4 production plays a role in gene expression through studies of the ipk2–3 mutant, which appears to be a 6-kinase-selective enzyme in cells (5). This work also demonstrates that production of I(1,4,5,6)P4, but not I(1,3,4,5)P4, was able to bypass a kinase-independent function of Ipk2 in transcriptional control as proposed by Messenguy and colleagues (30). Subsequent work of this group (31), and work of the O'Shea and Wu laboratories (6, 7), further support our initial claim that the kinase activity of Ipk2 was and still is required for its role in gene expression and biological processes (for example, ArgR-Mcm1 transcription, chromatin remodeling, growth on arginine or ornithine as a sole nitrogen source, and growth at high temperatures).

Perhaps the most surprising aspect of this work arose from the metabolic analysis of ipk2-deficient cells expressing dmIP3K. In these cells, we expected to observe conversion of I(1,4,5)P3 and stoichiometric accumulation of I(1,3,4,5)P4. However as Figs. 2 and 3 illustrate, we instead found over 11 new species of IPs and PP-IPs that were downstream metabolites of I(1,3,4,5)P4 production. Of interest, when we expressed dmIP3K in wild-type yeast, we did not observe stimulation of this pathway, and the metabolic profiles of these cells were identical to wild-type, having a signature major peak of IP6 (not shown). These data indicate that when Ipk2 was present the 6-kinase pathway was the major route of metabolism for I(1,4,5)P3. Having said this, the 3-kinase pathway was present in wild-type and ipk2-deficient cells, albeit at low levels as compared with IP6 synthesis.

Our data help interpret the observations that we and others made previously that several other low abundance IPs are present in ipk2 null and wild-type cells (5, 8, 18). In contrast to the report of Saiardi et al. (18), we found that ipk2{Delta} cells do not generate I(1,3,4,5,6)P5, IP6, PP-IP5{alpha}, and PP2-IP4{alpha}. Rather, they synthesize I(1,2,3,4,5)P5, PP-IP3{alpha}, PP-IP4{beta}, PP2IP2{alpha}, and PP2IP3{beta} (see Fig. 3). Of note, when using a Partisphere SAX HPLC column, the PP2-IP2{alpha} species that is generated co-elutes with IP6, which may have led to its misidentification. We, therefore, confirmed our results through treatment of the ipk2 null IP extracts with DIPP and found that the PP2-IP2{alpha} peak was completely hydrolyzed (not shown). Additionally, DIPP treatment of the extracts did not result in the formation of IP6, providing evidence that the PP-IPs were only synthesized from IP4 and/or I(1,2,3,4,5)P5. We note that the data presented in this study do not reveal the chemical structures of the PP-IP species produced in the pathway, specifically which ring positions harbor the pyrophosphate. However, their unique elution profiles and their synthesis via different precursors provided evidence that they appear structurally distinct (Fig. 8).



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  		FIG. 8.
Revised molecular map of IP and PP-IP pathways in budding yeast. The pathway designated by the thick bold arrows represents the originally described I(1,4,5)P3 6-kinase pathway. The novel 3-kinase-dependent IP/PP-IP pathway in S. cerevisiae is shown with thin arrows and may be initiated artificially by expression of dmIP3K or endogenously by a 3-kinase activity of Kcs1 and/or possibly Ipk2. The gene products involved in the synthesis of each reaction are designated. Symbols were assigned to the PP-IPs to distinguish between the different isomers. The PP-IP chemical structures and ring positions harboring the pyrophosphate have yet to be determined. Gray font illustrates portions of the model that are postulated from the data in this and/or previous studies.

 
The molecular and biochemical analysis of the 3-kinase pathway has allowed us to define most species and the gene products required for their synthesis. We now provide a revised genetic and metabolic map of IP/PP-IP metabolism in budding yeast (Fig. 8). Unlike the Ipk2/Ipk1-dependent pathway that synthesized IP6, the IP3K-dependent pathway required Kcs1 (possibly Ipk2), Ipk1, and Ips1 (possibly Ids1). Interestingly, despite that role of these kinases in both yeast pathways, their sequential order, substrates and, in at least one case, activities were unique to each pathway.

Through our studies we have assigned novel activities and specificities to Kcs1 and Ipk1. We assign an I(1,4,5,)P3 3-kinase activity to Kcs1 that was functional in cells. This extends previous work of Dubois and co-workers (13) that ascribed an undetermined I(1,4,5)P3 kinase activity to Kcs1. The metabolic phenocopy of dmIP3K and Kcs1 expression in ipk2 null cells, and the loss of I(1,3,4,5)P4 production observed in an ipk2 kcs1 double mutant demonstrate this new activity is present in cells. It is not entirely surprising that Kcs1 can function as an I(1,4,5)P3 kinase given that it is evolutionarily related to two I(1,4,5)P3 kinases, Ipk2/IPMKs and IP3Ks (17, 35), thus it may have retained I(1,4,5)P3 kinase activity, in addition to acquiring pyrophosphate synthase activities. Also, we note in the context of wild-type cells, our data do not exclude a role for Ipk2 in initiating the 3-kinase pathway. We show for the first time that Ipk1 from yeast acts as a 2-kinase on I(1,3,4,5)P4. Analysis of the kinetic properties of this reaction indicated that this is not as efficient as those described for other Ipk1 substrates, but clearly under conditions of I(1,3,4,5)P4 accumulations in cells this activity is relevant. Thus, it is possible that cellular alterations in Ipk2, Ipk1, and/or Kcs1 activity or specificity would regulate flux to either the 6- or 3-kinase pathways.

The discovery of the 3-kinase pathway has also enabled the identification of new IP phosphatase and inositol pyrophosphate synthase activities. Recently, we reported the existence of Ids1, an inositol pyrophosphate synthase present in cells lacking Kcs1 and DIPP whose activity appeared to convert IP6 to PP-IP5{beta} (10). Here we provide evidence of a inositol pyrophosphate synthase, designated Ips1, capable of phosphorylating I(1,2,3,4,5)P5 to generate PP-IP4{beta}. At this point we have not determined if these are two distinct enzymes or a single gene product, nor have we determined which phosphate position serves as an acceptor for the synthesis of the pyrophosphate. Lastly, we find evidence for an IP 1-phosphatase activity, designated Inp1, that does not appear to be related to lithium-inhibited inositol polyphosphate 1-phosphatase INPP1 (36, 37). Our previous studies of ipk2 mutant cells showed evidence for this activity toward I(1,4,5)P3 substrates (5, 8), and here we find evidence that such an activity also utilizes I(1,3,4,5)P4 to generate I(3,4,5)P3. The molecular identity of Inp1 has yet to be established. However, we have ruled out that this activity was encoded by Inp5s and SAC1-like inositol phosphatases (5, 8, 32, 33, 38) based on genetic and biochemical analysis.4

The discovery of the 3-kinase pathway may have important ramifications for interpreting genetic evidence. The 6-kinase and IP6 synthesis pathway has been implicated in the regulation of cellular functions, including transcription, chromatin remodeling, RNA export, vacuole function, DNA metabolism, and telomere maintenance (10, 15, 17). Clearly, the 3-kinase pathway that remains in ipk2-deficient cells does not appear to compensate for loss of 6-kinase, thereby supporting the parsimonious explanation that the 6-kinase pathway is most relevant to these functions. However, up-regulation of the 3-kinase activity by expression of dmIP3K or Kcs1 indicated that some of the IP/PP-IPs generated by this pathway were able to dosage compensate for at least some, but not all, of the functions attributed to 6-kinase metabolites. It is intriguing to speculate that the alterations in Ipk2 specificity (i.e. 6- versus 3-kinase) and/or Kcs1/Ipk1/Ids1/Ips1 activity may provide the yeast cell with a complex repertoire of signaling molecules to enable adaptation to changes in cellular environment.


   FOOTNOTES
 
* This work was supported by a grant from the Howard Hughes Medical Institute (to J. D. Y.), by National Institutes of Health (NIH) Grant R01 HL-55672 (to J. D. Y.), by a NIH minority supplement grant (to R. J. B.), and by NIH Grant R33 DK070272 (to J. D. Y). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Duke University Medical Center, DUMC BOX 3813, Durham, NC 27710. Tel.: 919-681-6414; Fax: 919-668-0991; E-mail: yorkj{at}duke.edu.

1 The abbreviations used are: I(1,4,5)P3, inositol 1,4,5-trisphosphate; I(1,3,4,5)P4, inositol 1,3,4,5-tetrakisphosphate; I(1,2,3,4,5)P5, inositol 1,2,3,4,5-pentakisphosphate; HPLC, high pressure liquid chromatography; Vmax, maximal velocity attained with excess substrate; Km, substrate concentration permitting a half-maximal velocity; IP, inositol polyphosphate; PP-IP, diphosphoinositol polyphosphate; IP3, inositol trisphosphate; IP4, inositol tetrakisphosphate; IP5, inositol pentakisphosphate; IP6, inositol hexakisphosphate; Ipk2, inositol polyphosphate kinase 2; dmIP3K, Drosophila I(1,4,5)P3 3-kinase {beta} isoform; DIPP, diphosphoryl inositol polyphosphate phosphohydrolase; IPK, inositol polyphosphate kinase; CM, complete minimal medium. Back

2 A. M. Seeds, J. Stevenson-Paulik, and J. D. York, unpublished results. Back

3 J. D. York and B. D. Spiegelberg, unpublished results. Back

4 B. Spiegelberg and J. York, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank members of the York laboratory for helpful discussions, especially Drs. Odom and Stevenson-Paulik for studies related to inositol polyphosphate "add-back" experiments.



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 ABSTRACT
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 RESULTS
 DISCUSSION
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