Discovery at the VICB
New Role for Inositol Phosphates Discovered
By: Carol A. Rouzer, VICB Communications
Published: December 15, 2015
Studies in the fruit fly demonstrate that inositol phosphates are required for normal development of external adult structures
One of the most important pathways by which cells send and receive information is initiated by the enzyme phospholipase C (PLC), which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to diacylglycerol and inositol 1,4,5-trisphosphate (IP3). Both diacylglycerol and IP3 serve as second messengers that activate downstream signaling pathways. Although first recognized for its ability to trigger intracellular Ca2+ release, IP3 also serves as substrate for a number of inositol kinases and phosphatases, leading to the formation of additional inositol phosphates (IPs) and inositol pyrophosphates. Of particular importance in this pathway is the enzyme inositol phosphate kinase 2 (Ipk2), which phosphorylates IP3 at the 3-, and 6-positions, yielding IP4 and IP5. Subsequently, inositol phosphate kinase 1 (Ipk1) converts IP5 to IP6 by adding a phosphate at the 2-position (Figure 1A). Studies in yeast and other organisms suggest that Ipk2 plays a role in the regulation of multiple biochemical processes; however relatively little is known about Ipk2’s function in complex, multicellular organisms. To address this question, Vanderbilt Institute of Chemical Biology member John York and his laboratory embarked on a detailed investigation of Ipk2’s role in fruit fly (Drosophila melanogaster) development. Their work demonstrates that Ipk2 is required for proper development of multiple structures in the adult fly through regulation of both cell proliferation and apoptosis [A. M. Seeds, et al., (2015) Proc. Natl. Acad. Sci. U.S.A., published online December 8, DOI: 10.1073/pnas.1514684112].
Figure 1. Effect of Ipk2 on D. melanogaster development. (A) Biochemical pathway for the formation of IP3 from PIP2 and its subsequent conversion to IP4, IP5, and IP6. (B-F) Pupae of various genotypes from the latest stage of development reached by ipk2 mutants. Arrows indicate malformed wings. (G-H) Eyes from wild-type (G) and ipk2 (H) larvae. (I-J) Wings from wild-type (I) and ipk2 (J) larvae. The deformed wing is indicated by the white arrow. (K-L) Notums from wild-type (K) and ipk2 (L) larvae. The yellow arrows indicate the location of degenerative holes. Figure reproduced by permission from A. M. Seeds, (2015) Proc. Natl. Acad. Sci. U.S.A., published online Dec. 8 DOI:10.1073/pnas.1514684112. Copyright 2015 A. M. Seeds, et. al.
The York lab investigators began their studies by creating ipk2 deletion mutant flies that completely lacked the protein. They found that 85% of the ipk2 homozygous mutants (simply designated as ipk2) survived through the larval stage but subsequently died during pupation. An examination of the ipk2 pupae revealed multiple defects, notably in the eyes, wings, and notums (the dorsal region of the insect thorax)(Figure 1C, H, J, and L). These defects did not occur in ipk2 mutants engineered to express ipk2 from D. melanogaster (Figure 1D), Saccharomyces cerevisiae (Figure 1F) or Arabidopsis thaliana transgenically. However, expression of D. melanogaster ipk2 bearing an inactivating point mutation failed to correct the defects in ipk2 mutant pupae (Figure 1E). From these results, the investigators concluded that Ipk2 is required for normal fruit fly development. Furthermore, since the S. cerevisiae and A. thaliana proteins, which exhibit low sequence identity (22% and 31%, respectively) to the enzyme from D. melanogaster, can substitute for the fruit fly enzyme, whereas an inactive mutant protein cannot, it appears that considerable structural variability is tolerated as long as activity is retained.
Because ipk2 homozygous mutants did not reach adulthood, all of the developing flies that the investigators had observed were derived from crossing heterozygous male and female flies. Consequently, the investigators hypothesized that the ability of ipk2 larvae to live into the pupal stage might be due to their retaining some ipk2 mRNA from their mothers. Indeed, northern blot analysis of ipk2 larvae confirmed the presence of significant levels of the ipk2 mRNA through the early larval stages. To enable the study of larval development in the complete absence of Ipk2, the researchers created ipk2 germ-line clones (GLCs), female flies that were heterozygous for the ipk2 deletion mutation with the exception of their germ-line tissue, which was homozygous for the mutation. From these flies, it was possible to obtain ipk2 homozygous mutant larvae completely lacking the enzyme from conception. The investigators discovered that, like the previously studied ipk2 homozygous mutants, the GLC larvae developed; however, upon pupation, they were distinguished by the total absence of an epidermis and its accompanying structures, such as developing legs, wings, or antennae (Figure 2).
Figure 2. Comparison of wild-type (A), conventional zygotic ipk2 homozygous mutant (B), and ipk2 germ line clone mutant pupae. Figure reproduced by permission from A. M. Seeds, (2015) Proc. Natl. Acad. Sci. U.S.A., published online Dec. 8 DOI:10.1073/pnas.1514684112. Copyright 2015 A. M. Seeds, et. al.
The defects observed in the GLC pupae suggested that Ipk2 is required for proper development of imaginal discs, clusters of cells that develop into external adult structures during pupation. The investigators found that disc precursor structures formed normally in GLC larvae, so they hypothesized that either excessive cell death, inadequate cell proliferation, or both could account for the failure of the imaginal discs to develop in the absence of Ipk2.
Apoptosis, the process of programmed cell death, plays an important role in tissue remodeling during development; however, excess apoptosis can lead to abnormalities. The York lab researchers discovered a higher number of apoptotic cells in imaginal discs ‐ but not in other tissues ‐ of ipk2 homozygous mutant pupae. As in other organisms, apoptosis in the fruit fly is mediated through the action of specialized proteases known as caspases. The major initiating caspase in Drosophila is Dronc. Selective expression of p35, a protein caspase inhibitor, in the wing imaginal discs of developing ipk2 flies reduced the number of dying cells in these structures. Consistently, ipk2 mutants that also carried a double-null mutation for dronc failed to exhibit increased imaginal disc cell death, confirming that the excessive death observed in the mutants was due to apoptosis. Further studies revealed that loss of the proapoptotic p53 protein did not rescue the ipk2 mutants’ imaginal disc apoptosis, and, although the proapoptotic JNK signaling pathway was clearly activated in the ipk2 mutants, it was also not required to initiate the observed increase in apoptosis in the imaginal discs.
Although the investigators had demonstrated increased apoptosis in the ipk2 imaginal discs, they quickly learned that this did not fully explain the developmental failure of these structures. Indeed, 5´-bromo-2´-deoxyuridine incorporation and clonal analysis studies revealed lower levels of cell proliferation in the imaginal discs of ipk2 mutants as compared to those of wild-type flies. Previous work had shown that Ipk2 is a regulator of the JAK/STAT (Janus kinase/signal transducers and activators of transcription) pathway (Figure 3), which plays an important role in the regulation of cell proliferation, differentiation, and survival. In Drosophila, the pathway comprises the extracellular ligand Unpaired (Upd), which binds to its transmembrane receptor, Domeless (Dome). This activates Hopscotch (Hop), a Dome-associated Janus tyrosine kinase, which then phosphorylates both itself and Dome. The resulting phosphotyrosines provide docking sites for Stat92E (Stat), which is then also phosphorylated by Hop. The phosphorylation promotes dimerization of Stat92E, and the dimers subsequently translocate to the cell nucleus where they bind to consensus DNA recognition sites and initiate transcription. The York lab investigators used Drosophila Kc167 cells transfected with a Stat-dependent luciferase reporter gene to test the effects of Ipk2 on the JAK/STAT pathway. They found that RNAi-mediated knockdown of Dome or Ipk2 expression both decreased Stat-dependent reporter activity, whereas knockdown of Ipk1 actually increased Stat-dependent transcription. Consistent with expectations, direct assay of IP levels revealed that Ipk2 knockdown resulted in reduced levels of IP4, IP5, and IP6, while Ipk1 knockdown resulted in reduced levels of IP6 only, accompanied by a build-up of IP4 and IP5. These findings suggested that IP4 and IP5, but not IP6, promote Stat-mediated reporter activity in the Kc167 cell model system.
Figure 3. JAK/STAT pathway in Drosophila. The Domeless transmembrane receptor (Dome) binds the extracellular ligand Unpaired (Upd), resulting in activation of the receptor-associated Janus tyrosine kinase, Hopscotch (Hop). This results in phosphorylation of both Dome and Hop, providing docking sites for the Stat92E (Stat) transcription factor. Phosphorylation of Stat leads to its dissociation from the receptor and dimerization followed by translocation to the nucleus where it binds to consensus DNA recognition sites and initiates transcription. Image courtesy Carol Rouzer.
Prior studies had shown that the effects of RNAi-mediated knockdown of Ipk2 on Stat-dependent transcription were ameliorated by culturing cells in Upd-conditioned medium. Consistently, the investigators found that Ipk2 knockdown in Kc167 cells led to a reduced secretion of Upd that was reversed by expression of Ipk2 from A. thaliana. These results suggested that at least part of the effects of Ipk2 on the JAK/STAT pathway are likely mediated by the ability of Ipk2 to promote Upd secretion.
To further investigate the role of Ipk2 in D. melanogaster development, the researchers ectopically expressed Upd in the eye disks of developing flies. They discovered that elevated Upd secretion resulted in enlarged eyes in contrast to those of wild-type flies; however, over-secretion of Upd did not ameliorate the small, dysmorphic eyes resulting from ipk2 mutation (Figure 4). Thus, although JAK/STAT signaling clearly has an effect on the developing eye disc, upregulation of signaling in this pathway alone could not counteract the effects of deficient Ipk2 expression in this tissue. In contrast, expression of a dominant gain-of-function mutant of Hop partially rescued the small wing phenotype found in ipk2 mutant flies (Figure 5). However, despite the beneficial effect of increased Hop activity, the ipk2 pupae still died before reaching adulthood.
Figure 4. Eyes of developing pupae with the following genetic manipulations: wild-type (1), ipk2 mutant (2), upd-overexpressing (3), and upd-overexpressing ipk2 mutant (4). Figure reproduced by permission from A. M. Seeds, (2015) Proc. Natl. Acad. Sci. U.S.A., published online Dec. 8 DOI:10.1073/pnas.1514684112. Copyright 2015 A. M. Seeds, et. al.
Figure 5. Wings of developing pupae with the following genetic manipulations: wild-type (top), ipk2 mutant (center), and ipk2 mutant expressing dominant gain-of-function Hop (bottom). Figure reproduced by permission from A. M. Seeds, (2015) Proc. Natl. Acad. Sci. U.S.A., published online Dec. 8 DOI:10.1073/pnas.1514684112. Copyright 2015 A. M. Seeds, et. al.
From their studies, the York lab investigators concluded that Ipk2 plays a critical role in D. melanogaster development. Enzyme activity is clearly required in this role, and the enzyme’s products, IP4 and/or IP5 appear to mediate at least some of the effects. Evidence indicates that Ipk2’s function is primarily focused on the imaginal discs, and that regulation of both cell death through apoptosis and cell proliferation through the JAK/STAT pathway are targets of IP-dependent modulation. The results demonstrate that D. melanogaster provides an excellent model system with which to explore the role of IPs in development, and we can expect further insights from the application of this model in the future.