InteractiveFly: GeneBrief

Growth-blocking peptide 1: Biological Overview | References

Gene name - Growth-blocking peptide 1

Synonyms -

Cytological map position - 54A1-54A1

Function - ligand

Keywords - cytokine, regulation of innate immunity and larval growth, regulates organism response to stress, activates Phospholipase C which promotes increases in the levels of inositol phosphates involved in intracellular signaling

Symbol - Gbp1

FlyBase ID: FBgn0034199

Genetic map position - chr2R:14,334,860-14,338,545

Classification - cytokine

Cellular location - secreted

NCBI link: EntrezGene

Gbp1 orthologs: Biolitmine

Antimicrobial peptides (AMPs), major innate immune effectors, are induced to protect hosts against invading microorganisms. AMPs are also induced under non-infectious stress; however, the signaling pathways of non-infectious stress-induced AMP expression are yet unclear. This study demonstrates that growth-blocking peptide (GBP) is a potent cytokine that regulates stressor-induced AMP expression in insects. GBP overexpression in Drosophila elevates expression of AMPs. GBP-induced AMP expression does not require Toll and immune deficiency (Imd) pathway-related genes, but imd and basket are essential, indicating that GBP signaling in Drosophila does not use the orthodox Toll or Imd pathway but uses the JNK pathway after association with the adaptor protein Imd. The enhancement of AMP expression by non-infectious physical or environmental stressors is apparent in controls but not in GBP-knockdown larvae. These results indicate that the Drosophila GBP signaling pathway mediates acute innate immune reactions under various stresses, regardless of whether they are infectious or non-infectious (Tsuzuki, 2012).

The innate immune system of animals provides the first and most primitive line of defense against invading microorganisms. Antimicrobial peptides (AMPs) are produced as immune effector molecules to fight pathogenic infection, and the induction of AMPs is regulated through activation of the Toll and immune deficiency (Imd) pathways in Drosophila. Although the activation of both signaling pathways in response to infection has been extensively investigated, it is also known that changes in innate immune activities are sometimes unrelated to microbial infection. Various physical and physiological factors such as temperature, starvation, and diapause also elevate AMP expression levels. It is also known that AMP expression is highly sensitive to developmental stage in mammals as well as insects. Further, it has been recently reported that AMP expression in starved Drosophila is enhanced in response to the transcription factor FOXO, a key regulator of stress resistance, metabolism, and ageing, independently of the immunoregulatory pathways (Becker, 2010). Insulin signaling is currently the only known pathway for the induction of AMP expression by non-infectious stress. However, it is unlikely that animals cope with various non-infectious stressors by using the same signaling pathway that manages the regulation of innate immunity (Tsuzuki, 2012).

To investigate extracellular signaling in the innate immune regulation under non-infectious stresses, focus was placed on insect cytokines because cytokines in general regulate many physiological events including stress resistance through transmission of signals from outside the cell to the inside. While a large number of cytokines have been identified and their roles in mammals studied extensively, the number of known insect cytokines is quite limited. In Drosophila, spätzle is known as the cytokine that activates Toll signaling after microbial infection, which leads to expression of the target AMPs. In lepidopteran insects, structurally similar bioactive peptides have been reported in the last 20 years and are now recognized as the insect cytokine family referred to as the ENF peptides on the basis of their common N-terminal sequence, Glu-Asn-Phe- (Strand, 2000). These peptides are typically 23-25 amino acids long, and growth-blocking peptide (GBP) was the first member of this peptide family discovered (Hayakawa, 1990). Other known insect-specific cytokines include Unpaired-3, which was first identified in Drosophila, and hemocyte chemotactic peptide (HCP), identified in the armyworm Pseudaletia separata. Stress-responsive peptide (SRP) has also recently been identified in the common cutworm Spodoptera litura. Drosophila eda-like cell death trigger (eiger) HAS been reported to modify AMP expression levels. Among these insect cytokines, this study focused on characterizing the functional role of GBP in innate immunity because GBP was initially identified as the factor responsible for the reduced growth exhibited by armyworm P. separata larvae under stress conditions such as parasitization by the parasitoid wasp Cotesia kariyai and exposure to low temperature (Ohnishi, 1995). NMR analysis of GBP showed that it consists of flexible N- and C-termini, and a structured core stabilized by a disulfide bridge and a short antiparallel β-sheet (β-hairpin) (Aizawa, 1999). Structural comparisons indicated that the core β-hairpin region adopts the C-terminal subdomain structure of human epidermal growth factor. Consistent with this structural similarity, GBP at concentrations of 10-1 to 102 pmol/ml induced proliferation of human keratinocytes as well as insect Sf 9 cells (Hayakawa, 1998; Tsuzuki, 2012 and references therein).

At least 16 members of the insect ENF cytokine family have been identified. They have diverse functions such as growth retardation, paralysis induction, cardioacceleration, embryogenic morphogenesis, and immune cell stimulation. Characterization of some of these peptide cDNAs demonstrated that the ENF peptides are synthesized as a precursor form in which the active peptide is located at the C-terminal region. Because it has been demonstrated that ENF family peptides stimulate insect immune cells like plasmatocytes to spread on foreign surfaces, this study first examined whether GBP affects humoral immune activity in a lepidopteran insect, the silkworm Bombyx mori. As expected, injection of B. mori GBP into B. mori larvae elevated the expression of AMPs. Further, GBP-induced elevation of AMP expression was demonstrated in silkworm larvae exposed to heat stress. Although this result demonstrated GBP-dependent induction of AMP expression in non-infected stressed silkworm larvae, elucidating in detail the pathway of GBP signaling in the immune system required analysis in Drosophila because little is known about the signaling pathways that activate AMP gene expression in non-Drosophila insects like B. mori. However, none of the ENF family cytokines have been identified in insect orders outside the Lepidoptera, making it necessary to identify the Drosophila GBP homolog. Database searches did not reveal any obvious homologs in the fly genome, which suggested either that the Diptera lack ENF genes or that members of this gene family might have diverged too much to be identified on a sequence level in Diptera. Therefore, a peptidergic factor with GBP-like activity was purified from the bluebottle fly Lucilia cuprina. Using the sequence of the bluebottle fly GBP homolog for motif and FASTA searches, five Drosophila melanogaster homologs were identified, among which CG15917 was most similar to lepidopteran GBPs in terms of the primary structure of its ORF. Overexpression and RNAi knockdown of GBP in the Drosophila larvae indicated that GBP regulates the expression of AMPs through a novel pathway associated with Imd and JNK. The Drosophila GBP signaling pathway stimulated AMP expression in Drosophila larvae in response to external stressors, whether they were infectious or non-infectious (Tsuzuki, 2012).

This study has revealed the innate immune activity of GBP in insects by demonstrating that it elevates expression levels of some AMPs in Drosophila larvae as well as in Bombyx larvae. In order to clarify the role and mechanism of GBP in inducing AMP expression in insects, its signaling pathway was analyzed in Drosophila because no detailed signaling pathway of an innate immune system had been recognized in non-Drosophila species. To perform the analyses, a GBP-like cytokine was isolated from bluebottle fly larvae, and then 46 Drosophila genes were identified encoding its homologous peptide by (a cysteine-based motif search) using the C-(X except for C)6-8-C-(X except for C)1-4 pattern. Among these genes, CG14069 and CG17244 were selected as candidate Drosophila GBP genes whose ORFs consist of 100-200 amino acid residues. Subsequent FASTA searches for the C-terminal 25-amino acid peptide sequence of CG14069 identified CG11395, CG15917, and CG12517. These five genes were found to encode GBP-like peptide sequences at the C-terminal ends. Among the five genes, the peptide gene most homologous to the bluebottle fly GBP and lepidopteran GBP genes were further selected based on the characteristics common to those GBP genes: the size (100-200 amino acids) of the ORF and the presence of an arginine residue at an appropriate position (7-9 residues upstream of the first cysteine). Further, after confirming the gene expression in the larval fat body, CG15917 remained as the most plausible candidate. The functional GBP peptide located in the C-terminal region of the CG15917 ORF stimulated aggregation of hemocytes from Drosophila y w larvae. Further, overexpression of CG15917 retarded larval development, creating a phenotype similar to that seen following injection of GBP into lepidopteran larvae. These results were interpreted as an indication that the CG15917 product has a physiological role identical to that of lepidopteran GBPs (Tsuzuki, 2012).

Overexpression of proGBP as well as GBP significantly elevated the expression of the antimicrobial peptides Mtk and Dpt with the aid of the adaptor protein Imd in Drosophila larvae. The results are partially consistent with previously published data on the Drosophila gene CG15917: the gene was detected, along with many other genes, by microarray analysis as being transcriptionally activated three hours after septic injury of Drosophila adults. It was further revealed that the GBP-dependent induction of Mtk expression was abolished in larvae whose bsk expression was reduced by RNAi, indicating that the GBP pathway stimulated AMP expression through JNK signaling after recruitment of Imd to the activated GBP receptor (GBPR). This interpretation was partially confirmed by showing that GBP induced Mtk expression independently of PGRP-LC, PGRP-LE, and Relish. The biological importance of GBP was demonstrated by the finding that RNAi targeting of GBP significantly repressed AMP expression during the initial phase of bacterial infection and consequently made the larvae more susceptible to the bacteria than control larvae. Moreover, GBP RNAi repression of AMP expression was found to occur when test larvae were subcutaneously damaged without bleeding by pinching. The GBP RNAi-induced repression of AMP expression was also observed in Drosophila larvae exposed to temperature stresses: AMP expression was significantly elevated in control larvae by transferring them from 4oC to 25oC or exposing to 4oC, but it was not seen in GBP RNAi larvae. The results clearly show that induction of GBP-dependent AMP expression does not require a pathogen-associated molecular pattern, and that such non-infectious or non-injurious stimuli-dependent AMP expression is probably mediated by the GBP signaling pathway (Tsuzuki, 2012).

In lepidopteran larvae, GBP is abundantly present in a precursor form (proGBP) in hemolymph (Kamimura, 2001; Oda, 2010). The present experiments showed that this is also true for Drosophila. Non-injurious stimuli instantly trigger activation of GBP-processing enzyme(s) in hemolymph, by which the proGBP is proteolytically activated to GBP. The active GBP that results from the proteolytic processing under various stresses should trigger GBP-dependent AMP expression. It is reasonable to expect that such processing of proGBP enables the production of AMPs for the swift supply of active GBP under stress conditions. This interpretation is consistent with the fact that AMP expression was enhanced more by overexpression of active GBP than that of proGBP. Therefore, it is reasonable to propose that GBP serves as a key cytokine in the enhancement of AMP gene expression through the Imd/JNK pathway in Drosophila larvae exposed to various stressors, regardless of whether they are infectious or non-infectious (Tsuzuki, 2012).

Although there have been reports of the cross-regulation or cross-modification of two signaling pathways, Toll and Imd or JNK (or JAK/STAT) and NF-kappaB, the current knowledge is not still adequate to fully understand all mechanisms controlling the regulatory system. Further, recent studies suggest the existence of an evolutionarily conserved mechanism of cross-regulation of metabolism and innate immunity. It might be worth emphasizing that GBP was initially identified as a growth inhibitory factor in armyworm larvae. Although this study confirmed that GBP overexpression retards normal larval development in Drosophila, it was confirmed that this GBP-induced growth retardation did not affect AMP expression levels in test larvae under the present experimental conditions. Further, GBP-induced AMP expression was observed in Drosophila adults. Therefore, it is reasonable to propose that the insect cytokine GBP contributes to regulation of both growth and innate immunity. It may be possible to solve important problems concerning the adaptation of organismal defense to environmental stresses if the contributions of the novel signaling pathway through GBP-JNK are taken into account there (Tsuzuki, 2012).

Activation of PLC by an endogenous cytokine (GBP) in Drosophila S3 cells and its application as a model for studying inositol phosphate signalling through ITPK1

Using immortalized [3H]inositol-labelled S3 cells, this study has demonstrated that various elements of the inositol phosphate signalling cascade are recruited by a Drosophila homologue from a cytokine family of so-called GBPs (growth-blocking peptides) (see The pathways of inositol phosphate metabolism). HPLC analysis revealed that dGBP (Drosophila GBP) elevates Ins(1,4,5)P3 levels 9-fold. By using fluorescent Ca2+ probes, it was determined that dGBP initially mobilizes Ca2+ from intracellular pools; the ensuing depletion of intracellular Ca2+ stores by dGBP subsequently activates a Ca2+ entry pathway. The addition of dsRNA (double-stranded RNA) to knock down expression of the Drosophila Ins(1,4,5)P3 receptor almost completely eliminates mobilization of intracellular Ca2+ stores by dGBP. Taken together, the results of the present study describe a classical activation of PLC (phospholipase C) by dGBP. The peptide also promotes increases in the levels of other inositol phosphates with signalling credentials: Ins(1,3,4,5)P4, Ins(1,4,5,6)P4 and Ins(1,3,4,5,6)P5. These results greatly expand the regulatory repertoire of the dGBP family, and also characterize S3 cells as a model for studying the regulation of inositol phosphate metabolism and signalling by endogenous cell-surface receptors. Therefore a cell-line (S3ITPK1) was created in which heterologous expression of human ITPK (inositol tetrakisphosphate kinase) was controlled by an inducible metallothionein promoter. It was found that dGBP-stimulated S3ITPK1 cells did not synthesize Ins(3,4,5,6)P4, contradicting a hypothesis that the PLC-coupled phosphotransferase activity of ITPK1 [Ins(1,3,4,5,6)P5+Ins(1,3,4)P3→Ins(3,4,5,6)P4+Ins(1,3,4,6)P4] is driven solely by the laws of mass action. This conclusion represents a fundamental breach in understanding of ITPK1 signalling (Zhou, 2012).

Stimulus-dependent activation of PLC (phospholipase C) hydrolyses PtdIns(4,5)P2 to generate two intracellular messengers: diacylglycerol which activates protein kinase C, and Ins(1,4,5)P3, which binds to specific receptors {IP3R [Ins(1,4,5)P3 receptor]; itpr in Drosophila} that gate intracellular Ca2+ stores. This is a ubiquitous signalling response that regulates diverse aspects of cellular biology in almost all animal cell types. As such, it is an intensively studied area of cell biology. Additionally, research into the metabolism of Ins(1,4,5)P3 has spurred the development of a largely separate signalling industry: the study of an elaborate network of interconnected metabolites, many of which have multiple biological functions. Thus the discovery that PLC is activated by an extracellular agonist endows that agent with a large number of new signalling activities (Zhou, 2012).

Drosophila melanogaster is a genetically-tractable eukaryotic model that has proved useful in unraveling the complexities of many aspects of Ca2+ signalling. For example, experiments with Drosophila mutants have demonstrated that the fly's single itpr gene is important for a range of physiological response. Cultured Drosophila cells have also been used to study the receptor-dependent activation of Ins(1,4,5)P3 production and Ca2+ mobilization. However, the range of opportunities offered by this model organism have not yet been fully exploited for studies into the function and agonist-regulated metabolism of the other inositol phosphate signals. Such studies, which typically rely on analysing cells that have been radiolabelled with [3H]inositol during several days of proliferation, are best suited to immortalized cell lines. Such cells have been derived from Drosophila. However, there is limited insight into the nature of peptides that act through endogenous receptors to activate PLC in these immortalized cells. The application of these cells to inositol phosphate research has therefore been restricted. To bypass this problem, some groups have heterologously expressed exogenous receptors into immortalized Drosophila cells. A more physiologically relevant model is described that utilizes endogenous receptors: the S3 imaginal-disc cell line, in which this study shows that PLC is activated by a recently discovered insect cytokine, dGBP [Drosophila GBP (growth-blocking peptide)](Zhou, 2012).

dGBP is a member of a large family of insect cytokines. These peptides range from 19-30 amino acid residues in length, and are produced upon proteolytic cleavage of longer prepropeptides. The first GBP to be identified was the factor responsible for the reduction in the rate of growth of lepidopteran larvae following their colonization by the larvae of a parasitic wasp, Costesia kariyai. The endocrinological perturbation that up-regulates GBP synthesis in the host stops it from forming the sclerotized pupal cuticle that would otherwise prevent the parasitic larvae from emerging. The GBP family also regulate morphogenesis, cell proliferation and innate immune responses, for example by stimulating plasmatocyte adhesion and spreading However, to date little progress has been made in understanding the molecular mechanisms of action of these cytokines. Recently, the discovery of dGBP has revealed that this peptide acts through c-Jun N-terminal kinase to regulate gene expression. However, in view of the multi-functionality of the GBP family, it has remained probable that these peptides recruit additional signalling pathways. The present study adds substantially to dGBP's signalling repertoire by demonstrating its ability to activate multiple facets of the PLC-dependent inositol phosphate/Ca2+ cascade (Zhou, 2012).

An increased insight into the molecular actions of insect cytokines such as dGBP is of interest in itself, but the importance of this field of research goes beyond the goal of expanding understanding of insect physiology. Knowledge of the roles of GBPs in immune responses in pest insects that impair human health or reduce crop yield may lead to the development of improved control programs. There is also considerable evolutionary conservation of innate immunity genes, pathways and effector mechanisms. Research into these defense mechanisms in Drosophila may ultimately improve understanding of human immune responses. Indeed, the identification of the human homologue of the Toll receptor was initially prompted by the discovery that this protein mediates antifungal immune responses in Drosophila (Zhou, 2012).

Another goal of the present study was to determine whether the discovery that dGBP activates PLC in S3 cells could be exploited to explore the signalling activities of inositol phosphate metabolizing enzymes from higher animals. In pursuit of this idea, it is noted that the Drosophila genome does not encode a homologue of the mammalian ITPK1 (inositol tetrakisphosphate 1-kinase). Therefore S3 cells offer a rare example of a model for characterizing any gain-of-function that might arise from the heterologous expression of human ITPK1. For example, the phosphorylation of Ins(1,3,4)P3 by ITPK1 in mammals has been proposed to be the rate-limiting step in the synthesis of Ins(1,3,4,5,6)P5 and InsP6. However, there continue to be differences of opinion as to the relative importance of this pathway compared with the alternative ITPK1-independent route to Ins(1,3,4,5,6)P5 and InsP6. This study aimed to address this debate by studying the impact of ITPK1 upon Ins(1,3,4,5,6)P5 and InsP6 synthesis in basal and PLC-activated S3 cells. Furthermore, ITPK1 has additional significance to mammalian cell signalling because this enzyme controls the synthesis of Ins(3,4,5,6)P4, which regulates the conductance of the Cl channel/transporter ClC-3 (chloride channel, voltage-sensitive 3). In this way, ITPK1 helps regulate a number of physiological processes, such as salt and fluid secretion, insulin secretion, and neurotransmission. However, it has proved difficult to characterize the mechanism by which Ins(3,4,5,6)P4 synthesis is accelerated following PLC activation. To explain this phenomenon, previous studies put forward a hypothesis that is based on the laws of mass action. This study proposes that an elevated rate of phosphorylation of Ins(1,3,4)P3 to Ins(1,3,4,6)P4 by ITPK1 is coupled through an unusual phosphotransferase reaction to an increased rate of dephosphorylation of Ins(1,3,4,5,6)P5 to Ins(3,4,5,6)P4. The present study has tested this hypothesis by determining whether PLC-dependent Ins(3,4,5,6)P4 synthesis could be heterologously introduced into S3 cells by the expression of ITPK1 (Zhou, 2012).

A central conclusion in the present study is that PLC is activated by dGBP, a member of a multi-functional family of insect cytokines. The Drosophila homologue of the GBP family was identified only quite recently, and it has been shown to stimulate transcription of antimicrobial peptides by activation of c-Jun NH2-terminal kinase. However, the GBP family is known to regulate other diverse aspects of insect cell physiology, including larval growth, morphogenesis and cell proliferation. Therefore it was thw goal of this study to search for additional signalling pathways that dGBP might recruit, in order to gain a more complete understanding of the mechanisms that underlie the various actions of these cytokines. The demonstration that dGBP activates PLC in S3 cells opens up a number of directions for further research into insect physiology. PLC-mediated hydrolysis of PtdIns(4,5)P2 yields diacylglycerol, which activates protein kinase C, and Ins(1,4,5)P3, which binds itpr and gates intracellular Ca2+ stores. Furthermore dGBP also promotes increases in a number of other inositol phosphates, several of which have their own distinct cellular functions (Zhou, 2012).

This study used several experimental approaches to ascertain that, in S3 cells, dGBP mobilizes Ca2+ from intracellular Ins(1,4,5)P3-releasable stores as a consequence of PLC activation. First, HPLC and chemical analyses were used to determine that dGBP promotes the increases in Ins(1,4,5)P3 and related metabolites that are typical of agonist-dependent stimulation of PLC activity in mammalian cells. Secondly, fluorescent Ca2+ probes were used to demonstrate that dGBP promotes the biphasic Ca2+ response that is typically observed following the activation of PLC-coupled receptors: depletion of intracellular Ca2+ stores, which then drives enhanced Ca2+ entry. Thirdly, dsRNA was used to knock down itpr expression, whereupon the ability of dGBP to mobilize intracellular Ca2+ stores was almost completely eliminated (Zhou, 2012).

The various elements of the inositol phosphate cascade that are activated by dGBP have independent functions, and so may contribute to the peptide's multi-functionality. The Ins(1,4,5)P3-mediated mobilization of Ca2+ probably mediates several physiological responses. The dGBP-mediated increases in Ins(1,3,4,5)P4 levels may also have some signalling significance. Changes in Ins(1,4,5,6)P4 levels also promise to be important, in the light of evidence that Ins(1,4,5,6)P4 regulates gene transcription. Ins(1,3,4,5,6)P5 levels in wild-type S3 cells were also elevated by 70% within 2 min of dGBP addition. Such a sizable Ins(1,3,4,5,6)P5 response to PLC activation is not usually observed in mammalian cells. However, in the murine F9 teratocarcinoma cell line, PLC activation has been associated with large increases in Ins(1,3,4,5,6)P5 levels, which regulate key aspects of the β-catenin signalling pathway. In F9 cells, Ins(1,3,4,5,6)P5 synthesis occurs primarily through direct phosphorylation of Ins(1,4,5)P3 by IPMK (inositol polyphosphate multikinase). This is also the case in Drosophila. Thus S3 cells may be of value for further exploring these signalling functions for Ins(1,3,4,5,6)P5 and their contribution to the physiology of dGBP (Zhou, 2012).

The genetic tractability of Drosophila S3 cells is also an advantage when using them to explore the mechanisms of agonist-dependent regulation of the metabolism and functions of the inositol phosphate family. The present study exploited the observation (Seeds, 2004) that the Drosophila genome is itpk1-null, and so human ITPK1 was heterologously expressed in S3 cells in order to gain insight into the signalling functions of that particular kinase. These experiments yielded three significant new conclusions with regards to the actions of ITPK1 (Zhou, 2012).

First, data was obtained that are relevant to the ongoing debate concerning the degree to which ITPK1 contributes to Ins(1,3,4,5,6)P5 synthesis in mammalian cells. Specifically, it was found that the expression of ITPK1 in S3 cells yielded several-fold increases in the steady-state levels of Ins(1,3,4,5,6)P5 and one of its metabolites, Ins(1,4,5,6)P4. Nevertheless, in both wild-type S3 and S3ITPK1 cells, the ratio of Ins(1,3,4,5,6)P5 to InsP6 was relatively low compared with that in mammalian cells. These results suggest that factors other than ITPK1 expression itself contribute to the synthesis of the relatively large quantities of Ins(1,3,4,5,6)P5 that are typically present in mammalian cells. For example, there is evidence that much of the mammalian cell's Ins(1,3,4,5,6)P5 resides in a metabolically-resistant pool. The mechanism by which Ins(1,3,4,5,6)P5 transfers into that pool in mammalian cells may be key to the accumulation of that inositol phosphate at relatively high levels (Zhou, 2012).

Secondly, the sizable accumulation of Ins(1,2,3,4,6)P5 in PLC-activated S3ITPK1 cells is also of interest. That result is attributed to the relatively high levels of Ins(1,3,4,6)P4 successfully out-competing the lower levels of Ins(1,3,4,5,6)P5 for phosphorylation by IP5K; such competition has previously been shown in vitro. Such a phenomenon could help explain a previously puzzling observation that the InsP6 pool in rat-1 cells is largely unaffected when Ins(1,3,4,5,6)P5 synthesis was compromised by knockdown of IPMK expression. An ITPK1-dependent alternative pathway of InsP6 synthesis in rat-1 in cells could solve that problem: phosphorylation of Ins(1,3,4)P3 into Ins(1,3,4,6)P4, then into Ins(1,2,3,4,6)P5 which then can be converted into InsP6 (Zhou, 2012).

The third, and arguably the most important, observation to emerge from these studies with ITPK1, was the discovery that the phosphotransferase activity of ITPK1 is not sufficient by itself to recapitulate PLC-dependent synthesis of Ins(3,4,5,6)P4, an intracellular signal that serves multiple biological roles through its regulation of Cl channel activity. Thus the results of the present study counter a previous hypothesis that Ins(3,4,5,6)P4 levels are solely regulated by the mass-action effects of substrate supply that are exerted upon ITPK1. This outcome was unexpected, because previous structural and metabolic data had indicated that the phosphotransferase activity of human ITPK1 is inherently well-adapted to regulation by mass-action effects in vitro. Perhaps the absence of Ins(3,4,5,6)P4 in PLC-activated S3ITPK1 cells is because metabolic compartmentalization prevents ITPK1 from accessing Ins(1,3,4,5,6)P5. It is also possible that there is a yet to be discovered mechanism, not active in S3 cells, by which the phosphotransferase activity of ITPK1 might be activated relative to its kinase activity. A potential mechanism might involve covalent modification of ITPK1, such as by its phosphorylation or acetylation. In any case, the present study indicates that the regulation of the Ins(3,4,5,6)P4 signalling cascade is more complex than hitherto was appreciated. As the number of physiological responses that are regulated by Ins(3,4,5,6)P4 increases, so the need to unravel these mechanisms becomes more urgent (Zhou, 2012).

In previous work, the heterologous expression in yeast of inositol phosphate kinases of animal and plant origin uncovered new aspects of inositol phosphate metabolism and signalling. However, the use of immortalized Drosophila cells as a host offers several advantages over the yeast model, including their more direct relevance to mammalian systems: unlike yeasts, flies have an Ins(1,4,5)P3-releasable Ca2+ pool. Genetic manipulation by dsRNA is also cheap and effective in cultured fly cells. Furthermore, the demonstration that dGBP stimulates PLC adds value to S3 cells as a resource for further studies into receptor-dependent regulation of inositol phosphate function and metabolism. This work with ITPK1 offers an example of how that model can usefully be exploited. Finally, this study adds substantially to the repertoire of the GBP family by demonstrating its ability to activate multiple facets of the PLC-dependent inositol phosphate signalling cascade (Zhou, 2012).


Search PubMed for articles about Drosophila Gbp

Aizawa, T., Fujitani, N., Hayakawa, Y., Ohnishi, A., Ohkubo, T., Kumaki, Y., Kawano, K., Hikichi, K. and Nitta, K. (1999). Solution structure of an insect growth factor, growth-blocking peptide. J Biol Chem 274: 1887-1890. PubMed ID: 9890941

Becker, T., Loch, G., Beyer, M., Zinke, I., Aschenbrenner, A. C., Carrera, P., Inhester, T., Schultze, J. L. and Hoch, M. (2010). FOXO-dependent regulation of innate immune homeostasis. Nature 463: 369-373. PubMed ID: 20090753

Hayakawa, Y. (1990). Juvenile hormone esterase activity repressive factor in the plasma of parasitized insect larvae. J Biol Chem 265: 10813-10816. PubMed ID: 2358440

Hayakawa, Y. and Ohnishi, A. (1998). Cell growth activity of growth-blocking peptide. Biochem Biophys Res Commun 250: 194-199. PubMed ID: 9753606

Kamimura, M., Nakahara, Y., Kanamori, Y., Tsuzuki, S., Hayakawa, Y. and Kiuchi, M. (2001). Molecular cloning of silkworm paralytic peptide and its developmental regulation. Biochem Biophys Res Commun 286: 67-73. PubMed ID: 11485309

Oda, Y., Matsumoto, H., Kurakake, M., Ochiai, M., Ohnishi, A. and Hayakawa, Y. (2010). Adaptor protein is essential for insect cytokine signaling in hemocytes. Proc Natl Acad Sci U S A 107: 15862-15867. PubMed ID: 20798052

Ohnishi, A., Hayakawa, Y., Matsuda, Y., Kwon, K. W., Takahashi, T. A. and Sekiguchi, S. (1995). Growth-blocking peptide titer during larval development of parasitized and cold-stressed armyworm. Insect Biochem Mol Biol 25: 1121-1127. PubMed ID: 8580912

Seeds, A. M., Sandquist, J. C., Spana, E. P. and York, J. D. (2004). A molecular basis for inositol polyphosphate synthesis in Drosophila melanogaster. J Biol Chem 279: 47222-47232. PubMed ID: 15322119

Strand, M. R., Hayakawa, Y. and Clark, K. D. (2000). Plasmatocyte spreading peptide (PSP1) and growth blocking peptide (GBP) are multifunctional homologs. J Insect Physiol 46: 817-824. PubMed ID: 10742531

Tsuzuki, S., Ochiai, M., Matsumoto, H., Kurata, S., Ohnishi, A. and Hayakawa, Y. (2012). Drosophila growth-blocking peptide-like factor mediates acute immune reactions during infectious and non-infectious stress. Sci Rep 2: 210. PubMed ID: 22355724

Zhou, Y., Wu, S., Wang, H., Hayakawa, Y., Bird, G. S. and Shears, S. B. (2012). Activation of PLC by an endogenous cytokine (GBP) in Drosophila S3 cells and its application as a model for studying inositol phosphate signalling through ITPK1. Biochem J 448: 273-283. PubMed ID: 22928859

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date revised: 15 March 2013

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