Abstract
Phosphatidylinositol-3,4,5-trisphosphate (PIP3) generation at the plasma membrane is a key event during activation of receptor tyrosine kinases such as the insulin receptor and is critical for normal growth and metabolism. The lipid kinases and phosphatases regulating PIP3 levels are described but mechanisms that control their activity remains unclear. We report that in Drosophila, phosphatidylinositol 5 phosphate 4-kinase (PIP4K) regulates PIP3 levels during insulin receptor activation. Depletion of PIP4K increases PIP3 levels and augments sensitivity to insulin through enhanced Class I phosphoinositide 3-kinase (PI3K) activity. Plasma membrane localized PIP4K was sufficient to control PIP3 levels. Animals lacking PIP4K show enhanced insulin dependent phenotypes in vivo and show resistance to the metabolic consequences of a high-sugar diet. Thus, PIP4K is required for normal metabolism and development. Our work defines PIP4Ks as regulators of receptor tyrosine kinase signalling with implications for growth factor dependent processes including tumour growth, T-cell activation and metabolism.
Introduction
Lipid kinases that can phosphorylate selected positions on the inositol head group of phosphatidylinositol (PI), generate second messengers that regulate multiple processes in eukaryotic cells. The generation of phosphatidylinositol 3,4,5-trisphosphate (PIP3) through the action of Class I PI3K following growth factor receptor (e.g Insulin receptor) stimulation, is a widespread signalling reaction (Hawkins et al., 2006) that regulates normal growth and development (Engelman et al., 2006). The role of Class I PI3K activation in response to insulin receptor signalling is evolutionarily conserved and has been widely studied in metazoan models such as the fly, worm and mammals (Barbieri et al., 2003). Robust control of the levels and the dynamics of PIP3 turnover is essential to maintain fidelity and sensitivity of information transfer during insulin signalling. This is achieved through a number of different molecular mechanisms. The Class I PI3K enzyme is a dimer of a catalytic subunit (p110) whose activity is inhibited under unstimulated conditions by the regulatory subunit (p85/50/55/60). Upstream receptor activation and subsequent binding to p-Tyr residues on the receptor and adaptor proteins relieves this inhibition. In addition, lipid phosphatases are also important in controlling PIP3 levels at the plasma membrane. PTEN, a 3-phosphatase, hydrolyzes PIP3 to produce PI(4,5)P2 (McConnachie et al., 2003) while SHIP2 is a 5-phosphate that generates PI(3,4)P2 from PIP3 (Pesesse et al., 1998). It is well documented that mutations in genes encoding any of these enzymes can be oncogenic or result in metabolic syndromes. Loss of function in PTEN or gain of function in Class I PI3K genes results in tumour development (Luo et al., 2003) while loss of SHIP2 results in altered insulin sensitivity in mammals (Clément et al., 2001; Kaisaki et al., 2004). Thus, the control of receptor-activated PIP3 levels is vital to the regulation of events that direct cell growth and metabolism.
Class I PI3K enzymes utilize phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] as substrate to generate PIP3. In animal cells, the major route of PI(4,5)P2 synthesis is the action of phosphatidylinositol 4 phosphate 5-kinase (PIP5K), enzymes that use phosphatidylinositol 4-phosphate (PI4P) as substrate and phosphorylate position 5 of the inositol headgroup (Stephens et al., 1991). More recently, Cantley and colleagues have described a distinct class of lipid kinases, the phosphatidylinositol 5 phosphate 4-kinases (PIP4K), enzymes that utilize phosphatidylinositol 5-phosphate (PI5P) as substrate and phosphorylate position 4 to generate PI(4,5)P2 (Rameh et al., 1997). Loss of PIP4Ks does not result in a drop in the mass of total cellular PI(4,5)P2 but the levels of its preferred substrate, PI5P are elevated [(Gupta et al., 2013), reviewed in (Kolay et al., 2016)]. In mammalian cells, three isoforms of PIP4Ks occur, viz. PIP4K2A, PIP4K2B and PIP4K2C. The phenotypes of mouse knockouts in each of these genes suggest a role for PIP4Ks in regulating receptor tyrosine kinase and PI3K signaling; deletion of PIP4K2A and PIP4K2B is able to slow tumor growth in p53-/- mice (Emerling et al., 2013); depletion of PIP4K2C results in excessive T-cell activation (Shim et al., 2016) and loss of PIP4K2B in mice results in hyper-responsiveness to insulin and a progressive loss of body weight in adults (Lamia et al., 2004). Previous studies have linked PIP4K2B to insulin and PI3K signalling. Overexpression of PIP4K2B in CHO-IR cells (expressing extremely low levels of endogenous PIP4K2B) results in reduced PIP3 production following insulin stimulation (Carricaburu et al., 2003). Similarly, in U2oS cells, acute doxycycline-induced overexpression of PIP4K2A reduces AKT activation seen on insulin stimulation although changes in PIP3 levels were not reported under these conditions (Jones et al., 2013). By contrast, a recent study has reported that in immortalized B-cells that carry a deletion of PIP4K2A, there is a reduction in PIP3 levels following insulin stimulation (Bulley et al., 2016). Thus, although there are multiple lines of evidence suggesting a link between PIP4K and Class I PI3K signaling during insulin stimulation, the impact of the PIP4K function on PIP3 levels remains unresolved.
It has been reported that loss of the only PIP4K in Drosophila results in a larval growth deficit and developmental delay. These phenotypes were associated with an overall reduction in the levels of pS6KT398 and pAKTS505, both outputs of mechanistic Target Of Rapamycin (mTOR) signalling. The systemic growth defect in the dPIP4K mutants (dPIP4K29) could be rescued by enhancing mTOR complex 1(TORC1) activity through pan-larval overexpression of its activator Rheb (Durán and Hall, 2012; Gupta et al., 2013). Since then it has also been shown in mice that PIP4K2C can regulate TORC1-mediated signalling in immune cells (Shim et al., 2016). The loss of PIP4K2C was also shown to enhance TORC1 outputs in Tsc1/2 deficient MEFs during starvation (Mackey et al., 2014). mTOR signalling can transduce multiple developmental and environmental cues including growth factor signalling, amino acid and cellular ATP levels into growth responses (Wullschleger et al., 2006). However, the relationship between PIP4K function and its role in regulating TORC1 activity and Class I PI3K signaling remains unclear.
During Drosophila development, larval stages are accompanied by a dramatic increase in body size. Much of this growth occurs without increases in cell number but via an increase in cellular biomass that occurs in polyploid larval tissues such as the salivary gland and fat body (Church and Robertson, 1966). One major mechanism that drives this form of larval growth is the ongoing insulin signalling; characterized by the endocrine secretion of insulin-like peptides (dILPs) from insulin-producing cells (IPCs) in the larval brain and their action on peripheral tissues through the single insulin receptor in flies (Brogiolo et al., 2001). Removal of insulin receptor (dInR) activity (Shingleton et al., 2005) or the insulin receptor substrate (chico) (Bohni et al., 1999) results in reduced growth and delayed development through multiple mechanisms. In flies, cell size in the salivary glands can be tuned by enhancing cell-specific Class I PI3K-dependent PIP3 production (Georgiev et al., 2010). In this study, we use salivary glands and fat body cells of Drosophila larvae to study the effect of dPIP4K on insulin receptor activated, Class I PI3K signalling. We find that in Drosophila larval salivary gland cells, loss of dPIP4K enhanced the growth-promoting effects of overexpressing components of the insulin signalling pathway. dPIP4K regulates the levels of PIP3 and the intrinsic sensitivity to insulin at the plasma membrane. Insulin signalling activity is regulated through negative feedback from TORC1 in cells (Gual et al., 2005; Kockel et al., 2010). This TORC1 dependent reduction in insulin-stimulated PIP3 production is rendered ineffective in the absence of dPIP4K. Finally, we show that these cellular changes in insulin signalling have consequences on circulating sugar metabolism in larvae and also their susceptibility to insulin resistance on a high-sugar diet. Altogether, we demonstrate an important physiological role for dPIP4K as a negative regulator of Class I PI3K signaling during insulin stimulation in Drosophila in vivo.
Results
dPIP4K genetically interacts with the insulin receptor signalling pathway
Salivary glands are endo-replicative organs in Drosophila larvae that are composed of large polarized polyploid cells. Previously, we have demonstrated the use of this organ as a model to study changes in cell size (Georgiev et al., 2010; Gupta et al., 2013). Prior studies on insulin receptor signalling have revealed a role for this pathway in the autonomous control of both cell size and proliferation (Bohni et al., 1999; Brogiolo et al., 2001). However, direct evidence for such regulation in salivary glands has not been demonstrated. Therefore, as proof of principle, we depleted dInR levels through RNA interference (RNAi) selectively in the salivary glands of 3rd instar larvae using the driver AB1Gal4. As expected, this resulted in a reduction of the average size of salivary gland cells without a change in the number of cells (Fig. 1A, B, C). Likewise, overexpression of dInR (Fig. 1D) and chico (Fig. 1E) selectively in the salivary glands also results in an increase in cell size. Thus, insulin receptor signalling regulates cell size in the salivary gland.
We then compared the effect of overexpressing dInR in wild-type and dPIP4K29 cells. When dInR was over-expressed in dPIP4K29 (AB1>dInR; dPIP4K29), we also found an increase in salivary gland cell size; but the increase in cell size elicited was significantly greater than that seen in wild-type cells (AB1>dInR) (Compare Fig. 1H(i) and (ii)). Similar results were seen when comparing the effect of chico overexpression in wild-type and dPIP4K29 cells; i.e. chico overexpression elicited a larger increase in cell size in dPIP4K29 compared to wild-type (compare Fig. 1I(i) and (ii)). We reasoned that if dPIP4K specifically interacted with the early, plasma membrane components of the insulin signalling cascade, then bypassing these by constitutively activating a downstream step will abolish the differences between wild-type and dPIP4K29 cells. In order to test this, we expressed a constitutively active form of Phosphoinositide-Dependent Kinase-1 (PDK1) (PDK1A467V) which is normally activated by PIP3 downstream of insulin receptor activation and regulates cell growth (Paradis et al., 1999; Rintelen et al., 2001). Expression of PDK1A467V in salivary glands results in an increase in cell size (Fig. 1F) and this was also seen when PDK1A467V was expressed in dPIP4K29 (Fig. 1G). However, in contrast to dInR and chico manipulations, the effect of overexpressing PDK1A467V resulted in an equivalent cell size increase in wild-type and dPIP4K29 (Fig. 1J(i) and (ii)). These findings suggest that in Drosophila larval cells dPIP4K modulates insulin receptor signalling at a step that is likely to be prior to PDK1 activation.
PIP3 levels are elevated in dPIP4K depleted larval tissues
An essential early event in InR signal transduction is the activation of Class I PI3K leading to the production of PIP3 at the plasma membrane (Hawkins et al., 2006). Therefore, we measured PIP3 levels at the plasma membrane by imaging salivary glands from wandering third instar larvae expressing a PIP3-specific probe (GFP::PH-GRP1) (Britton et al., 2002). We observed that under basal conditions, plasma membrane PIP3 in dPIP4K29 showed a small but significant elevation compared to wild-type cells (Fig. 2A(i)and (ii)). Similar results were observed in experiments with fat body cells, i.e. PIP3 levels in dPIP4K29 fat body cells were elevated compared to wild type (Fig. 2B(i) and (ii)).
During larval development in Drosophila, nutritional cues and other signals result in the release of Drosophila Insulin-like peptides (dILPs) (Nässel and Broeck, 2016) that bind to and activate dInR triggering Class I PI3K activation and PIP3 production. The elevated PIP3 levels observed in dPIP4K29 tissues could, therefore, result from (i) enhanced production and release of dILPs (ii) upregulation in insulin receptor levels (iii) increase in activity of insulin receptor or events downstream of receptor activation. To distinguish between these possibilities, we performed Q-PCR analysis to measure the levels of dILP2, 3, 5 mRNAs [the levels of these are known to be transcriptionally regulated] (Brogiolo et al., 2001). We found that the transcript levels for these dILPs were not upregulated in dPIP4K29 compared to wildtype (Fig 2C). To check for enhanced dILP release, we measured the levels of dILP2 within the neurosecretory insulin-producing cells (IPCs) from the brains of wandering third instar larvae. Immunoreactivity for dILP2 produced in IPCs is expected to be lower when more of it is released into the hemolymph. We found that the average intensity of dILP2 immunostaining in the IPCs was not lower in dPIP4K29 compared to controls; instead, it showed a small but significant increase (Fig. 2E(i) and (ii)). Thus, we found no evidence of elevated production or release of dILPs in 3rd instar larvae that might explain the increased PIP3 levels observed in dPIP4K29. Further, we observed that InR receptor mRNA levels were also not different between dPIP4K29 and wildtype indicating that levels of InR that are activated by dILPs are also not likely to be different between the two genotypes (Fig. 2D). Collectively, our experiments show plasma membrane PIP3 levels to be elevated in cells lacking dPIP4K without an increase in dILP secretion or cellular insulin receptor levels.
dPIP4K29 cells are intrinsically more sensitive to insulin stimulation
We developed ex-vivo assays to test the sensitivity of tissues dissected from 3rd instar larvae to stimulation with bovine insulin. It has previously been shown that Drosophila cells stimulated with bovine insulin respond using signal transduction elements conserved with those proposed for the canonical mammalian insulin signalling pathway (Lizcano et al., 2003). We observed that in salivary glands and fat body dissected from 3rd instar larvae, ex-vivo insulin stimulation triggered a rise in plasma membrane PIP3 levels, measured using the GFP::PH-GRP1 probe. Following insulin stimulation (10 min, 10 µM), the rise in PIP3 levels in dPIP4K29 was higher than in wild type (Fig. 3A(i), A(ii)). This increased PIP3 production was also seen in salivary gland cells (Fig. 3C(ii)) where the dPIP4K protein had been selectively depleted using salivary gland specific RNAi (Fig. 3C(i)). The increased sensitivity of dPIP4K29 cells to ex-vivo insulin stimulation could be reverted by specifically reconstituting dPIP4K in salivary gland cells (Fig. 3D). Overexpression of dPIP4K in wild-type salivary gland cells resulted in reduced levels of insulin stimulated PIP3 levels (Fig. 3E). A similar observation was made in fat body cells where PIP3 production increased with stimulation over a wide range of insulin concentrations used. Fat body lobes dissected from starved larvae were stimulated with over a 100-fold range of insulin concentrations. While 100 nM of insulin barely elicited an increase in plasma membrane PIP3 levels, we observed that dPIP4K29 fat cells show a larger rise in PIP3 levels compared to the controls at higher concentrations(Fig. 3C(i)-(iii)).
Quantitative measurements of PIP3 mass in Drosophila larvae
To test if the probe-based imaging of PIP3 in single cells indeed reflects in vivo changes across the animal, we refined and adapted existing protocols (Clark et al., 2011) to perform mass spectrometric measurements of PIP3 from Drosophila whole larval lipid extracts. The amount of PIP3 that has been detected and quantified from biological samples is in the range of a few tens of picomoles (Malek et al., 2017). We coupled liquid chromatography to high sensitivity mass spectrometry (LCMS) and used a Multiple Reaction Monitoring (MRM) method to detect PIP3 standards for reliable quantification down to a few femtomoles (ca. 10 fmol, the lowest point in the figure inset on the standard curve in Fig. 3, Supplement 1, (A). Since cellular lipids are composed of molecular species with varying acyl chain lengths, we first characterized the PIP3 species from Drosophila whole larval extracts through use of neutral loss scans and thereafter quantified the abundance of these species. Fig. 3, Supplement 1, (B) depicts the elution profiles of the different PIP3 species that were reproducibly detected across samples and Fig. 3, supplement 2, (A) shows the relative abundance of various PIP3 species. The 34:2 PIP3 species was found to be the most abundant. In a pilot experiment, we bisected whole larvae, stimulated them with insulin and measured the levels of various PIP3 species between samples with and without insulin stimulation. Our LCMS method could clearly detect an increase in the levels of several PIP3 species upon insulin stimulation (Fig. 3, supplement 2, (B)).
Using this method, we compared PIP3 levels from whole larval lipid extracts of various genotypes following insulin stimulation. We observed that compared to controls, dPIP4K29 larvae showed higher PIP3 levels upon insulin stimulation (Fig. 3F(i), F(ii)). Similarly, upon pan-larval knockdown of dPIP4K by RNAi (Fig. 3, supplement 2, C(i)) enhanced PIP3 levels were observed following insulin stimulation (Fig. 3, supplement 2, C(ii) and (iii)) although the differences were not as striking as seen in dPIP4K29; presumably this reflects the residual and variable amounts of dPIP4K protein seen during RNAi based knockdown (Fig. 3, supplement 2, C(i)). We also performed pan-larval rescue of dPIP4K protein in dPIP4K29 larvae and observed a rescue in levels of various PIP3 species (Fig. 3, supplement 2, D(i) and D(ii)). Finally, we also depleted dPIP4K in Drosophila S2 cells (Fig. 3G(ii)) in culture using two independent dsRNA treatments and found that on insulin stimulation of serum starved cells, the total level of PIP3 was enhanced compared to that in control cells (Fig. 3G(i)); the levels of individuals species of PIP3 underlying this elevation broadly reflected those seen in experiments with Drosophila larval extracts (Fig. 3G (iii)). Together, the observations from these two independent assays (fluorescent probe based PIP3 measurement and mass spectrometry) suggests that in dPIP4K depleted cells, increased amounts of PIP3 are produced at the plasma membrane during insulin stimulation, thus implying that dPIP4K negatively regulates PIP3 production in this setting.
dPIP4K supports TORC1-mediated feedback inhibition on insulin receptor signalling
We had previously reported a systemic reduction in TORC1 activity in dPIP4K29 larvae. It is well understood in mammalian cells that TORC1 activation can mediate feedback inhibition on insulin receptor substrate (IRS) through phosphorylation to suppress insulin signalling. In Drosophila, such feedback inhibition has been partly demonstrated (Kockel et al., 2010), though its precise mechanism is unclear. To understand if there was a relationship between reduced TORC1 output (Gupta et.al, 2013) and the increased insulin-stimulated PIP3 production in dPIP4K29 cells (this study), we studied the effect of tissue-specific manipulation of TORC1 activity on insulin-stimulated PIP3 production in salivary gland cells. For this, we down-regulated Rheb, the GTPase that directly binds and activates TORC1 (Tee et al., 2003). In AB1>RhebRNAi glands, cell size is substantially reduced consistent with the known requirement for TORC1 signalling in regulating cell size (Fig. 4A (i)). Following insulin stimulation of AB1>RhebRNAi glands, PIP3 levels at the plasma membrane were elevated compared to controls (Fig. 4A ii). Conversely, we compared PIP3 production in control cells and those selectively overexpressing Rheb (AB1>dRheb) that is expected to enhance TORC1 signalling activity. Following insulin stimulation, the levels of PIP3 generated were significantly lower in AB1>dRheb glands compared to controls (Fig. 4B (i), (ii)). Similarly, knockdown of TSC, the GTPase activating protein (GAP) for Rheb, expected to result in hyperactivation of Rheb (Zhang et al., 2003), also reduces the PIP3 levels seen post insulin stimulation (Fig. 4C (i), (ii)). Thus, TORC1 output can control plasma membrane PIP3 levels during insulin signaling in salivary gland cells.
We also tested the requirement for dPIP4K in TORC1-mediated control of PIP3 levels during insulin stimulation. When dPIP4K function is reconstituted in salivary glands (AB1>dPIP4K; dPIP4K29), as expected, normal levels of insulin-stimulated PIP3 production were restored (refer Fig. 3D). Knockdown of dRheb in salivary glands resulted in a further elevation of insulin-stimulated PIP3 levels over that seen in dPIP4K29 (Fig. 4D(i), (ii)). However, when dRheb was overexpressed in dPIP4K29 salivary glands; (AB1>dRheb; dPIP4K29), surprisingly, we found that insulin-stimulated PIP3 levels were not lower than in AB1>; dPIP4K29 (Fig. 4E (i), (ii)). Likewise, depletion of TSC in dPIP4K29 (AB1>TscRNAi; dPIP4K29) did not lower insulin stimulated PIP3 levels (Fig. 4F (i), (ii)). Thus, dPIP4K function facilitates TORC1-mediated feedback inhibition of PIP3 levels during insulin stimulation. Upon loss of dPIP4K, the inhibitory action of TORC1-mediated feedback upon insulin signalling is insufficient to generate normal levels of PIP3.
PIP4K is required at the plasma membrane to control of insulin-stimulated PIP3 production
We and others have previously shown that PIP4Ks localize to multiple subcellular membrane compartments (Clarke et al., 2010; Gupta et al., 2013). It is also reported that the substrate for this enzyme i.e. PI5P is present on various organellar membranes inside cells (Sarkes and Rameh, 2010). To further probe the mechanism of regulation of PIP3 levels by dPIP4K, we decided to identify the sub-cellular compartment at which dPIP4K function is required to regulate PIP3 levels. We generated transgenic flies to target dPIP4K to specific subcellular compartments (Fig. 5A). Using unique signal sequences, we targeted dPIP4K specifically to the plasma membrane (Fig. 5B (ii)), endomembrane compartments viz. the ER and Golgi (Fig. 5B (iii)) and the lysosomes (Fig. 5B (iv)). Lysates from S2R+ cells expressing these constructs for assayed for PIP4K activity and we found that all of the targeted dPIP4K enzymes were active (Fig. 5C(i), C(ii)); activity was proportional to the amount of protein expressed. Each of these targeted dPIP4K constructs were selectively reconstituted into dPIP4K null (dPIP4K29) cells and tested for its ability to revert the enhanced insulin-stimulated PIP3 production of dPIP4K29. For this, we stimulated dissected salivary glands ex-vivo with insulin and measured PIP3 production using the GFP::PH-GRP1 probe. Under these conditions, while endomembrane (Fig. 5E) and lysosome-targeted (Fig. 5F) dPIP4K failed to revert the elevated PIP3 levels of dPIP4K29, reconstitution with the plasma-membrane targeted dPIP4K completely restored the elevated PIP3 levels in dPIP4K29 to that of controls (Fig. 5D). Further, overexpression of plasma-membrane targeted dPIP4K in wildtype salivary gland cells resulted in lower insulin stimulated PIP3 levels compared to the controls at 5 mins post insulin stimulation (Fig. 5G). These observations suggest that plasma membrane localized dPIP4K is sufficient to regulate insulin-stimulated PIP3 production.
We also tested the ability of plasma membrane localized PIP4K to regulate PIP3 production during insulin signalling. In a previous study, overexpression of human PIP4K2B in CHO-IR cells was shown to reduce the levels of pAKTT308, an important PIP3 dependent signalling event during insulin stimulation (Carricaburu et al., 2003). We tested the effect of overexpressing plasma membrane restricted PIP4K2B in these cells on pAKTT308 during insulin stimulation. We generated a PIP4K2B construct with a CAAX-motif at its C-terminus (PIP4K2B::mCherryCAAX) that localized the enzyme to the plasma membrane as expected, while the wildtype PIP4K2B (PIP4K2B::eGFP) can be seen at various subcellular compartments (Fig. 5H(i)). CHO-IR cells transiently overexpressing either PIP4K2B::eGFP or PIP4K2B::mCherryCAAX were serum starved, stimulated with insulin and pAKTT308 was measured through immunoblotting. As previously reported, we found that PIP4K2B::eGFP overexpression resulted in a small but significant decrease in pAKTT308 (Fig. 5H’). Interestingly, consistent with our findings in Drosophila larval cells, we observed that over-expressed PIP4K2B::mCherryCAAX also caused a decrease in pAKTT308. In fact, this decrease was achieved despite lower levels of expression of PIP4K2B::mCherryCAAX compared to the wildtype protein. Thus, PIP4K2B activity at the plasma membrane is sufficient to negatively regulate PIP3 dependent pAKTT308 levels in mammalian cells.
dPIP4K alters PIP3 turnover by modifying Class I PI3K activity
PIP3 levels at the plasma membrane upon insulin stimulation also depend on the length of time the receptor remains activated. Our 10-min stimulation protocol was based on earlier studies performed on Drosophila S2 cell-cultures where the response to insulin was maximal at 10 min (Lizcano et al., 2003). However, in order to check for any differences in the dynamics of response to insulin, we also studied the time course of PIP3 elevation following increasing times of insulin stimulation ex-vivo. Comparison of fixed preparations of salivary glands expressing GFP-PH-GRP1 probe showed a comparable time course of PIP3 elevation but higher PIP3 levels at every time point in dPIP4K29 than in control glands (Fig. 6, Supplement 1, (i) and (ii)). To understand the effect of dPIP4K on insulin signaling at the plasma membrane with increased temporal resolution, we developed a live-imaging assay to follow the dynamics of PIP3 turnover using the PH-GRP1 probe in salivary gland cells. A schematic of the reactions involved in the process and the assay protocol is depicted in Fig. 6A(i) and (ii). In this assay, during insulin stimulation, the dynamics of PIP3 turnover has three phases – (i) Rise phase – PIP3 levels increase after a stimulus owing to the activation of PI3K and relatively lower phosphatase activity (ii) Steady-state phase – the opposing kinase and phosphatase activities regulating PIP3 levels balance out each other (iii) Decay phase - PI3K activity is irreversibly inhibited by wortmannin while PIP3 phosphatases remain active. A single experimental trace is shown in Fig. 6B (i); insulin stimulation triggers a rise in PIP3 levels that peak and subsequently decline. Addition of wortmannin prior to addition of insulin abolished insulin stimulated PIP3 production establishing the effectiveness of Class I PI3K inhibition in this assay (Fig. 6B(ii)).
We tested the effect of loss of dPIP4K and tissue-specific overexpression of dPIP4K on PIP3 turnover. Loss of dPIP4K resulted in higher steady state levels of PIP3 in salivary gland cells compared to controls (Fig. 6C) while overexpression of dPIP4K resulted in lower steady-state levels of PIP3 (Fig. 6D). These findings are consistent with the results from our imaging of PIP3 levels from fixed salivary glands of these genotypes (see Fig 3 A(ii) and E). We also analyzed the rate of change in PIP3 levels during the initial phase following insulin stimulation. This analysis clearly revealed an enhanced rate of PIP3 production on loss of dPIP4K29 relative to controls and a reduced rate of PIP3 production in cells overexpressing this enzyme (Fig. 6E(i)). Thus, dPIP4K has the ability to modulate the rate of PIP3 production during insulin stimulation. A similar analysis of the rate of decrease in PIP3 levels during the phase after wortmannin addition (i.e when Class I PI3K activity has been inhibited) showed a marginally slower rate of decay in PIP3 levels in both dPIP4K depleted cells relative to controls but also in cells overexpressing dPIP4K (Fig. 6E(ii)). This finding implies that dPIP4K function is also able to modulate the PIP3 phosphatase activity operative during insulin signalling in Drosophila salivary gland cells although less substantially than its effect on Class I PI3K activity.
dPIP4K function regulates sugar metabolism during larval development
We tested if increased sensitivity to insulin seen in dPIP4K29 had any impact on the physiological response of the animals to sugar intake. It has previously been reported that larvae raised on a high sugar diet (HSD) develop an insulin resistance phenotypes reminiscent of Type II diabetes (Musselman et al., 2011; Pasco and Léopold, 2012). At the level of the organism, this includes reduced body weight, a developmental delay and elevated levels of hemolymph trehalose, the main circulating sugar in insect hemolymph. As previously reported, we found that when grown on HSD (1M sucrose), wild-type larvae show ca. 9 days delay in development compared to animals grown on normal food (0.1M Sucrose) (Fig. 7A). However, interestingly, in dPIP4K29 larvae grown on HSD a delay of only 5 days was seen compared to the same genotype grown on 0.1M sucrose (Fig. 7A). We also biochemically measured the levels of circulating trehalose in the hemolymph of wandering third instar larvae. It was observed that dPIP4K29 larvae raised on normal food, showed circulating trehalose levels are ca. 40% lower compared to controls. Further, when wild-type animals were grown on HSD, circulating trehalose levels in larvae were elevated by ca. 25 % compared to that on normal food (Fig. 7B). However, when dPIP4K29 larvae were raised on HSD, circulating trehalose levels remained essentially unchanged (Fig. 7B) compared to that in animals grown on normal food. Together, these observations suggest that loss of dPIP4K in larvae confers partial protection against phenotypes that arise when challenged with a high sugar diet.
Discussion
The generation of PIP3 is a conserved element of signal transduction by many growth factor receptors. The enzymes that control PIP3 levels during this process, namely Class I PI3K and the lipid phosphatases PTEN and SHIP2 are well studied and the biological consequences of mutations in genes encoding these enzymes underscore the importance of tight regulation of PIP3 levels during growth factor signalling. While the roles of many of the core enzymes that are directly involved in PIP3 metabolism have been studied extensively, the function of proteins that regulate their activity remains less understood; to date regulation of Class I PI3K activity by small GTPases (Ras, Rac) and Gβγ subunits has been described [reviewed in (Hawkins and Stephens, 2015)]. Although a role for PIP4K enzymes in regulating growth factor signalling through PIP3 generation has been reported by several studies (Bulley et al., 2016; Carricaburu et al., 2003; Lamia et al., 2004), the biochemical mechanism and cell-biological context in which they do so has remained obscure. PIP4Ks convert PI5P to PI(4,5)P2 but to date no study has found a role for PIP4K in regulating overall cellular PI(4,5)P2 levels [reviewed in (Kolay et al., 2016)]. One possibility that has been raised is that PIP4Ks may generate the PI(4,5)P2 pool from which PIP3 is produced by Class I PI3K activity. Although PI5P, the preferred substrate of PIP4K, is a low abundance lipid, in principle, it is possible that a small, local pool of PI(4,5)P2 is generated from PI5P by PIP4K an d the loss of this small pool of PI(4,5)P2 is not detected by the mass assays for estimating total cellular levels of this lipid. Quantitatively, based on their relative abundance, the small PIP4K generated pool of PI(4,5)P2 is likely to be sufficient to serve as the substrate for PIP3 generation by Class I PI3K. A recent study (Bulley et al., 2016) has reported that PIP3 levels are reduced in immortalized B-cells in which PIP4K2A activity is down regulated. By contrast, it has been previously reported that loss of PIP4K2B in mice results in increased levels of insulin signalling readouts such as pAKT308 that are direct correlates of PIP3 levels (Lamia et al., 2004). The exact reasons for these conflicting results is unclear and may include the different cell types used in each study; a key reason is likely to be the overlapping function of the three PIP4K isoforms present in mammalian genomes. In this study, we found that in Drosophila, that contains only a single gene encoding PIP4K activity (dPIP4K)(Balakrishnan et al., 2015; Gupta et al., 2013), the levels of plasma membrane PIP3 in cells lacking dPIP4K were elevated compared to controls. We established this finding using both a fluorescent reporter for plasma membrane PIP3 in single cell assays using multiple cell types and also using lipid mass spectrometry across larval tissues and cultured, dPIP4K depleted Drosophila S2 cells. Thus, our study clearly demonstrates that in Drosophila, dPIP4K function is a negative regulator of PIP3 production during growth factor stimulation. The elevated PIP3 levels seen when dPIP4K is depleted are not consistent with a role for this enzyme in generating the PI(4,5)P2 at the plasma membrane used by Class I PI3K as substrate to generate PIP3 during insulin signalling. Therefore, is likely that dPIP4K regulates PIP3 levels through its ability to control the function of proteins that themselves regulate PIP3 levels during Class I PI3K signalling.
In an earlier study (Gupta et al., 2013), we had observed dPIP4K29 larvae to have systemically reduced levels of TOR activation. In mammalian cells, reducing TOR activity through the use of rapamycin or a loss of S6K, a direct target of TORC1, leads to increased activation of insulin signalling pathway and obesity resistance which was associated with increased insulin sensitivity (Haruta et al., 2000; Reilly et al., 2011; Um et al., 2004). It is also reported that S6K inactivates IRS-1 by phosphorylating it on multiple serine residues (Gual et al., 2005; Tremblay et al., 2007). Therefore, it is reasonable to hypothesize a scenario where the reduced TORC1 activity in dPIP4K29 cells may be the defect that drives the increase in the levels of PIP3 in dPIP4K29. An alternative explanation of our observations is that loss of dPIP4K could uncouple the negative feedback from TORC1 activity to PIP3 generation at the plasma membrane. In this study, we found that in wild-type larval cells, modulating TORC1 activity could tune PIP3 levels during insulin stimulation (Fig 4 A-C); enhancing TORC1 output resulted in lower levels of insulin-induced PIP3 whereas reducing TORC1 activity caused higher levels of PIP3. By contrast, overexpression of Rheb or the down-regulation of Tsc1/2 was not able to revert the elevated plasma membrane PIP3 levels in dPIP4K29 cells (Fig 4 E-F) although they were able to restore the reduced cell size in dPIP4K29(Fig. 4, Supplement 1, (A) and (B)). These results imply two conclusions: 1) Decreased TORC1 activity is not sufficient to explain the enhanced PIP3 levels in dPIP4K29 larval cells and 2) Efficient feedback regulation of PIP3 levels by TORC1 outputs following Rheb activation requires intact dPIP4K function.
Binding of insulin to its receptor triggers a signalling cascade where the initial events occur at the plasma membrane. These involve interaction of the activated insulin receptor-ligand complex with IRS followed by the recruitment and activation of Class I PI3K at the plasma membrane. At which sub-cellular location is dPIP4K activity required to regulate this process? Fractionation and immunolocalization studies in mammalian cells (Clarke et al., 2010) and Drosophila (Gupta et al., 2013) have indicated that PIP4K isoforms are distributed across multiple subcellular compartments including the plasma membrane, nucleus and internal vesicular compartments. In this study, using selective reconstitution of the dPIP4K to specific membrane compartments, in cells devoid of any endogenous PIP4K protein, we found that plasma membrane targeted dPIP4K could rescue the elevated PIP3 levels in dPIP4K null cells. This observation strongly suggests that the plasma membrane localized dPIP4K is sufficient to control PIP3 production during insulin stimulation. Our observation that dPIP4K29 cells were hypersensitive to overexpression of dINR or chico compared to wild-type cells likely reflects the loss of a dPIP4K dependent event in the control of PIP3 levels at the plasma membrane. Overexpression of plasma-membrane localized PIP4K2B was able to reduce pAKT308 phosphorylation upon insulin stimulation in mammalian cells just as well as the wildtype PIP4K2B enzyme. Our finding of a role for the plasma membrane localized PIP4K in regulating PIP3 levels in both Drosophila and mammalian cells underscores the evolutionarily conserved nature of this mechanism. Previous studies have shown that levels of PI5P, the substrate for PIP4Ks, increases upon insulin stimulation and importantly, addition of exogenous PI5P can stimulate glucose uptake in a PI3K-dependent manner (Grainger et al., 2011; Jones et al., 2013). Therefore, plasma membrane localized PIP4K and the levels of its substrate PI5P could be a mechanism by which early events during insulin signalling are regulated.
What molecular event involved in PIP3 turnover might dPIP4K regulate at the plasma membrane? Using live cell imaging studies of PIP3 turnover at the plasma membrane coupled with chemical inhibition of Class I PI3K, we were able to observe that dPIP4K function has a substantial impact on the rate of PIP3 production following insulin stimulation whereas the rate of PIP3 degradation was only marginally affected. This finding suggests that dPIP4K likely regulates the activity of Class I PI3K either directly or by controlling its coupling to the activated insulin receptor complex at the plasma membrane; the mechanism by which it does so remains to be established.
What might be the physiological consequence of losing dPIP4K mediated feedback control on PIP3 production in the context of insulin signalling? Previous studies in mouse and human cells have reported that excessive activation of TORC1 signalling leads to inactivation of insulin signalling pathway and development of insulin resistance (Harrington et al., 2004; Shah et al., 2004; Tzatsos and Kandror, 2006). Since TORC1 activity is reduced (Gupta et al., 2013) and PIP3 were elevated (this study) in animals lacking dPIP4K, it is likely that loss of dPIP4K impacts sugar metabolism in Drosophila larvae. Using a recently reported high-sugar induced obesity and Type II diabetes-like disease model in Drosophila (Musselman et al., 2011), we found that dPIP4K29 larvae appear resistant to a high sugar diet as measured by the elevation in the hemolymph trehalose levels and they were relatively resistant to the developmental delay seen when wild-type larvae are reared on a high-sugar diet. This observation is reminiscent of that reported for the PIP4K2B-/- mice that have a reduced adult body weight compared to controls and clear blood glucose faster following a sugar bolus than control animals (Lamia et al., 2004). Our observation that dPIP4K at the plasma-membrane controls sensitivity to insulin receptor activation suggests a molecular basis for the physiological phenotypes observed in dPIP4K29 larvae and PIP4K2B-/- mice. These observations also raise the possibility that inhibition of PIP4K activity may offer a route to reducing insulin resistance in the context of Type II diabetes. Such a mechanism may explain the hyperactivation of the T-cell receptor responses in mice lacking PIP4K2C (Shim et al., 2016), since the activation of Class I PI3K is a key element of T-cell receptor signal transduction. More generally, PIP4K activity likely offers a novel element of regulation for Class I PI3K activity in the context of receptor tyrosine kinase signalling.
Materials and Methods
Drosophila strains and rearing
Unless indicated, flies were grown on standard fly medium containing corn meal, yeast extract, sucrose, glucose, agar and antifungal agents. For all experiments, crosses were setup at 25°C in vials/bottles under non-crowded conditions.
Fly medium composition:
The following stocks were used in the study: wildtype strain Red Oregon R (ROR), AB1-Gal4 (Bloomington # 1824), UAS-dInR (Bloomington # 8262), UAS-RhebRNAi (Bloomington TRiP # 33966), UAS-Rheb (Bloomington # 9688), UAS-TSCRNA i(Bloomington TRiP # 52931), P{tGPH}4 (Bloomington # 8164), UAS-dPIP4KRNAi (Bloomington TRiP # 65891). UAS-dPIP4K::eGFP and dPIP4K29 were generated in the lab and described in (Gupta et al., 2013). For PIP3 measurements in the dPIP4K29 rescue experiment (Fig. 4F) using GFP-PH-GRP1 probe, we cloned dPIP4K cDNA (BDGP clone# LD10864) into pUAST-attB between EcoRI and XhoI sites without the GFP tag. The generation of flies expressing dPIP4K::-mCherry-CAAX is described in Kumari K et.al, 2017. For targeting dPIP4K to the endomembranes, the sequence QGSMGLPCVVM (Sato.M et. al., 2006) replaced the CAAX motif in the dPIP4K::mCherry-CAAX construct. To generate Lysosomal-dPIP4K::eGFP, the 39 amino-acid sequence from p18/LAMTOR (Menon S et.al., 2014) was used as a signal sequence. The signal sequence was commercially synthesized with a C-terminal flag tag and introduced upstream of dPIP4K::eGFP. The entire fusion construct was cloned into pUAST-attB by GIBSON assembly using NotI and XbaI sites. All transgenic lines were generated using insertions that were performed using site-specific recombination. The level of GFP fluorescence from lysosomal-dPIP4K::eGFP was observed to be very low in the salivary glands and did not interfere with our analysis PIP3 measurements using the GFP-PH-GRP1 probe in Fig. 6F.
Cell Culture, dsRNA treatment and Insulin stimulation assays
CHO cell line stably expressing insulin receptor (isoform A) was a kind gift from Dr Nicholas Webster, UCSD. These were maintained at standard conditions in HF12 culture medium supplemented with 10% Fetal bovine serum and under G418 selection (400 µg/ml). Transfections were done 48 hrs. before the assay using FuGene, Promega Inc. as per manufacturer’s protocols when the cultures were 50% confluent. For insulin stimulation assays, cells were starved overnight in HF12 medium without serum. Thereafter, the cells were de-adhered, collected into eppendorf tubes and stimulated with 1 µM insulin for indicated times. Post stimulation, cells were spun down and immediately lysed. For dsRNA treatments, 0.5 X 106 cells were seeded into a 24-well plate. Once observed to be settled, cells were incubated with incomplete medium containing 1.875 µg of dsRNA. After 1 hour, an equal amount of complete medium was added to each well. The same procedure was repeated on each well 48 hours after initial transfection after removal of the spent medium from each well. Cells were harvested by trypsinization after a total of 96 hours of dsRNA treatment. For mass spectrometric estimation of PIP3, S2R+ cells were pelleted down and stimulated with 1 µM insulin for 10 min. The reaction was stopped by the addition of ice-cold initial organic mix (described later in the section) and used for lipid extraction.
Larval growth Curve Analysis -
Adult flies were made to lay eggs within a span of 4-6 hrs on normal food. After 24 hrs, newly hatched first instar larvae were collected and transferred in batches of about 15-25 larvae per into vials containing either 0.1/1M Sucrose in the fly media with other components unaltered. The vials were then observed to count the number of pupae.
Hemolymph Trehalose Measurements
The measurements were done exactly as described in (Musselman et al., 2011). In brief, hemolymph was pooled from five to eight larvae to obtain 1 μl for assay. The reagents used porcine trehalase (SIGMA, T8778) and GO kit (SIGMA, GAGO20)
Cell size analysis in salivary glands-
Salivary glands were dissected from wandering third instar larvae and fixed in 4% paraformaldehyde for 30 min at 4°C. Post fixation, glands were washed thrice with 1X PBS and incubated in BODIPY-FL-488 for 3 hours at room temperature. The glands were washed thrice in 1X PBS following which nuclei were labelled (using either DAPI or TOTO3) for 10 mins at room temperature and washed with 1X PBS again. The glands were then mounted in 70% glycerol and imaged within a day of mounting. Imaging was done on Olympus FV1000 Confocal LSM using a 20x objective. The images were then stitched into a 3D projection using an ImageJ plugin. These reconstituted 3D z-stacks were then analyzed for nuclei numbers (correlate for cell number) and volume of the whole gland using Volocity Software (version 5.5.1, Perkin Elmer Inc.). The average cell size was calculated as the ratio of the average volume of the gland to the number of nuclei.
Ex vivo insulin stimulation and PIP3 measurements in salivary glands and fat body-
For experiments with salivary glands, wandering third instar larvae were dissected one larva at a time and glands were immediately dropped into a well of a 96-well plate containing either only PBS or PBS + 10 µM Insulin (75 µl) and incubated for 10 min at RT. Following this, 25 µl of 16% PFA was added into the same well to yield a final conc. of 4% PFA. The glands were fixed in this solution for 18 min at room temperature and then transferred sequentially to wells containing PBS every 10 min for 3 washes. Finally, glands were mounted in 80% glycerol in PBS containing antifade (0.4% propyl-gallate). For experiments with fat body lobes, late third instar feeding larvae were starved by placing them on a filter paper soaked in 1X PBS for 2 hrs. Thereafter, the incubation, fixation and mounting steps were done exactly as described for salivary glands. Imaging was done on LSM 780 inverted confocal microscope with a 20X/0.8 NA Plan Apochromat objective. For quantification, confocal slices were manually curated to generate maximum z-projections of middle few planes of cells. Thereafter, line profiles were drawn across clearly identifiable plasma membrane regions and their adjacent cytosolic regions and ratios of mean intensities for these line profiles were calculated for each cell. For salivary glands, about 10-15 cells from multiple glands were analyzed and used to generate statistics. For fat body, about 50 cells each from multiple animals were used for analysis.
For live imaging, salivary glands from wandering third instar larvae were dissected (glands from one larva imaged in one imaging run) and placed inside a drop of imaging buffer (1X PBS containing 2mg/ml glucose) on a coverglass. The buffer was carefully and slowly soaked out with a paper tissue to let the glands settle and adhere to the surface. Thereafter, the glands were immediately rehydrated with 25 µl of imaging buffer. The imaging was done on Olympus FV3000 LSM confocal system using a 10X objective. A total of 80 frames of a single plane were acquired, with 10s intervals. While imaging, 25 µl of 20uM (2X) bovine insulin was used to stimulate the glands. After the steady state was achieved, 50 µl of 800nM (2X) of wortmannin was added on top to inhibit PI3K activity.
PIP3 measurement by LC-MS/MS
The method was adopted and modified as required from (Clark et al., 2011).
Lipid extraction
5 larvae were dissected in 1X PBS and transferred immediately into 37.5 µl of 1X PBS in a 2 ml Eppendorf. For insulin stimulation, to this, 37.5 µl of 100 µM Insulin (final concentration – 50 µM) was added and the tube was incubated on a mix mate shaker for 10 min at 500 rpm. At the end of incubation time, 750 µl of ice-cold 2:1 MeOH:CHCl3 organic mix was added to stop the reaction. Part of this solution was decanted and the rest of the mix containing larval tissues was transferred into a homogenization tube. Larval tissues were homogenized in 4 cycles of 10 secs with 30 sec intervals at 6000 rpm in a homogenizer (Precellys, Bertin Technologies). The tubes were kept on ice at all intervals. The entire homogenate was then transferred to a fresh eppendorf and the homogenization tube was then washed with the decanted mix kept aside earlier. 120 µl of water was added to the homogenate collected in eppendorf, followed by addition of 5 ng of 17:0, 20:4 PIP3 internal standard (ISD). The mixture was vortexed and 725 µl of chloroform was added to it. After vortexing again for 2 min at around 1000-1500 rpm, the phases were separated by centrifugation for 3 min at 1500g. 1ml of lower organic phase was removed and stored in a fresh tube. To the remaining aqueous upper phase, again 725 µl of chloroform was added. The mixture was vortexed and spun down to separate the phases. Again, 1 ml of the organic phase was collected and pooled with the previous collection (total of 2ml). This organic phase was used for measuring total organic phosphate. To the aqueous phase, 500 µl of the initial organic mix was added followed by 170 µl of 2.4M HCl and 500 µl of CHCl3. This mixture was vortexed for 5 min at 1000-1500 rpm and allowed to stand at room temperature for 5 minutes. The phases were separated by centrifugation (1500g, 3 min). The lower organic phase was collected into a fresh tube by piercing through the protein band sitting at the interface. To this, 708 µl of lower phase wash solution was added, the mixture was vortexed and spun down (1500g, 3 min). The resultant lower organic phase was completely taken out carefully into an Eppendorf tube and used for derivatization reaction.
Extraction solvent mixtures
Initial organic mix: MeOH/Chloroform in the ratio of 484/242 ml, Lower Phase Wash Solution: Methanol/1 M hydrochloric acid/ chloroform in a ratio of 235/245/15 ml. All ratios are expressed as vol/vol/vol.
Derivatization of Lipids
To the organic phase of the sample, 50 µl of 2M TMS-Diazomethane was added (TO BE USED WITH ALL SAFETY PRECAUTIONS!). The reaction was allowed to proceed at room temperature for 10 min at 600 rpm. After 10 min, 10 µl of Glacial acetic acid was added to quench the reaction, vortexed briefly and spun down. 700 µl of post derivatization wash solvent was then added to the sample, vortexed (2 min, 1000-1500 rpm) and spun down. The upper aqueous phase was discarded and the wash step was repeated. To the final organic phase, 100 µl of 9:1 MeOH:H2O mix was added and the sample was dried down to about 10-15 µl in a speedvac under vacuum.
Chromatographic separation and Mass spectrometric detection
The larval lipid extracts were re-suspended in 170 µl LC-MS grade methanol and 30 µl LC-MS grade water. Samples were injected as duplicate runs of 3.5 µl. Chromatographic separation was performed on an Acquity UPLC BEH300 C4 column (100 x 1.0 mm; 1.7 µm particle size) purchased from Waters Corporation, USA on a Waters Aquity UPLC system and detected using an ABSCIEX 6500 QTRAP mass spectrometer. The flow rate was 100 µL/min. Gradients were run starting from 55% Buffer A (Water + 0.1% Formic Acid)- 45% Buffer B (Acetonitrile + 0.1% Formic acid) to 42% B from 0-5 min; thereafter 45% B to 100% B from 5-10 min; 100% B was held from 10-15 min; brought down from 100% B to 45% B between 15-16 min and held there till 20th min to re-equilibrate the column. On the mass spectrometer, in pilot standardization experiments, we first employed Neutral Loss Scans on biological samples to look for parent ions that would lose neutral fragments of 598 a.m.u indicative of PIP3 lipid species (as described in (Clark et al., 2011)). Thereafter, these PIP3 species were quantified in biological samples using the selective Multiple Reaction Monitoring (MRM) method in the positive mode. Only those MRM transitions that showed an increase upon insulin stimulation of biological samples were used for the final experiments (depicted in figure S3B). The MRM transitions for the different PIP3 species quantified are listed out in the table below. Area of all the peaks was calculated on Sciex MultiQuant software. The area of the internal standard peak was used to normalize for lipid recovery during extraction. The normalized for each of the species was then divided by the amount of organic phosphate measured in each of the biological samples. The other mass spectrometer parameters are as follows: ESI voltage: +4500V; Dwell time: 40 ms; DP (De-clustering Potential): 35.0 V; EP: (Entrance Potential): 10.1 V, CE (Collision Energy): 47.0 V; CXP (Collision cell Exit Potential): 11.6 V, Source Temperature : 450 C, Ion Spray Voltage – 4000 V, Curtain Gas : 35.0, GS1: 15, GS2: 16. The area under the peaks was extracted using MultiQuant v1.1 software (ABSCIEX). Numerical analysis was done in Microsoft Excel.
Total Organic Phosphate measurement
1 ml of the organic phase from each sample was taken into phosphate-free tubes and dried completely at 90°C. The remaining steps were performed as described in Thakur et.al., 2016.
Preparation of S2R+ cell lysate for in vitro PI5P 4- kinase assay
The S2R+ cells were pelleted at 1000g for 10 min and washed with ice-cold PBS Twice. Cells were thereafter homogenized in lysis buffer containing 50mM Tris-Cl, pH – 7.5, 1mM EDTA, 1mM EGTA, 1% Triton-X-100, 50mM NaF, 0.27 M Sucrose, 0.1% β- Mercaptoethanol and freshly added protease and phosphatase inhibitors (Roche). The lysate was then centrifuged at 1000g for 15 min at 4 °C. Protein estimation was performed using the Bradford reagent according to the manufacturer’s instructions.
PI5P4-kinase Assay
Vacuum-dried substrate lipid (6 μM PI5P) and 20 μM of phosphatidylserine were resuspended in 10 mM Tris pH 7.4 and micelles were formed by sonication for 2 min in a bath-sonicator. 50 µl of 2× PIPkinase reaction buffer (100 mM Tris pH 7.4, 20 mM MgCl2, 140 mM KCl, and 2 mM EGTA) containing 20 µM ATP, 5 µCi [γ-32P] ATP and cell lysates containing ∼10 µg total protein was added to the micelles. The reaction mixture was incubated at 30 °C for 16 h. Lipids were extracted and resolved by one dimensional TLC (45:35:8:2 chloroform: methanol: water: 25% ammonia). The resolved lipids were imaged using phosphorImager.
Western Blotting
For larval western blots, lysates were prepared by homogenizing 3 wandering third instar larvae or 5 pairs of salivary glands from third instar larvae. In the case of CHO-IR cells, pelleted cells were lysed by repeated pipetting in lysis buffer (same as described above). Thereafter, the samples were heated at 95°C with Laemli loading buffer for 5 min and loaded onto an SDS-Polyacrylamide gel. The proteins were subsequently transferred onto a nitrocellulose membrane and incubated with indicated antibodies overnight at 4°C (for actin/tubulin incubation was done at room temperature for 3 hrs.). Primary antibody concentrations used were – anti-α-actin (SIGMA A5060) 1:1000; anti-dPIP4K 1:1000, anti – GAPDH (Novus Biologicals, #IM-5143A), anti-PIP4KB (Cell Signaling, #9694), anti – pAKTT308 (Cell Signaling, #9275), anti-AKT (Cell Signaling, # 9272). The blots were then washed thrice with Tris Buffer Saline containing 0.1% Tween-20 (0.1% TBS-T) and incubated with 1:10000 concentration of appropriate HRP-conjugated secondary antibodies (Jackson Laboratories, Inc.) for 1.5 hrs. After three washes with 0.1% TBS-T, blots were developed using Clarity Western ECL substrate on a GE ImageQuant LAS 4000 system.
Acknowledgements
This work was supported by the National Centre for Biological Sciences, TIFR, Department of Biotechnology, Ministry of Science and Technology (India); and a Wellcome Trust-DBT India Alliance Senior Fellowship to PR. S.S is a recipient of the S.P Mukherji Fellowship from CSIR and S.M an ICMR Fellowship. We thank the NCBS Drosophila, Imaging and Lipidomics Facility for support.