Type II phosphatidylinositol 4-kinases function sequentially in cargo delivery from early endosomes to melanosomes.

Melanosomes are pigment cell-specific lysosome-related organelles in which melanin pigments are synthesized and stored. Melanosome maturation requires delivery of melanogenic cargoes via tubular transport carriers that emanate from early endosomes and that require BLOC-1 for their formation. Here we show that phosphatidylinositol-4-phosphate (PtdIns4P) and the type II PtdIns-4-kinases (PI4KIIα and PI4KIIß) support BLOC-1-dependent tubule formation to regulate melanosome biogenesis. Depletion of either PI4KIIα or PI4KIIß with shRNAs in melanocytes reduced melanin content and misrouted BLOC-1-dependent cargoes to late endosomes/lysosomes. Genetic epistasis, cell fractionation, and quantitative live-cell imaging analyses show that PI4KIIα and PI4KIIß function sequentially and non-redundantly downstream of BLOC-1 during tubule elongation toward melanosomes by generating local pools of PtdIns4P. The data show that both type II PtdIns-4-kinases are necessary for efficient BLOC-1-dependent tubule elongation and subsequent melanosome contact and content delivery during melanosome biogenesis. The independent functions of PtdIns-4-kinases in tubule extension are downstream of likely redundant functions in BLOC-1-dependent tubule initiation.

PI4KIIIß Activity Regulates Lateral Root Formation Driven by Endocytic Trafficking to the Vacuole.

Lateral roots (LRs) increase the contact area of the root with the rhizosphere and thereby improve water and nutrient uptake from the soil. LRs are generated either via a developmentally controlled mechanism or through induction by external stimuli, such as water and nutrient availability. Auxin regulates LR organogenesis via transcriptional activation by an auxin complex receptor. Endocytic trafficking to the vacuole positively regulates LR organogenesis independently of the auxin complex receptor in Arabidopsis (Arabidopsis thaliana). Here, we demonstrate that phosphatidylinositol 4-phosphate (PI4P) biosynthesis regulated by the phosphatidylinositol 4-kinases PI4KIIIß1 and PI4KIIIß2 is essential for the LR organogenesis driven by endocytic trafficking to the vacuole. Stimulation with Sortin2, a biomodulator that promotes protein targeting to the vacuole, altered PI4P abundance at both the plasma membrane and endosomal compartments, a process dependent on PI4K activity. These findings suggest that endocytic trafficking to the vacuole regulated by the enzymatic activities of PI4KIIIß1 and PI4KIIIß2 participates in a mechanism independent of the auxin complex receptor that regulates LR organogenesis in Arabidopsis. Surprisingly, loss-of-function of PI4KIIIß1 and PI4KIIIß2 induced both LR primordium formation and endocytic trafficking toward the vacuole. This LR primordium induction was alleviated by exogenous PI4P, suggesting that PI4KIIIß1 and PI4KIIIß2 activity constitutively negatively regulates LR primordium formation. Overall, this research demonstrates a dual role of PI4KIIIß1 and PI4KIIIß2 in LR primordium formation in Arabidopsis.

Bioactive lipids are altered in the kidney of chronic undernourished rats: is there any correlation with the progression of prevalent nephropathies?

BACKGROUND: Undernutrition during childhood leads to chronic diseases in adult life including hypertension, diabetes and chronic kidney disease. Here we explore the hypothesis that physiological alterations in the bioactive lipids pattern within kidney tissue might be involved in the progression of chronic kidney disease. METHODS: Membrane fractions from kidney homogenates of undernourished rats (RBD) were submitted to lipid extraction and analysis by thin layer chromatography and cholesterol determination. RESULTS: Kidneys from RBD rats had 25% lower cholesterol content, which disturb membrane microdomains, affecting Ca2+ homeostasis and the enzymes responsible for important lipid mediators such as phosphatidylinositol-4 kinase, sphingosine kinase, diacylglicerol kinase and phospholipase A2. We observed a decrease in phosphatidylinositol(4)-phosphate (8.8 ± 0.9 vs. 3.6 ± 0.7 pmol.mg-1.mim-1), and an increase in phosphatidic acid (2.2 ± 0.8 vs. 3.8 ± 1.3 pmol.mg-1.mim-1), being these lipid mediators involved in the regulation of key renal functions. Ceramide levels are augmented in kidney tissue from RBD rats (18.7 ± 1.4 vs. 21.7 ± 1.5 fmol.mg-1.min-1) indicating an ongoing renal lesion. CONCLUSION: Results point to an imbalance in the bioactive lipid generation with further consequences to key events related to kidney function, thus contributing to the establishment of chronic kidney disease.

Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231.

Golgi phosphoprotein 3 (GOLPH3) has been implicated in the development of carcinomas in many human tissues, and is currently considered a bona fide oncoprotein. Importantly, several tumor types show overexpression of GOLPH3, which is associated with tumor progress and poor prognosis. However, the underlying molecular mechanisms that connect GOLPH3 function with tumorigenicity are poorly understood. Experimental evidence shows that depletion of GOLPH3 abolishes transformation and proliferation of tumor cells in GOLPH3-overexpressing cell lines. Conversely, GOLPH3 overexpression drives transformation of primary cell lines and enhances mouse xenograft tumor growth in vivo. This evidence suggests that overexpression of GOLPH3 could result in distinct features of GOLPH3 in tumor cells compared to that of non-tumorigenic cells. GOLPH3 is a peripheral membrane protein mostly localized at the trans-Golgi network, and its association with Golgi membranes depends on binding to phosphatidylinositol-4-phosphate. GOLPH3 is also contained in a large cytosolic pool that rapidly exchanges with Golgi-associated pools. GOLPH3 has also been observed associated with vesicles and tubules arising from the Golgi, as well as other cellular compartments, and hence it has been implicated in several membrane trafficking events. Whether these and other features are typical to all different types of cells is unknown. Moreover, it remains undetermined how GOLPH3 acts as an oncoprotein at the Golgi. Therefore, to better understand the roles of GOLPH3 in cancer cells, we sought to compare some of its biochemical and cellular properties in the human breast cancer cell lines MCF7 and MDA-MB-231 with that of the non-tumorigenic breast human cell line MCF 10A. We found unexpected differences that support the notion that in different cancer cells, overexpression of GOLPH3 functions in diverse fashions, which may influence specific tumorigenic phenotypes.

Biphasic regulation of type II phosphatidylinositol-4 kinase by sphingosine: cross talk between glycero- and sphingolipids in the kidney.

Phosphatidylinositol-4 kinase (PI-4K) is responsible for the generation of phosphatidylinositol-4 phosphate (PtdIns(4)P), a bioactive signaling molecule involved in several biological functions. In this study, we show that sphingosine modulates the activity of the PI-4K isoform associated with the basolateral membranes (BLM) from kidney proximal tubules. Immunoblotting with an anti-α subunit PI-4K polyclonal antibody revealed the presence of two bands of 57 and 62kDa in the BLM. BLM-PI-4K activity retains noteworthy biochemical properties; it is adenosine-sensitive, not altered by wortmanin, and significantly inhibited by Ca(2+) at the µM range. Together, these observations indicate the presence of a type II PI-4K. Endogenous phosphatidylinositol (PI) alone reaches PI-4K half-maximal activity, revealing that even slight modifications in PI levels at the membrane environment promote significant variations in BLM-associated-PI-4K activity. ATP-dependence assays suggested that the Mg.ATP(2-) complex is the true substrate of the enzyme and that free Mg(2+) is an essential cofactor. Another observation indicated that higher concentrations of free ATP are inhibitory. BLM-associated-PI-4K activity was ~3-fold stimulated in the presence of increasing concentration of sphingosine, while in concentrations higher than 0.4mM, in which S1P is pronouncedly formed, there was an inhibitory effect on PtdIns(4)P formation. We propose that a tightly coupled regulatory network involving phosphoinositides and sphingolipids participate in the regulation of key physiological processes in renal BLM carried out by PI-4K.

Cloning of a phosphatidylinositol 4-kinase gene based on fiber strength transcriptome QTL mapping in the cotton species Gossypium barbadense.

Sea Island cotton (Gossypium barbadense) is highly valued for its superior fiber qualities, especially fiber strength. Based on a transcript-derived fragment originated from transcriptome QTL mapping, a fiber strength related candidate gene of phosphatidylinositol 4-kinase cDNA, designated as GbPI4K, was first cloned, and its expression was characterized in the secondary cell wall thickening stage of G. barbadense fibers. The ORF of GbPI4K was found to be 1926 bp in length and encoded a predicted protein of 641 amino acid residues. The putative protein contained a clear PI3/4K kinase catalytic domain and fell into the plant type II PI4K cluster in phylogenetic analysis. In this study, the expression of cotton PI4K protein was also induced in Escherichia coli BL21 (DE3) as a fused protein. Semi-quantitative RT-PCR analysis showed that the gene expressed in the root, hypocotyl and leaf of the cotton plants. Real-time RT-PCR indicated that this gene in Sea Island cotton fibers expressed 10 days longer than that in Upland cotton fibers, and the main expression difference of PI4K between Sea Island cotton and Upland cotton in fibers was located in the secondary cell wall thickening stage of the fiber. Further analysis indicated that PI4K is a crucial factor in the ability of Rac proteins to regulate phospholipid signaling pathways.

Comparative phytohormone profiles, lipid kinase and lipid phosphatase activities in barley aleurone, coleoptile, and root tissues.

We analyzed lipid kinase and lipid phosphatase activities and determined endogenous phytohormone levels by liquid chromatography-tandem mass spectrometry in root and coleoptile tissues following germination of barley (Hordeum vulgare) seeds. The enzymes showing highest activity in aleurone cells were diacylglycerol kinase (DAG-k, EC 2.7.1.107) and phosphatidate kinase (PA-k). The ratio of gibberellins (GAs) to abscisic acid (ABA) was 2-fold higher in aleurone than in coleoptile or root tissues. In coleoptiles, phosphatidylinositol 4-kinase (PI4-k, EC 2.7.1.67) showed the highest enzyme activity, and jasmonic acid (JA) level was higher than in aleurone. In roots, activities of PI4-k, DAG-k, and PA-k were similar, and salicylic acid (SA) showed the highest concentration. In the assays to evaluate the hydrolysis of DGPP (diacylglycerol pyrophosphate) and PA (phosphatidic acid) we observed that PA hydrolysis by LPPs (lipid phosphate phosphatases) was not modified; however, the diacylglycerol pyrophosphate phosphatase (DGPPase) was strikingly higher in coleoptile and root tissues than to aleurone. Relevance of these findings in terms of signaling responses and seedling growth is discussed.

CaV2.1 channels are modulated by muscarinic M1 receptors through phosphoinositide hydrolysis in neostriatal neurons.

In adult neostriatal projection neurons, the intracellular Ca(2+) supplied by Ca(V)2.1 (P/Q) Ca(2+) channels is in charge of both the generation of the afterhyperpolarizing potential (AHP) and the release of GABA from their synaptic terminals, thus being a major target for firing pattern and transmitter release modulations. We have shown that activation of muscarinic M(1)-class receptors modulates Ca(V)2.1 channels in these neurons in rats. This modulation is reversible, is not membrane delimited, is blocked by the specific M(1)-class muscarinic antagonist muscarine toxin 7 (MT-7), and is neither mediated by protein kinase C (PKC) nor by protein phosphatase 2B (PP-2B). Hence, the signaling mechanism of muscarinic Ca(V)2.1 channel modulation has remained elusive. The present paper shows that inactivation of phospholipase C (PLC) abolishes this modulation while inhibition of phosphoinositide kinases, PI-3K and PI-4K, prevents its reversibility, suggesting that the reconstitution of muscarinic modulation depends on phosphoinositide rephosphorylation. In support of this hypothesis, the supply of intracellular phosphatidylinositol (4,5) bisphosphate [PI(4,5)P(2)] blocked all muscarinic modulation of this channel. The results indicate that muscarinic M(1) modulation of Ca(V)2.1 Ca(2+) channels in these neurons involves phosphoinositide hydrolysis.

Phosphatidylinositol kinases as regulators of GA-stimulated alpha-amylase secretion in barley (Hordeum vulgare).

Phosphorylated derivatives of phosphatidylinositol, in association with phosphatidylinositol 3-kinase (PI3 kinase, EC 2.7.1.137) and phosphatidylinositol 4-kinase (PI4 kinase, EC 2.7.1.67), play a key role in regulation of fundamental cell processes. We present evidence for a relationship between alpha-amylase (EC 3.2.1.1) secretion regulated by GA and levels of phosphatidylinositol 3-phosphate and phosphatidylinositol 4-phosphate (PtdIns(4)P) in barley (Hordeum vulgare). Microsomal membranes were incubated in the presence of [gamma-(32)P]ATP, and radiolabeled membrane lipids were extracted and separated by TLC using a boric acid system. Treatment of aleurone layers with GA for short or long periods of time increased PI4 kinase activity. To evaluate the effect of PtdIns(4)P levels on GA signaling, we used phenylarsine oxide (PAO), an inhibitor of PI4 kinase activity. PAO reversibly reduced the alpha-amylase secretion and protoplast cell vacuolation in a dose-dependent manner. Wortmannin showed a similar inhibitory effect on alpha-amylase secretion and PI4 kinase activity. GA evoked only a long-term increase in PI3 kinase activity, which was also affected by PAO. The effect of PAO was suppressed by the reducing agent 2,3-dimercapto-1-propanol (BAL), leading to restoration of secretion, vacuolation and PI4 kinase activity. In contrast, the effect of PAO on PI3 kinase activity was not abolished by BAL, suggesting that PI3 kinase is not involved in the secretion process. Likewise, the compound LY294002 inhibited PI3 kinase but had no effect on the secretion process. These findings indicate that PI4 kinase acts as a positive regulator of early GA signaling in aleurone.

Organophosphorous and organochlorine pesticides affect human placental phosphoinositides metabolism and PI-4 kinase activity.

The objective of this work was to describe the effect of organophosphorous and organochlorine pesticides on phosphoinositides metabolism in human placenta. Pesticides concentration (10 microM) was used for in vitro incubations of cell-free homogenates labelled with (32)P orthophosphate. Heptachlor (HC) and dichloro-diphenyl-trichloroethane (o-p' DDT) increased phosphatidyl-inositol, phosphatidylinositolphosphate, and phosphatidyl-inositolbiphosphate phosphorylation while azinphosmethyl (AM) increased phosphatidylinositolbiphosphate labeling. Decreased (32)P incorporation in phosphatidylinositol was found with phosmet (PM), AM, and chlorpyriphos (CHL). The effects of these xenobiotics on PI4-kinase activity using different subcellular fractions were also examined. Both type of pesticides affected the postmembrane supernatant enzyme activity. A biphasic effect on membrane and nuclear PI4-kinase activity was seen with HC. The strongest effect found was seen with o-p' DDT in nuclear kinase activity while substantial changes were also observed in membrane. These data demonstrate the sensitivity of human placental PI4-kinase to pesticides currently found in human tissues and suggest deleterious consequences in different processes regulated by 4-phosphoinositides.

Sphingosine-1-phosphate formation activates phosphatidylinositol-4 kinase in basolateral membranes from kidney cells: crosstalk in cell signaling through sphingolipids and phospholipids.

Sphingosine-1-phosphate (S1P) and phosphatidylinositol-4 phosphate [PtdIns(4)P] are important second messengers in various cellular processes. Here, we show that S1P and PtdIns(4)P are formed in purified basolateral membranes (BLM) derived from kidney proximal tubules, indicating the presence of a plasma membrane associated SPK (BLM-SPK) and phosphatidylinositol-4 kinase (PI-4K). We observed that S1P synthesis is linear with time, dependent on protein concentration, and saturable in the presence of increasing concentrations of sphingosine. Different from the observations on cytosolic SPKs, the formation of S1P by BLM-SPK is Mg(2+)-independent and insensitive to the classical inhibitor of the cytosolic SPKs, DL-threo-dihydrosphingosine. With sphingosine as substrate, the enzyme shows cooperative kinetics (n = 3.4) with a K(0.5) value of 0.12 mM, suggesting that BLM-SPK is different from the previously characterized cytosolic SPK. The formation of PtdIns(4)P markedly inhibits BLM-SPK activity. Conversely, a strong activation of PtdIns(4)P synthesis by the formation of S1P is observed. Taken together, these results indicate that (i) basolateral membranes from kidney cells harbor a SPK activity that potentially regulates renal epithelium function, and (ii) the formation of S1P mediated by SPK enhances PI-4K activity, while PtdIns(4)P in turn inhibits SPK, suggesting an interplay between these lipid signaling molecules. These findings suggest the possibility of crosstalk between sphingolipids and glycerolipids, which might be involved in the regulation of transepithelial fluxes across the BLM of kidney cells.