D-3 phosphoinositide metabolism in cells treated with platelet-derived growth factor

Whiteford, Craig C.; Best, C.; Kazlauskas, Andrius; Ulug, Emin T.

doi:10.1042/bj3190851

Cited by 29 publications

(25 citation statements)

References 59 publications

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“…In quiescent cells stimulated with growth factors, PI3K has been shown to be activated twice, as cells transition from G0 into G1 phase 99,101,102 and then later in G1 phase. However, the use of PI3K inhibitors has established that it is only during the later stages of G1 phase that PI3K activity promotes entry into S phase.…”

Section: Monographsmentioning

confidence: 99%

RAS Interaction with PI3K: More Than Just Another Effector Pathway

2011

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RAS proteins are small GTPases known for their involvement in oncogenesis: around 25% of human tumors present mutations in a member of this family. RAS operates in a complex signaling network with multiple activators and effectors, which allows them to regulate many cellular functions such as cell proliferation, differentiation, apoptosis, and senescence. Phosphatidylinositol 3-kinase (PI3K) is one of the main effector pathways of RAS, regulating cell growth, cell cycle entry, cell survival, cytoskeleton reorganization, and metabolism. However, it is the involvement of this pathway in human tumors that has attracted most attention. PI3K has proven to be necessary for RAS-induced transformation in vitro, and more importantly, mice with mutations in the PI3K catalytic subunit p110α that block its ability to interact with RAS are highly resistant to endogenous oncogenic KRASinduced lung tumorigenesis and HRAS-induced skin carcinogenesis. These animals also have a delayed development of the lymphatic vasculature. Many PI3K inhibitors have been developed that are now in clinical trials. However, it is a complex pathway with many feedback loops, and interactions with other pathways make the results of its inhibition hard to predict. Combined therapy with another RAS-regulated pathway such as RAF/MEK/ ERK may be the most effective way to treat cancer, at least in animal models mimicking the human disease. In this review, we will summarize current knowledge about how RAS regulates one of its best-known effectors, PI3K.

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Section: Monographsmentioning

confidence: 99%

RAS Interaction with PI3K: More Than Just Another Effector Pathway

2011

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“…3 H]inositol (PerkinElmer Life Sciences) following our protocols for adipocytes (14) with a slight modification (38). Briefly, cells (35-mm dishes) maintained for 22 h in glucose-and inositol-free DMEM containing 10% dialyzed FBS, 5 mg/ml insulin, 5 mg/ml transferrin, 2 mM pyruvate, 25 mM HEPES, pH 7.4, 100 units/ml penicillin, and 100 g/ml streptomycin were labeled for 26 h with 25 Ci/ml myo- [2][3] H]inositol in glucose-and inositol-free DMEM containing 5 mg/ml transferrin, 2 mM pyruvate, and 25 mM HEPES, pH 7.4.…”

Section: Myo-[2-3 H]inositol Labeling Lipid Extraction and Hplcmefsmentioning

confidence: 99%

The Phosphoinositide Kinase PIKfyve Is Vital in Early Embryonic Development

Ikonomov¹,

Sbrissa²,

Delvecchio³

et al. 2011

Journal of Biological Chemistry

128

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Gene mutations in the phosphoinositide-metabolizing enzymes are linked to various human diseases. In mammals, PIKfyve synthesizes PtdIns(3,5)P 2 and PtdIns5P lipids that regulate endosomal trafficking and responses to extracellular stimuli. The consequence of pikfyve gene ablation in mammals is unknown. To clarify the importance of PIKfyve and PIKfyve lipid products, in this study, we have characterized the first mouse model with global deletion of the pikfyve gene using the Cre-loxP approach. We report that nearly all PIKfyve KO/KO mutant embryos died before the 32-64-cell stage. Cultured fibroblasts derived from PIKfyve flox/flox embryos and rendered pikfyve-null by Cre recombinase expression displayed severely reduced DNA synthesis, consistent with impaired cell division causing early embryo lethality. The heterozygous PIKfyve WT/KO mice were born at the expected Mendelian ratio and developed into adulthood. PIKfyve WT/KO mice were ostensibly normal by several other in vivo, ex vivo, and in vitro criteria despite the fact that their levels of the PIKfyve protein and in vitro enzymatic activity in cells and tissues were 50 -55% lower than those of wild-type mice. Consistently, steady-state levels of the PIKfyve products PtdIns(3,5)P 2 and PtdIns5P selectively decreased, but this reduction (35-40%) was 10 -15% less than that expected based on PIKfyve protein reduction. The nonlinear decrease of the PIKfyve protein versus PIKfyve lipid products, the potential mechanism(s) discussed herein, may explain how one functional allele in PIKfyve WT/KO mice is able to support the demands for PtdIns(3,5)P 2 /PtdIns5P synthesis during life. Our data also shed light on the known human disorder linked to PIKFYVE mutations.Reversible phosphorylation by kinases and phosphatases at positions 3, 4, and/or 5 of the inositol head group in phosphatidylinositol (PtdIns) 2 generates a family of seven phosphoinositide species (1-6). They all are now found to function as versatile membrane-anchored signals that control diverse and essential cellular processes. Consequently, mutations in the genes encoding the phosphoinositide-metabolizing enzymes are associated with an increasing number of human diseases (1-6). The mammalian enzyme that makes PtdIns(3,5)P 2 from PtdIns3P and PtdIns5P from PtdIns is PIKfyve (7,8). It is an evolutionarily conserved large protein of ϳ230 kDa, a product of a single gene in the animal kingdom, whose function is required for proper performance of the endosomal system and certain signaling pathways (9 -11). PIKfyve harbors a PtdIns3P-binding module, i.e. the FYVE finger domain that associates with the PtdIns3P-enriched endosomal membranes, assuring a rapid PIKfyve recruitment to this low abundance substrate of its catalytic activity (12). PIKfyve interacts physically or functionally with multiple partner proteins; the ones involved in PtdIns(3,5)P 2 homeostasis are the best studied (9). Thus, PIKfyve physically associates with the antagonistic enzyme, i.e. the PtdIns(3,5)P 2 -specific phosphatase Sac3, which ...

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“…Cells were harvested by centrifugation and resuspended in 1 ml ice-cold 4.5% perchloric acid (Whiteford et al, 1996). After 15 min on ice, 0.5 g of acid-washed glass beads were added and the cells were lysed by vortexing for 5 min at maximum speed.…”

Section: In Vivo Phosphoinositide Analysismentioning

confidence: 99%

Roles of Phosphoinositides and of Spo14p (phospholipase D)-generated Phosphatidic Acid during Yeast Sporulation

Rudge

Sciorra

Iwamoto

et al. 2004

MBoC

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During yeast sporulation, internal membrane synthesis ensures that each haploid nucleus is packaged into a spore. Prospore membrane formation requires Spo14p, a phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ]-stimulated phospholipase D (PLD), which hydrolyzes phosphatidylcholine (PtdCho) to phosphatidic acid (PtdOH) and choline. We found that both meiosis and spore formation also require the phosphatidylinositol (PtdIns)/PtdCho transport protein Sec14p. Specific ablation of the PtdIns transport activity of Sec14p was sufficient to impair spore formation but not meiosis. Overexpression of Pik1p, a PtdIns 4-kinase, suppressed the sec14-1 meiosis and spore formation defects; conversely, pik1-ts diploids failed to undergo meiosis and spore formation. The PtdIns(4)P 5-kinase, Mss4p, also is essential for spore formation. Use of phosphoinositide-specific GFP-PH domain reporters confirmed that PtdIns(4,5)P 2 is enriched in prospore membranes. sec14, pik1, and mss4 mutants displayed decreased Spo14p PLD activity, whereas absence of Spo14p did not affect phosphoinositide levels in vivo, suggesting that formation of PtdIns(4,5)P 2 is important for Spo14p activity. Spo14p-generated PtdOH appears to have an essential role in sporulation, because treatment of cells with 1-butanol, which supports Spo14p-catalyzed PtdCho breakdown but leads to production of Cho and Ptd-butanol, blocks spore formation at concentrations where the inert isomer, 2-butanol, has little effect. Thus, rather than a role for PtdOH in stimulating PtdIns(4,5)P 2 formation, our findings indicate that during sporulation, Spo14p-mediated PtdOH production functions downstream of Sec14p-, Pik1p-, and Mss4p-dependent PtdIns(4,5)P 2 synthesis.

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D-3 phosphoinositide metabolism in cells treated with platelet-derived growth factor

Cited by 29 publications

References 59 publications

RAS Interaction with PI3K: More Than Just Another Effector Pathway

RAS Interaction with PI3K: More Than Just Another Effector Pathway

The Phosphoinositide Kinase PIKfyve Is Vital in Early Embryonic Development

Roles of Phosphoinositides and of Spo14p (phospholipase D)-generated Phosphatidic Acid during Yeast Sporulation

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