Dual activation of phospholipase C-epsilon by Rho and Ras GTPases.
ABSTRACT Phospholipase C-epsilon (PLC-epsilon) is a highly elaborated PLC required for a diverse set of signaling pathways. Here we use a combination of cellular assays and studies with purified proteins to show that activated RhoA and Ras isoforms directly engage distinct regions of PLC-epsilon to stimulate its phospholipase activity. Purified PLC-epsilon was activated in a guanine nucleotide- and concentration-dependent fashion by purified lipidated K-Ras reconstituted in PtdIns(4,5)P(2)-containing phospholipid vesicles. Whereas mutation of two critical lysine residues within the second Ras-association domain of PLC-epsilon prevented K-Ras-dependent activation of the purified enzyme, guanine nucleotide-dependent activation by RhoA was retained. Deletion of a loop unique to PLC-epsilon eliminated its activation by RhoA but not H-Ras. In contrast, removal of the autoinhibitory X/Y-linker region of the catalytic core of PLC-epsilon markedly activates the enzyme (Hicks, S. N., Jezyk, M. R., Gershburg, S., Seifert, J. P., Harden, T. K., and Sondek, J. (2008) Mol. Cell, 31, 383-394), but PLC-epsilon lacking this regulatory region retained activation by both Rho and Ras GTPases. Additive activation of PLC-epsilon by RhoA and K- or H-Ras was observed in intact cell studies, and this additivity was recapitulated in experiments in which activation of purified PLC-epsilon was quantified with PtdIns(4,5)P(2)-containing phospholipid vesicles reconstituted with purified, isoprenylated GTPases. A maximally effective concentration of activated RhoA also increased the sensitivity of purified PLC-epsilon to activation by K-Ras. These results indicate that PLC-epsilon can be directly and concomitantly activated by both RhoA and individual Ras GTPases resulting in diverse upstream control of signaling cascades downstream of PLC-epsilon.
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ABSTRACT: The definition of the number and nature of signal transduction pathways networking in the pathogenesis of osteosarcoma raised great interest. Intracellular calcium ions are important second messengers implicated in the control of cell death. The calcium concentration is regulated by signal transduction pathways, including the Phosphoinositides (PI) signaling. Phosphatydil inositol (4,5) bisphosphate (PIP2) is critical for many cellular activities. The levels of PIP2 are regulated by means of Phosphoinositide-specific Phospholipase C (PI-PLC) family of enzymes. We delineated the panel of expression of PI-PLC enzymes in four human osteosarcoma cell lines. In MG-63 cell line, PI-PLC β1, β2, β3, β4, γ1, γ2, δ1, δ3 and ε resulted expressed. In 143B cell line, PI-PLC β1, β2, β3, β4, γ1, γ2, δ1, δ3 and ε were expressed. In SaOS-2 cell line, PI-PLC β1, β3, β4, γ1, γ2, δ1, δ3, ε and η1. In Hs888 cell line, PI-PLC β1, β3, β4, γ1, δ1, δ3, δ4, ε and η1 the administration of U-73122 to cultures briefly modifies the levels of PI-PLC transcripts. The obtained complete expression panel of PI-PLC isoforms will be a useful tool for further functional studies about the role of the PI signal transduction pathway in osteosarcoma.Journal of Cell Communication and Signaling 02/2013; 8(3). DOI:10.1007/s12079-013-0194-6
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ABSTRACT: The physiological effects of many extracellular stimuli are initiated through receptor-promoted activation of phospholipase C and inositol lipid signaling pathways. The historical view that phospholipase C-promoted signaling primarily occurs through activation of heterotrimeric G proteins or tyrosine kinases has expanded in recent years with the realization that at least three different mammalian phospholipase C isozymes are directly activated by members of the Ras superfamily of GTPases. Thus, Ras, Rap, Rac, and Rho GTPases all specifically regulate certain phospholipase C isozymes, and insight into the physiological significance of these signaling responses is beginning to accrue. High resolution three-dimensional structures of phospholipase C isozymes also are beginning to shed light on their mechanism of activation.The Journal of Lipid Research 12/2008; 50 Suppl(Supplement):S243-8. DOI:10.1194/jlr.R800045-JLR200 · 4.73 Impact Factor
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ABSTRACT: Insect-pathogenic fungi penetrate their hosts directly through the cuticle. To better understand this process, we identified genes that were up-regulated by Metarhizium anisopliae germinating and differentiating on Locusta migratoria wings using suppression subtractive hybridization (SSH). A total of 78 unique expressed sequence tags (ESTs) up-regulated more than twofold during fungal growth on locust wings were identified. Among these 78 ESTs, 30 (38.5%) shared significant similarity with NCBI annotated hypothetical proteins, 16 (20.5%) shared low similarity to known or predicted genes, might represent novel genes, and 32 (41.0%) shared significant similarity with known proteins that are involved in various cell and molecular processes such as cell metabolism, protein metabolism, stress response and defense, and cell structure and function. Semi-quantitative RT-PCR analysis of six randomly selected genes confirmed the SSH results, verifying the fidelity of the SSH data. The results of this study provide novel information on genes expressed during early stages of infection with M. anisopliae, and improve current understanding of fungal pathogenesis.Current Microbiology 03/2011; 62(5):1649-55. DOI:10.1007/s00284-011-9909-1 · 1.36 Impact Factor