4.6 Article

Plasma membrane phosphatidylinositol 4-phosphate and 4,5-bisphosphate determine the distribution and function of K-Ras4B but not H-Ras proteins

期刊

JOURNAL OF BIOLOGICAL CHEMISTRY
卷 292, 期 46, 页码 18862-18877

出版社

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M117.806679

关键词

bioluminescence resonance energy transfer (BRET); G protein-coupled receptor (GPCR); Golgi; GTPase Kras (KRAS); phosphoinositide

资金

  1. Intramural Research Program of the Eunice Kennedy Shriver NICHD, National Institutes of Health
  2. National Research, Development and Innovation Fund [NKFIH K105006]

向作者/读者索取更多资源

Plasma membrane (PM) localization of Ras proteins is crucial for transmitting signals upon mitogen stimulation. Post-translational lipid modification of Ras proteins plays an important role in their recruitment to the PM. Electrostatic interactions between negatively charged PM phospholipids and basic amino acids found in K-Ras4B (K-Ras) but not in H-Ras are important for permanent K-Ras localization to the PM. Here, we investigated how acute depletion of negatively charged PM polyphosphoinositides (PPIns) from the PM alters the intracellular distribution and activity of K- and H-Ras proteins. PPIns depletion from the PM was achieved either by agonist-induced activation of phospholipase C or with a rapamycin-inducible system in which various phosphatidylinositol phosphatases were recruited to the PM. Redistribution of the two Ras proteins was monitored with confocal microscopy or with a recently developed bioluminescence resonance energy transfer-based approach involving fusion of the Ras C-terminal targeting sequences or the entire Ras proteins to Venus fluorescent protein. We found that PM PPIns depletion caused rapid translocation of K-Ras but not H-Ras from the PM to the Golgi. PM depletion of either phosphatidylinositol 4-phosphate (PtdIns4P) or PtdIns(4,5)P-2 but not PtdIns(3,4,5)P-3 was sufficient to evoke K-Ras translocation. This effect was diminished by deltarasin, an inhibitor of the Ras-phosphodiesterase interaction, or by simultaneous depletion of the Golgi PtdIns4P. The PPIns depletion decreased incorporation of [H-3]leucine in K-Ras-expressing cells, suggesting that Golgi-localized K-Ras is not as signaling-competent as its PM-bound form. We conclude that PPIns in the PM are important regulators of K-Ras-mediated signals.

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