Abstract
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 β 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)P2 but not PtdIns(3,4,5)P3 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 [3H]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.
Highlights
Plasma membrane (PM) localization of Ras proteins is crucial for transmitting signals upon mitogen stimulation
Using a bioluminescence resonance energy transfer (BRET)-based method suitable to monitor the movement of PM-bound proteins after they are released from the PM, we show the translocation of K-Ras or its isolated targeting sequence to either the endoplasmic reticulum (ER) or Golgi upon rapid PM PPIns depletion
As for C-terminal signal sequences, we used the C-terminal 22 residues of K-Ras modified by prenylation (Venus-K-Ras-CAAX) or the C-terminal 22 amino acid signal sequence of H-Ras, which possesses double palmitoylation and prenylation (Venus-H-Ras-CAAX)
Summary
We designed several constructs to target the Venus protein to the inner leaflet of the PM using targeting signal sequences either added to the N or C termini of the fluorescent protein (Fig. 1A). The robust PtdIns[3,4,5]P3 depletion in the PM achieved by wortmannin (Wm) treatment (see Fig. 2C) did not cause Venus-K-Ras-CAAX to appear in the Golgi compartment nor did it affect the Golgi translocation response following PJ recruitment to the PM. When cells were cotransfected with the EGF receptor, EGF stimulation caused translocation of Venus-K-Ras-CAAX from the PM to the Golgi in a similar manner as did M3R stimulation (0.665 ϫ 10Ϫ2 Ϯ 0.293 ϫ 10Ϫ2, p ϭ 0.002) (Fig. 7C) This effect was even larger when PI4KA was inhibited with A1, consistent with the notion that depletion of plasma membrane.
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