Rapid Communication Activation of Protein Kinase C Inhibits Kainate-Induced Currents in Oocytes Expressing Glutamate Receptor Subunits

Abstract: The effect of protein kinase C (PKC) activation on maximal kainate (KA)-induced currents was studied in Xenopus oocytes expressing the glutamate receptor (GluR) subunits GluR3, GluR1+3, GluR2+3, and GluR6. The PKC activator phorbol 12- myristate 13-acetate (PMA) inhibited peak KA responses in a time-dependent manner. The magnitude of inhibition was greatest in GluR6-expressing oocytes. Desensitizing KA currents characterized by a peak, transient current followed by a slower, desensitizing current were observed in oocytes expressing GluR3 and GluR 1+3 receptors. PMA inhibited the desensitization, and this effect could be observed before PMA's inhibition of peak current amplitude. PMA-mediated inhibition of both desensitization and peak current amplitude was prevented by intracellular injection of the protein kinase C (PKC) inhibitor peptide. These results suggest that the function of GluRs is regulated by PKC-dependent phosphorylation     

Involvement of myosin Vb in glutamate receptor trafficking

Myosin V motors mediate cargo transport; however, the identity of neuronal molecules transported by these proteins remains unknown. Here we show that myosin Vb is expressed in several neuronal populations and associates with the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate-type glutamate receptor subunit GluR1. In developing hippocampal neurons, expression of the tail domain of myosin Vb, but not myosin Va, enhanced GluR1 accumulation in the soma and reduced its surface expression. These changes were accompanied by reduced GluR1 clustering and diminished frequency of excitatory but not inhibitory synaptic currents. Similar effects were observed upon expression of full-length myosin Vb lacking a C-terminal region required for binding to the small GTPase Rab11. In contrast, mutant myosin Vb did not change the localization of several other neurotransmitter receptors, including the glutamate receptor subunit NR1. These results reveal a novel mechanism for the transport of a specific glutamate receptor subunit in neurons mediated by a member of the myosin V family.     

Functional polymorphism in the carboxyl terminus of the alpha-subunit of the human epithelial sodium channel

A common human epithelial sodium channel (ENaC) polymorphism, alphaT663A, is present in the cytoplasmic C terminus of the alpha-subunit, although it is unclear whether this polymorphism segregates with blood pressure. We examined whether this polymorphism was associated with differences in functional Na(+) channel expression. Whole cell amiloride-sensitive currents in Xenopus oocytes expressing wild type channels (alphaT663betagamma) were significantly approximately 1.3-2.0-fold higher than currents measured in oocytes expressing channels with an Ala, Gly or Leu, or Lys at position alpha663. In contrast, differences in functional human ENaC expression were not observed with oocytes expressing channels having Thr (wild type), Ser, or Asp at this position. The surface expression of channels, measured using an epitope-tagged beta-subunit, was significantly reduced in oocytes expressing alphaT663Abetagamma when compared with oocytes expressing alphaT663betagamma. The corresponding polymorphism was generated in the mouse alpha-subunit (malphaA692T) and was not associated with differences in functional alphabetagamma-mouse ENaC expression. The polymorphism is present in a region that is not well conserved between human and mouse. We generated a mouse/human chimera by replacement of the distal C terminus of the mouse alpha-subunit with the distal C terminus of the human alpha-subunit. Co-expression of this m(1-678)/h(650-669)T663A chimera with mouse betagamma led to a significant reduction in whole cell Na(+) currents and surface expression when compared with m(1-678)/h(650-669)T663-mbetagamma. Our results suggest that halphaT663A is a functional polymorphism that affects human ENaC surface expression.     

Rapid Germination of a Barley Mutant Is Correlated with a Rapid Turnover of Abscisic Acid Outside the Embryo

In our study of the role of abscisic acid (ABA) in controlling the germination of barley grains, we tested a barley mutant line with a gigantum appearance (Hordeum distichum cv Quantum) for an ABA-insensitive phenotype by assaying germination in the presence of 10-4 M ABA. Dissected embryos of the mutant germinated at least 10 h earlier than did those of the wild type. The half-maximal concentrations of ABA inhibitory for germination were determined to be 5 x 10-4 M for the mutant and 10-6 M for the wild type. Expression of an ABA-induced Rab gene was studied to determine ABA responsiveness. The ABA concentration required for a half-maximal induction of Rab gene expression was 4 x 10-6 M in isolated embryos of both the mutant and wild type. This result suggests that ABA signal transduction pathways were not affected in the mutant. When isolated embryos were allowed to imbibe in water, ABA was released from the mutant and wild-type embryos at the same rate. However, the free ABA level in the incubation medium of the mutant showed a much faster decrease than that of the wild type, as demonstrated by two independent ABA assay methods (high-performance liquid chromatography and enzyme-linked immunosorbent assay). Our results suggest that turnover of ABA outside the embryo is a determining factor in the germination of barley seeds.     

Epithelial sodium channel inhibition by AMP-activated protein kinase in oocytes and polarized renal epithelial cells

The epithelial Na(+) channel (ENaC) regulates epithelial salt and water reabsorption, processes that require significant expenditure of cellular energy. To test whether the ubiquitous metabolic sensor AMP-activated kinase (AMPK) regulates ENaC, we examined the effects of AMPK activation on amiloride-sensitive currents in Xenopus oocytes and polarized mouse collecting duct mpkCCD(c14) cells. Microinjection of oocytes expressing mouse ENaC (mENaC) with either active AMPK protein or an AMPK activator inhibited mENaC currents relative to controls as measured by two-electrode voltage-clamp studies. Similarly, pharmacological AMPK activation or overexpression of an activating AMPK mutant in mpkCCD(c14) cells inhibited amiloride-sensitive short circuit currents. Expression of a degenerin mutant beta-mENaC subunit (S518K) along with wild type alpha and gamma increased the channel open probability (P(o)) to approximately 1. However, AMPK activation inhibited currents similarly with expression of either degenerin mutant or wild type mENaC. Single channel recordings under these conditions demonstrated that neither P(o) nor channel conductance was affected by AMPK activation. Moreover, expression of a Liddle's syndrome-type beta-mENaC mutant (Y618A) greatly enhanced ENaC whole cell currents relative to wild type ENaC controls and prevented AMPK-dependent inhibition. These findings indicate that AMPK-dependent ENaC inhibition is mediated through a decrease in the number of active channels at the plasma membrane (N), presumably through enhanced Nedd4-2-dependent ENaC endocytosis. The AMPK-ENaC interaction appears to be indirect; AMPK did not bind ENaC in cells, as assessed by in vivo pull-down assays, nor did it phosphorylate ENaC in vitro. In summary, these results suggest a novel mechanism for coupling ENaC activity and renal Na(+) handling to cellular metabolic status through AMPK, which may help prevent cellular Na(+) loading under hypoxic or ischemic conditions.     

MARK2/EMK1/Par-1Balpha phosphorylation of Rab11-family interacting protein 2 is necessary for the timely establishment of polarity in Madin-Darby canine kidney cells

Rab11a, myosin Vb, and the Rab11-family interacting protein 2 (FIP2) regulate plasma membrane recycling in epithelial cells. This study sought to characterize more fully Rab11-FIP2 function by identifying kinase activities modifying Rab11-FIP2. We have found that gastric microsomal membrane extracts phosphorylate Rab11-FIP2 on serine 227. We identified the kinase that phosphorylated Rab11-FIP2 as MARK2/EMK1/Par-1Balpha (MARK2), and recombinant MARK2 phosphorylated Rab11-FIP2 only on serine 227. We created stable Madin-Darby canine kidney (MDCK) cell lines expressing enhanced green fluorescent protein-Rab11-FIP2 wild type or a nonphosphorylatable mutant [Rab11-FIP2(S227A)]. Analysis of these cell lines demonstrates a new role for Rab11-FIP2 in addition to that in the plasma membrane recycling system. In calcium switch assays, cells expressing Rab11-FIP2(S227A) showed a defect in the timely reestablishment of p120-containing junctional complexes. However, Rab11-FIP2(S227A) did not affect localization with recycling system components or the normal function of apical recycling and transcytosis pathways. These results indicate that phosphorylation of Rab11-FIP2 on serine 227 by MARK2 regulates an alternative pathway modulating the establishment of epithelial polarity.     

Covalent Modification of Mutant Rat P2X2 Receptors with a Thiol-Reactive Fluorophore Allows Channel Activation by Zinc or Acidic pH without ATP

Rat P2X2 receptors open at an undetectably low rate in the absence of ATP. Furthermore, two allosteric modulators, zinc and acidic pH, cannot by themselves open these channels. We describe here the properties of a mutant receptor, K69C, before and after treatment with the thiol-reactive fluorophore Alexa Fluor 546 C₅-maleimide (AM546). Xenopus oocytes expressing unmodified K69C were not activated under basal conditions nor by 1,000 µM ATP. AM546 treatment caused a small increase in the inward holding current which persisted on washout and control experiments demonstrated this current was due to ATP independent opening of the channels. Following AM546 treatment, zinc (100 µM) or acidic external solution (pH 6.5) elicited inward currents when applied without any exogenous ATP. In the double mutant K69C/H319K, zinc elicited much larger inward currents, while acidic pH generated outward currents. Suramin, which is an antagonist of wild type receptors, behaved as an agonist at AM546-treated K69C receptors. Several other cysteine-reactive fluorophores tested on K69C did not cause these changes. These modified receptors show promise as a tool for studying the mechanisms of P2X receptor activation.     

Regulation of KCNQ4 potassium channel prepulse dependence and current amplitude by SGK1 in Xenopus oocytes.

The KCNQ gene family comprises voltage-gated potassium channels expressed in epithelial tissues (KCNQ1, KCNQ5), inner ear structures (KCNQ1, KCNQ4) and the brain (KCNQ2-5). KCNQ4 is expressed in inner and outer hair cells of the inner ear where it determines electrical excitability. Accordingly, loss of function mutations of the KCNQ4 gene cause hearing loss. Several K+ channels including the closely related KCNQ1/KCNE1 channel are regulated by the serum- and glucocorticoid-inducible kinase (SGK) family. The present study utilized the Xenopus oocyte system to explore effects of SGK isoforms on KCNQ4 mediated K(+)-currents: KCNQ4 channels activated in a voltage dependent manner with half maximal activation at -10 mV. The peak channel activity was significantly increased by prepulsing. Coexpression of wild type SGK1 but not coexpression of the inactive mutant (K127N)SGK1 significantly increased current amplitudes (by 67 %) and significantly increased the resting potential of KCNQ4 expressing oocytes. Here we describe for the first time a prepulse dependence of KCNQ4 channels with increased currents after hyperpolarizing prepulses. Coexpression of SGK1 significantly attenuated the effect of prepulsing on peak currents. Mutation of Ser to Asp or Ala in the putative phosphorylation consensus sequence in KCNQ4 significantly decreased the sensitivity to SGK1-coexpression. In conclusion, SGK1 regulates current amplitudes and kinetic properties of KCNQ4 channel activity, an effect sensitive to mutations in the SGK1 consensus sequence of the channel.     

Regulation of anterograde transport of adrenergic and angiotensin II receptors by Rab2 and Rab6 GTPases

Three Rab GTPases, Rab1, Rab2 and Rab6, are involved in protein transport between the endoplasmic reticulum (ER) and the Golgi. Whereas Rab1 regulates the anterograde ER-to-Golgi transport, Rab2 and Rab6 coordinate the retrograde Golgi-to-ER transport. We have previously demonstrated that Rab1 differentially modulates the export trafficking of distinct G protein-coupled receptors (GPCRs). In this report, we determined the role of Rab2 and Rab6 in the cell-surface expression and signaling of alpha(2B)-adrenergic (alpha(2B)-AR), beta(2)-AR and angiotensin II type 1 receptors (AT1R). Expression of the GTP-bound mutant Rab2Q65L significantly attenuated the cell-surface expression of both alpha(2B)-AR and beta(2)-AR, whereas the GTP-bound mutant Rab6Q72L selectively inhibited the transport of beta(2)-AR, but not alpha(2B)-AR. Similar results were obtained by siRNA-mediated selective knockdown of endogenous Rab2 and Rab6. Consistently, Rab2Q65L and Rab2 siRNA inhibited alpha(2B)-AR and beta(2)-AR signaling measured as ERK1/2 activation and cAMP production, respectively, whereas Rab6Q72L and Rab6 siRNA reduced signaling of beta(2)-AR, but not alpha(2B)-AR. Similar to the beta(2)-AR, AT1R expression at the cell surface and AT1R-promoted inositol phosphate accumulation were inhibited by Rab6Q72L. Furthermore, the nucleotide-free mutant Rab6N126I selectively attenuated the cell-surface expression of beta(2)-AR and AT1R, but not alpha(2B)-AR. These data demonstrate that Rab2 and Rab6 differentially influence anterograde transport and signaling of GPCRs. These data also provide the first evidence indicating that Rab6-coordinated retrograde transport selectively modulates intracellular trafficking and signaling of GPCRs.     

The ubiquitin-like protein FAT10 forms covalent conjugates and induces apoptosis.

FAT10 is a ubiquitin-like protein that is encoded in the major histocompatibility complex class I locus and is synergistically inducible with interferon-gamma and tumor necrosis factor alpha. The molecule consists of two ubiquitin-like domains in tandem arrangement and bears a conserved diglycine motif at its carboxyl terminus commonly used in ubiquitin-like proteins for isopeptide linkage to conjugated proteins. We investigated the function of FAT10 by expressing murine FAT10 in a hemagglutinin-tagged wild type form as well as a diglycine-deficient mutant form in mouse fibroblasts in a tetracycline-repressible manner. FAT10 expression did not affect major histocompatibility complex class I cell surface expression or antigen presentation. However, we found that wild type but not mutant FAT10 caused apoptosis within 24 h of induction in a caspase-dependent manner as indicated by annexin V cell surface staining and DNA fragmentation. Wild type FAT10, but not its diglycine mutant, was covalently conjugated to thus far unidentified proteins, indicating that specific FAT10 activating and conjugating enzymes must be operative in unstimulated fibroblasts. Because FAT10 expression causes apoptosis and is inducible with tumor necrosis factor alpha, it may be functionally involved in the programmed cell death mediated by this cytokine.     

Up-regulation of the betaine/GABA transporter BGT1 by JAK2

Janus-activated kinase-2 JAK2 is activated by hyperosmotic shock and modifies the activity of several Na(+) coupled transporters. Carriers up-regulated by osmotic shock include the Na(+) coupled osmolyte transporter BGT1 (betaine/GABA transporter 1), which accomplishes the concentrative cellular uptake of γ-amino-butyric acid (GABA). The present study thus explored whether JAK2 participates in the regulation of BGT1 activity. To this end, cRNA encoding BGT1 was injected into Xenopus oocytes with or without cRNA encoding wild type JAK2, constitutively active (V617F)JAK2 or inactive (K882E)JAK2, and electrogenic GABA transport determined by dual electrode voltage clamp. In oocytes injected with cRNA encoding BGT1 but not in oocytes injected with water or with cRNA encoding JAK2 alone, the addition of 1mM GABA to the extracellular fluid generated an inward current (I(BGT)). In BGT1 expressing oocytes I(BGT) was significantly increased by coexpression of JAK2 or (V617F)JAK2, but not by coexpression of (K882E)JAK2. According to kinetic analysis coexpression of JAK2 increased the maximal I(BGT) without significantly modifying the concentration required for halfmaximal I(BGT) (K(M)). In oocytes expressing BGT1 and (V617F)JAK2 I(BGT) was gradually decreased by JAK2 inhibitor AG490 (40 μM). The decline of I(BGT) following disruption of carrier insertion with brefeldin A (5 μM) was similar in the absence and presence of the JAK2 inhibitor AG490 (40 μM). In conclusion, JAK2 is a novel regulator of the GABA transporter BGT1. The kinase up-regulates the carrier presumably by enhancing the insertion of carrier protein into the cell membrane.     

Small GTPase Rab11b regulates degradation of surface membrane L-type Cav1.2 channels

L-type Ca(2+) channels (LTCCs) play a critical role in Ca(2+)-dependent signaling processes in a variety of cell types. The number of functional LTCCs at the plasma membrane strongly influences the strength and duration of Ca(2+) signals. Recent studies demonstrated that endosomal trafficking provides a mechanism for dynamic changes in LTCC surface membrane density. The purpose of the current study was to determine whether the small GTPase Rab11b, a known regulator of endosomal recycling, impacts plasmalemmal expression of Ca(v)1.2 LTCCs. Disruption of endogenous Rab11b function with a dominant negative Rab11b S25N mutant led to a significant 64% increase in peak L-type Ba(2+) current (I(Ba,L)) in human embryonic kidney (HEK)293 cells. Short-hairpin RNA (shRNA)-mediated knockdown of Rab11b also significantly increased peak I(Ba,L) by 66% compared when with cells transfected with control shRNA, whereas knockdown of Rab11a did not impact I(Ba,L). Rab11b S25N led to a 1.7-fold increase in plasma membrane density of hemagglutinin epitope-tagged Ca(v)1.2 expressed in HEK293 cells. Cell surface biotinylation experiments demonstrated that Rab11b S25N does not significantly impact anterograde trafficking of LTCCs to the surface membrane but rather slows degradation of plasmalemmal Ca(v)1.2 channels. We further demonstrated Rab11b expression in ventricular myocardium and showed that Rab11b S25N significantly increases peak I(Ba,L) by 98% in neonatal mouse cardiac myocytes. These findings reveal a novel role for Rab11b in limiting, rather than promoting, the plasma membrane expression of Ca(v)1.2 LTCCs in contrast to its effects on other ion channels including human ether-a-go-go-related gene (hERG) K(+) channels and cystic fibrosis transmembrane conductance regulator. This suggests Rab11b differentially regulates the trafficking of distinct cargo and extends our understanding of how endosomal transport impacts the functional expression of LTCCs.     

Regulation of cloned cardiac L-type calcium channels by cGMP-dependent protein kinase

We have studied the effect of 8-bromo-cyclic GMP (8-Br-cGMP) on cloned cardiac L-type calcium channel currents to determine the site and mechanism of action underlying the functional effect. Rabbit cardiac alpha(1C) subunit, in the presence or absence of beta(1) subunit (rabbit skeletal muscle) or beta(2) subunit (rat cardiac/brain), was expressed in Xenopus oocytes, and two-electrode voltage-clamp recordings were made 2 or 3 days later. Application of 8-Br-cGMP caused decreases in calcium channel currents in cells expressing the alpha(1C) subunit, whether or not a beta subunit was co-expressed. No inhibition of currents by 8-Br-cGMP was observed in the presence of the protein kinase G inhibitor KT5823. Substitutions of serine residues by alanine were made at residues Ser(533) and Ser(1371) on the alpha(1C) subunit. As for wild type, the mutant S1371A exhibited inhibition of calcium channel currents by 8-Br-cGMP, whereas no effect of 8-Br-cGMP was observed for mutant S533A. Inhibition of calcium currents by 8-Br-cGMP was also observed in the additional presence of the alpha(2)delta subunit for wild type channels but not for the mutant S533A. These results indicate that cGMP causes inhibition of L-type calcium channel currents by phosphorylation of the alpha(1C) subunit at position Ser(533) via the action of protein kinase G.     

Pacemaker channels produce an instantaneous current.

Spontaneous rhythmic activity in mammalian heart and brain depends on pacemaker currents (I(h)), which are produced by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Here, we report that the mouse HCN2 pacemaker channel isoform also produced a large instantaneous current (I(inst(HCN2))) in addition to the well characterized, slowly activating I(h). I(inst(HCN2)) was specific to expression of HCN2 on the plasma membrane and its amplitude was correlated with that of I(h). The two currents had similar reversal potentials, and both were modulated by changes in intracellular Cl(-) and cAMP. A mutation in the S4 domain of HCN2 (S306Q) decreased I(h) but did not alter I(inst(HCN2)), and instantaneous currents in cells expressing either wild type HCN2 or mutant S306Q channels were insensitive to block by Cs(+). Co-expression of HCN2 with the accessory subunit, MiRP1, decreased I(h) and increased I(inst(HCN2)), suggesting a mechanism for modulation of both currents in vivo. These data suggest that expression of HCN channels may be accompanied by a background conductance in native tissues and are consistent with at least two open states of HCN channels: I(inst(HCN2)) is produced by a Cs(+)-open state; hyperpolarization produces an additional Cs(+)-sensitive open state, which results in I(h).     

Stimulation of the creatine transporter SLC6A8 by the protein kinase mTOR

Cellular accumulation of creatine is accomplished by the Na(+), Cl(-), and creatine transporter CreaT (SLC6A8). The mammalian target of rapamycin (mTOR) is a kinase stimulating cellular nutrient uptake. The present experiments explored whether SLC6A8 is regulated by mTOR. In Xenopus oocytes expressing SLC6A8 but not in water injected oocytes, creatine-induced a current which was significantly enhanced by coexpression of mTOR. Kinetic analysis revealed that mTOR enhanced maximal current without significantly altering affinity. Preincubation of the oocytes for 32 h with rapamycin (50 nM) decreased the creatine-induced current and abrogated its stimulation by mTOR. The effect of mTOR on CreaT was blunted by additional coexpression of the inactive mutant of the serum and glucocorticoid-inducible kinase (K119N)SGK1 and mimicked by coexpression of wild type SGK1. In conclusion, mTOR stimulates the creatine transporter SLC6A8 through mechanisms at least partially shared by the serum and glucocorticoid-inducible kinase SGK1.     

Regulation of vesicle trafficking in madin-darby canine kidney cells by Rab11a and Rab25

Polarized epithelial cells maintain the polarized distribution of basolateral and apical membrane proteins through a process of receptor-mediated endocytosis, sorting, and then recycling to the appropriate membrane domain. We have previously shown that the small GTP-binding proteins, Rab11a and Rab25, are associated with the apical recycling system of Madin-Darby canine kidney cells. Here we have utilized inducible expression of wild-type, dominant negative, and constitutively active mutants to directly compare the functions of Rab25 and Rab11a in postendocytic vesicular transport. We found that a Rab11a mutant deficient in GTP binding, Rab11aS25N, potently inhibited both transcytosis and apical recycling yet failed to inhibit transferrin recycling. Similarly, expression of either wild type Rab25 or the active mutant Rab25S21V inhibited both apical recycling and transcytosis of IgA by greater than 50% but had no effect on basolateral recycling of transferrin. Interestingly, the GTPase-deficient mutant Rab11aS20V inhibited basolateral to apical transcytosis of IgA, but had no effect on either apical or basolateral recycling. These results indicate that neither Rab11a nor Rab25 function in the basolateral recycling of transferrin in polarized Madin-Darby canine kidney cells cells, consistent with recent morphological observations by others. Thus, transferrin receptors must be recycled to the plasma membrane prior to sorting of apically directed cargoes into Rab11a/Rab25-positive apical recycling endosomes.     

Endocytic trafficking routes of wild type and DeltaF508 cystic fibrosis transmembrane conductance regulator

Intracellular trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) is a focus of attention because it is defective in most patients with cystic fibrosis. DeltaF508 CFTR, which does not mature conformationally, normally does not exit the endoplasmic reticulum, but if induced to do so at reduced temperature is short-lived at the surface. We used external epitope-tagged constructs to elucidate the itinerary and kinetics of wild type and DeltaF508 CFTR in the endocytic pathway and visualized movement of CFTR from the surface to intracellular compartments. Modulation of different endocytic steps with low temperature (16 degrees C) block, protease inhibitors, and overexpression of wild type and mutant Rab GTPases revealed that surface CFTR enters several different routes, including a Rab5-dependent initial step to early endosomes, then either Rab11-dependent recycling back to the surface or Rab7-regulated movement to late endosomes or alternatively Rab9-mediated transit to the trans-Golgi network. Without any of these modulations DeltaF508 CFTR rapidly disappears from and does not return to the cell surface, confirming that its altered structure is detected in the distal as well as proximal secretory pathway. Importantly, however, the mutant protein can be rescued at the plasma membrane by Rab11 overexpression, proteasome inhibitors, or inhibition of Rab5-dependent endocytosis.     

A unique platform for H-Ras signaling involving clathrin-independent endocytosis

Trafficking of H-Ras was examined to determine whether it can enter cells through clathrin-independent endocytosis (CIE). H-Ras colocalized with the CIE cargo protein, class I major histocompatibility complex, and it was sequestered in vacuoles that formed upon expression of an active mutant of Arf6, Q67L. Activation of Ras, either through epidermal growth factor stimulation or the expression of an active mutant of Ras, G12V, induced plasma membrane ruffling and macropinocytosis, a stimulated form of CIE. Live imaging of cells expressing H-RasG12V and fluorescent protein chimeras with pleckstrin homology domains that recognize specific phosphoinositides showed that incoming macropinosomes contained phosphatidylinositol 4,5-bisphosphate (PIP(2)) and phosphatiylinositol 3,4,5-trisphosphate (PIP(3)). PIP(2) loss from the macropinosome was followed by the recruitment of Rab5, a downstream target of Ras, and then PIP(3) loss. Our studies support a model whereby Ras can signal on macropinosomes that pass through three distinct stages: PIP(2)/PIP(3), PIP(3)/Rab5, and Rab5. Vacuoles that form in cells expressing Arf6Q67L trap Ras signaling in the first stage, recruiting the active form of the Ras effectors extracellular signal-regulated kinase and protein kinase B (Akt) but not Rab5. Arf6 stimulation of macropinocytosis also involves passage through the distinct lipid phases, but recruitment of Akt is not observed.