scholarly journals Rac1 Nanoscale Organization on the Plasma Membrane Is Driven by Lipid Binding Specificity Encoded in the Membrane Anchor

2018 ◽  
Vol 38 (18) ◽  
Author(s):  
Kelsey N. Maxwell ◽  
Yong Zhou ◽  
John F. Hancock
Keyword(s):  
Plasma Membrane ◽  
Bound States ◽  
Point Mutations ◽  
Binding Specificity ◽  
Bound State ◽  
Lipid Binding ◽  
Membrane Anchor ◽  
Guanine Nucleotide Binding Protein ◽  
Guanine Nucleotide Binding ◽  
And Migration

ABSTRACT Rac1 is a small guanine nucleotide binding protein that cycles between an inactive GDP-bound and active GTP-bound state to regulate cell motility and migration. Rac1 signaling is initiated from the plasma membrane (PM). Here, we used high-resolution spatial mapping and manipulation of PM lipid composition to define Rac1 nanoscale organization. We found that Rac1 proteins in the GTP- and GDP-bound states assemble into nonoverlapping nanoclusters; thus, Rac1 proteins undergo nucleotide-dependent segregation. Rac1 also selectively interacts with phosphatidic acid (PA) and phosphoinositol (3,4,5)-trisphosphate (PIP3), resulting in nanoclusters enriched in these lipids. These lipids are structurally important because depleting the PM of PA or PIP3 impairs both Rac1 PM binding and Rac1 nanoclustering. Lipid binding specificity of Rac1 is encoded in the amino acid sequence of the polybasic domain (PBD) of the C-terminal membrane anchor. Point mutations within the PBD, including arginine-to-lysine substitutions, profoundly alter Rac1 lipid binding specificity without changing the electrostatics of the protein and result in impaired macropinocytosis and decreased cell spreading. We propose that Rac1 nanoclusters act as lipid-based signaling platforms emulating the spatiotemporal organization of Ras proteins and show that the Rac1 PBD-prenyl anchor has a biological function that extends beyond simple electrostatic engagement with the PM.

scholarly journals Targeting plasma membrane phosphatidylserine content to inhibit oncogenic KRAS function

2019 ◽  
Vol 2 (5) ◽  
pp. e201900431 ◽  
Author(s):  
Walaa E Kattan ◽  
Wei Chen ◽  
Xiaoping Ma ◽  
Tien Hung Lan ◽  
Dharini van der Hoeven ◽  
...  
Keyword(s):  
Biological Activity ◽  
Plasma Membrane ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Lines ◽  
Lipid Binding ◽  
Small Gtpase ◽  
Membrane Anchor ◽  
Normal Cells

The small GTPase KRAS, which is frequently mutated in human cancers, must be localized to the plasma membrane (PM) for biological activity. We recently showed that the KRAS C-terminal membrane anchor exhibits exquisite lipid-binding specificity for select species of phosphatidylserine (PtdSer). We, therefore, investigated whether reducing PM PtdSer content is sufficient to abrogate KRAS oncogenesis. Oxysterol-related binding proteins ORP5 and ORP8 exchange PtdSer synthesized in the ER for phosphatidyl-4-phosphate synthesized in the PM. We show that depletion of ORP5 or ORP8 reduced PM PtdSer levels, resulting in extensive mislocalization of KRAS from the PM. Concordantly, ORP5 or ORP8 depletion significantly reduced proliferation and anchorage-independent growth of multiple KRAS-dependent cancer cell lines, and attenuated KRAS signaling in vivo. Similarly, functionally inhibiting ORP5 and ORP8 by inhibiting PI4KIIIα-mediated synthesis of phosphatidyl-4-phosphate at the PM selectively inhibited the growth of KRAS-dependent cancer cell lines over normal cells. Inhibiting KRAS function through regulating PM lipid PtdSer content may represent a viable strategy for KRAS-driven cancers.

Dual function of Ras in Raf activation

Development ◽  
1998 ◽  
Vol 125 (24) ◽  
pp. 4999-5008 ◽  
Author(s):  
W. Li ◽  
M. Melnick ◽  
N. Perrimon
Keyword(s):  
Plasma Membrane ◽  
Nucleotide Binding ◽  
Dual Function ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding Protein ◽  
Raf Kinase ◽  
P21 Ras ◽  
Guanine Nucleotide Binding ◽  
Drosophila Embryos

The small guanine nucleotide binding protein p21(Ras) plays an important role in the activation of the Raf kinase. However, the precise mechanism by which Raf is activated remains unclear. It has been proposed that the sole function of p21(Ras)in Raf activation is to recruit Raf to the plasma membrane. We have used Drosophila embryos to examine the mechanism of Raf (Draf) activation in the complete absence of p21(Ras) (Ras1). We demonstrate that the role of Ras1 in Draf activation is not limited to the translocation of Draf to the membrane through a Ras1-Draf association. In addition, Ras1 is essential for the activation of an additional factor which in turn activates Draf.

scholarly journals The role of a guanine nucleotide-binding protein in the activation of rat liver plasma-membrane adenylate cyclase by forskolin

1983 ◽  
Vol 216 (3) ◽  
pp. 753-759 ◽  
Author(s):  
S K F Wong ◽  
B R Martin
Keyword(s):  
Plasma Membrane ◽  
Adenylate Cyclase ◽  
G Protein ◽  
Rat Liver ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding Protein ◽  
Liver Plasma Membrane ◽  
Guanine Nucleotide Binding ◽  
The Individual

The effects of the photoreactive GTP analogue GTP-gamma-azidoanilide on rat liver plasma-membrane adenylate cyclase are described. U.v. irradiation in the presence of the analogue abolished activation by any effector or combination of effectors that function via the activatory G protein. Partial protection against this inhibition was given by F- and guanosine 5′-[gamma-thio]triphosphate. It is concluded that GTP-gamma-azidoanilide acts by a light-induced covalent reaction with the G protein. In the dark the effects of the analogue were similar to those of GTP. Irradiation in the presence of GTP-gamma-azidoanilide was found to reduce but not to abolish activation of rat liver plasma membrane adenylate cyclase by forskolin. The activation by forskolin and GTP together were greater than the sum of the individual activations. Forskolin doubled adenylate cyclase activity in the presence of glucagon and guanosine 5′-[beta, gamma-imido]triphosphate, which might be expected to activate to the maximum possible extent via the G protein. It is concluded that there are two components to the forskolin activation, a guanine nucleotide-dependent and a guanine nucleotide-independent component.

scholarly journals Cholera toxin treatment produces down-regulation of the α-subunit of the stimulatory guanine-nucleotide-binding protein (Gs)

1989 ◽  
Vol 262 (2) ◽  
pp. 643-649 ◽  
Author(s):  
G Milligan ◽  
C G Unson ◽  
M J Wakelam
Keyword(s):  
Plasma Membrane ◽  
Cholera Toxin ◽  
Binding Protein ◽  
Nucleotide Binding ◽  
Guanine Nucleotide ◽  
Down Regulation ◽  
Guanine Nucleotide Binding Protein ◽  
Adp Ribosylation ◽  
Guanine Nucleotide Binding ◽  
Nucleotide Binding Protein

The effect of activation of the alpha-subunit(s) of the stimulatory guanine-nucleotide-binding protein, Gs, on levels of this polypeptide(s) associated with the plasma membrane of L6 skeletal myoblasts was ascertained. Incubation of these cells with cholera toxin led to a time- and concentration-dependent ‘down-regulation’ of both 44 and 42 kDa forms of Gs alpha as assessed by immunoblotting with an anti-peptide antiserum (CS1) able to identify the extreme C-terminus of Gs. The effect of cholera toxin was specific for Gs; levels of Gi alpha in membranes of cholera toxin-treated cells were not different from untreated cells. Down-regulation of Gs was absolutely dependent upon prior ADP-ribosylation, and hence activation of Gs and was not mimicked by other agents which elevate intracellular levels of cyclic AMP. Pretreatment with pertussis toxin, which catalyses ADP-ribosylation of Gi but not of Gs, did not down-regulate either Gi or Gs, demonstrating that covalent modification by ADP-ribosylation is alone not a signal for removal of G-proteins from the plasma membrane.

scholarly journals Persistent activation of the α subunit of Gs promotes its removal from the plasma membrane

1989 ◽  
Vol 260 (3) ◽  
pp. 837-841 ◽  
Author(s):  
G Milligan ◽  
C G Unson
Keyword(s):  
Plasma Membrane ◽  
Membrane Fraction ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding Protein ◽  
Α Subunit ◽  
Rat Glioma ◽  
Glioma C6 ◽  
Membrane Attachment ◽  
Guanine Nucleotide Binding ◽  
Gs Alpha

As assessed both by cholera-toxin-catalysed ADP-ribosylation and by immunoblotting with an anti-peptide antiserum raised against the C-terminal decapeptide of forms of Gs alpha (the alpha subunit of the stimulatory guanine nucleotide-binding protein), rat glioma C6 BU1 cells express two forms of Gs alpha: a major 44 kDa form and a much less prevalent 42 kDa form. We examined the effects of guanine nucleotides on the interaction of the 44 kDa form with the plasma membrane. Incubation of membranes of C6 BU1 cells with poorly hydrolysed analogues of GTP, but not with analogues of either ATP or GDP, caused the release of this Gs alpha from the membrane fraction. Release of Gs alpha was observed within 5 min, and continued throughout the incubation period. After treatment with guanosine 5′-[beta gamma-imido]triphosphate for 60 min, some 75% of this polypeptide had been released from its site of membrane attachment. These experiments demonstrate that Gs alpha need not remain associated invariantly with the plasma membrane.

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