A novel prenyl-polybasic domain code determines lipid-binding specificity of the K-Ras membrane anchor

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.

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Identification of Key Residues in the A-Raf Kinase Important for Phosphoinositide Lipid Binding Specificity

Biochemistry ◽

10.1021/bi0487692 ◽

2005 ◽

Vol 44 (9) ◽

pp. 3432-3440 ◽

Cited By ~ 18

Author(s):

Lindsey M. Johnson ◽

Kristy M. James ◽

M. Dean Chamberlain ◽

Deborah H. Anderson

Keyword(s):

Binding Specificity ◽

Lipid Binding ◽

Raf Kinase ◽

Key Residues

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Targeting plasma membrane phosphatidylserine content to inhibit oncogenic KRAS function

Life Science Alliance ◽

10.26508/lsa.201900431 ◽

2019 ◽

Vol 2 (5) ◽

pp. e201900431 ◽

Cited By ~ 6

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.

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Lipid binding specificity of bovine α-lactalbumin: A multidimensional approach

Biochimica et Biophysica Acta (BBA) - Biomembranes ◽

10.1016/j.bbamem.2014.04.027 ◽

2014 ◽

Vol 1838 (8) ◽

pp. 2078-2086 ◽

Cited By ~ 23

Author(s):

Arunima Chaudhuri ◽

Amitabha Chattopadhyay

Keyword(s):

Binding Specificity ◽

Lipid Binding ◽

Multidimensional Approach

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The Role of Phosphatidylglycerol as a Functional Effector and Membrane Anchor of the D1-Core Peptide from Photosystem II-Particles of the Cyanobacterium Oscillatoria chalybea

Zeitschrift für Naturforschung C ◽

10.1515/znc-1995-5-608 ◽

1995 ◽

Vol 50 (5-6) ◽

pp. 380-390 ◽

Cited By ~ 37

Author(s):

O. Kruse ◽

G. H. Schmid

Keyword(s):

Amino Acid ◽

Photosystem Ii ◽

Binding Sites ◽

Hydrophobic Interactions ◽

Covalent Binding ◽

Spatial Separation ◽

Lipid Binding ◽

Membrane Anchor ◽

Oscillatoria Chalybea ◽

Eukaryotic Organisms

Abstract The intrinsic polypeptide D1, isolated from photosystem (PS) Il-particles of the cyanobacterium Oscillatoria chalybea, was obtained by electroelution and fractionated extraction with organic solvents. Purification was demonstrated by Western blotting and amino acid sequencing. By carrying out D1 -immunization in rabbits a polyclonal monospecific D1-antiserum was obtained.For the qualitative characterization of D1 as a lipid-binding peptide, the effect of the lipids phosphatidylglycerol (PG), monogalactosyldiacylglyceride (MGDG) and phosphatidylcho line (PC) on PSII-oxygen evolution was analysed in reconstitution experiments. In these experiments purified photosystem II (PSII)-particle preparations were treated with the enzyme phospholipase A2 and supplemented with lipid emulsions. We were able to show that the inhibition of electron transport, as the consequence of this lipase treatment, was only relieved, if phosphatidylglycerol was added to the preparation. A model was proposed, in which phosphatidylglycerol is a functional effector for the optimal conformation of D1 in the PSII core complex. Phosphatidylglycerol molecules are unusually tightly bound to the D1 peptide by hydrophobic interactions. A covalent binding seems not probable. The localisation of phosphatidylglycerol binding sites was found by trypsin treatment of D1 and analysis of the obtained oligopeptides with HPLC and immunoblotting. The binding sites could be confined to the hydrophobic amino acid section between arginine 27 and arginine 225, which is known to be the membrane anchor of D1. This has led us to the conclusion that the phospholipid phosphatidylglycerol plays an important role for anchoring the D1-peptide and for its orientation in the thylakoid membrane. Phosphatidylglycerol with its high amount of palmitic acid has in prokaryotic cyanobacteria apparently a role in stabilization and orientation.The high turn-over of D1 and the spatial separation of the synthesis-and incorporation-site in the membrane, developed during evolution in eukaryotic organisms, might have changed the requirement on the mobility and the orientation of D1 in photosynthetic membranes.

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