Neutrophils are critical for host defense against microorganisms and are the cellular mediators of acute inflammation. Neutrophils share with other cells signal transduction pathways, many of which are regulated by ras-related GTPases, including the rho proteins recently shown to regulate the actin cytoskeleton. These molecular switches are targeted to membranes by a series of post-translational modifications that include prenylation, proteolysis, and carboxyl methylation. We have established that reversible carboxyl methylation of rho proteins is GTP-dependent and is associated with neutrophil activation. We now propose to identify myeloid proteins that activate rho GTPases, to clone the gene for human myeloid prenylcysteine-directed carboxyl methyltransferase (pcCMT), and to test the hypothesis that carboxyl methylation regulates protein- protein interactions involving rho GTPases.
Specific Aim 1 - Upstream regulators of rho proteins: identification of guanine nucleotide exchange factors (GEFs) for rho proteins in human neutrophils. We will identify novel myeloid rho GEFs by affinity purification using nucleotide-free (transition state) recombinant GST-rho proteins. We will identify activation-dependent neutrophil rho GEFs as activities that promote release of [3H]GDP from recombinant GST-rho proteins, with or without in vitro prenylation.
Specific Aim 2 - Modification of rho proteins: characterization of neutrophil pcCMT. Multiple approaches will be taken: PCR cloning from an HL60 cDNA library based on homology to conserved regions of two yeast pcCMT genes, expression cloning in E. coli using a novel filter-based pcCMT assay for screening, conventional enzymatic purification using a PC/PA liposomal reconstitution assay to follow pcCMT activity, photoaffinity labeling with an artificial substrate to obviate the need to follow labile enzymatic activity, affinity purification with a crosslinkable biotinylated peptide substrate, and developing an anti- pcCMT monoclonal Ab.
Specific Aim 3 - Downstream targets of rho proteins: identification of neutrophil proteins that interact with prenylated cdc42Hs/rac.GTP. We will characterize three novel proteins that, in preliminary experiments, we have found interact with the GTP-bound form of partially purified, neutrophil cdc42Hs and rac with greater affinity than recombinant, unprocessed cdc42Hs and rac, suggesting that the interactions are affected by prenylation, carboxyl methylation, and/or association with rhoGDI. Elucidation of the signaling pathways of rho proteins in neutrophils may identify targets for novel antiinflammatory drugs and is likely to have wide-ranging biological implications.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM055279-02
Application #
2655029
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1997-02-01
Project End
2001-01-31
Budget Start
1998-02-01
Budget End
1999-01-31
Support Year
2
Fiscal Year
1998
Total Cost
Indirect Cost
Name
New York University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
City
New York
State
NY
Country
United States
Zip Code
10016
Fehrenbacher, Nicole; Tojal da Silva, Israel; Ramirez, Craig et al. (2017) The G protein-coupled receptor GPR31 promotes membrane association of KRAS. J Cell Biol 216:2329-2338
Zhou, Mo; Philips, Mark R (2017) Nitrogen Cavitation and Differential Centrifugation Allows for Monitoring the Distribution of Peripheral Membrane Proteins in Cultured Cells. J Vis Exp :
Court, Helen; Ahearn, Ian M; Amoyel, Marc et al. (2017) Regulation of NOTCH signaling by RAB7 and RAB8 requires carboxyl methylation by ICMT. J Cell Biol 216:4165-4182
Zhou, Mo; Wiener, Heidi; Su, Wenjuan et al. (2016) VPS35 binds farnesylated N-Ras in the cytosol to regulate N-Ras trafficking. J Cell Biol 214:445-58
Tsai, Frederick D; Lopes, Mathew S; Zhou, Mo et al. (2015) K-Ras4A splice variant is widely expressed in cancer and uses a hybrid membrane-targeting motif. Proc Natl Acad Sci U S A 112:779-84
Lynch, Stephen J; Snitkin, Harriet; Gumper, Iwona et al. (2015) The differential palmitoylation states of N-Ras and H-Ras determine their distinct Golgi subcompartment localizations. J Cell Physiol 230:610-9
Cox, Adrienne D; Der, Channing J; Philips, Mark R (2015) Targeting RAS Membrane Association: Back to the Future for Anti-RAS Drug Discovery? Clin Cancer Res 21:1819-27
Su, Wenjuan; Wynne, Joseph; Pinheiro, Elaine M et al. (2015) Rap1 and its effector RIAM are required for lymphocyte trafficking. Blood 126:2695-703
Tsai, Frederick D; Wynne, Joseph P; Ahearn, Ian M et al. (2014) Metabolic labeling of Ras with tritiated palmitate to monitor palmitoylation and depalmitoylation. Methods Mol Biol 1120:33-41
Court, Helen; Amoyel, Marc; Hackman, Michael et al. (2013) Isoprenylcysteine carboxylmethyltransferase deficiency exacerbates KRAS-driven pancreatic neoplasia via Notch suppression. J Clin Invest 123:4681-94

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