The Tumor Procurement Core (TPC) within the Department of Pathology for
The Tumor Procurement Core (TPC) in the Department of Pathology for supplying the HNSCC tumor samples. The authors also thank Dr. Sushma Shivaswamy and Mr. John Simard for kindly supplying the human neutralizing IL-1 antibody for use in our in vivo research. Finally, we thank Nicholas Borcherding and Drs. GlyT2 Gene ID Weizhou Zhang, Fayyaz Sutterwala and Hasem Habelhah for their helpful suggestions and discussions concerning this function. Grant Help This work was supported by grants NIH R01DE024550, NIH K01CA134941 and IRG-77-004-34 in the American Cancer Society, administered by way of the Holden Complete Cancer Center at the University of Iowa.
H-Ras forms dimers on membrane DP Storage & Stability surfaces through a protein rotein interfaceWan-Chen Lina,b,1, Lars Iversena,b,1,two, Hsiung-Lin Tua,b, Christopher Rhodesa,b, Sune M. Christensena,b, Jeffrey S. Iwiga,c, Scott D. Hansena,b, William Y. C. Huanga,b, and Jay T. Grovesa,b,d,a Howard Hughes Health-related Institute and Departments of bChemistry and cMolecular and Cell Biology, University of California, Berkeley, CA 94720; and dPhysical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CAEdited by Michael K. Rosen, University of Texas Southwestern Health-related Center, Dallas, TX, and accepted by the Editorial Board January 15, 2014 (received for critique November 15, 2013)The lipid-anchored modest GTPase Ras is an critical signaling node in mammalian cells. Numerous observations suggest that Ras is laterally organized within the cell membrane, and this may well play a regulatory part in its activation. Lipid anchors composed of palmitoyl and farnesyl moieties in H-, N-, and K-Ras are broadly suspected to become responsible for guiding protein organization in membranes. Here, we report that H-Ras forms a dimer on membrane surfaces through a protein rotein binding interface. A Y64A point mutation inside the switch II area, known to stop Son of sevenless and PI3K effector interactions, abolishes dimer formation. This suggests that the switch II area, near the nucleotide binding cleft, is either part of, or allosterically coupled to, the dimer interface. By tethering H-Ras to bilayers through a membrane-miscible lipid tail, we show that dimer formation is mediated by protein interactions and will not demand lipid anchor clustering. We quantitatively characterize H-Ras dimerization in supported membranes making use of a mixture of fluorescence correlation spectroscopy, photon counting histogram analysis, time-resolved fluorescence anisotropy, single-molecule tracking, and step photobleaching analysis. The 2D dimerization Kd is measured to become 1 103 moleculesm2, and no higher-order oligomers had been observed. Dimerization only happens around the membrane surface; H-Ras is strictly monomeric at comparable densities in resolution. Analysis of quite a few H-Ras constructs, like crucial changes to the lipidation pattern in the hypervariable area, suggest that dimerization is really a general home of native H-Ras on membrane surfaces.Ras signaling| Ras assayIn addition to biochemical proof for communication involving the C-terminal membrane binding region and the nucleotide binding pocket, NMR and IR spectroscopic observations suggest that the HVR and lipid anchor membrane insertion impacts Ras structure and orientation (157). Molecular dynamics (MD) modeling of bilayer-induced H-Ras conformations has identified two nucleotide-dependent states, which differ in HVR conformation, membrane contacts, and G-domain orientation (18). In vivo FRET meas.