It is proposed to characterize the proteins involved in guanine nucleotide regulation of hormone-sensitive adenylate cyclase. Cholera toxin is used to alter the guanine nucleotide regulation of adenylate cyclase, and to identify specific regulatory proteins of the adenylate cyclase system. Cholera toxin, using (32 P) NAD ion, specifically radiolabels at least two proteins in membranes of wild-type S49 lymphoma cells. A genetic variant of S49 cells (cyc-) which is deficient in guanine nucleotide regulatory proteins also lacks the specific cholera toxin substrates. Differences in the number of specific cholera toxin substrates is apparent in cell types expressing different guanine nucleotide regulatory properties suggesting that this may be related to the complexity of the hormone-sensitive adenylate cyclase system. The differences in functional properties of the regulatory proteins will be correlated with their differences in structure and number. Functional differences will be determined using three procedures: 1. Reconstitution assays using cyc- membranes and detergent extracts from membranes of cell types having different adenylate cyclase regulatory properties. 2. Membrane-cell fusion techniques to build adenylate cyclase systems having specific regulatory proteins to examine their role in agonist-induced desensitization. 3. Heterokaryon and cell hybrid formation using avian erythrocytes and cyc-cells to examing the re-expression of avian erythrocyte regulatory proteins. Structural differences will be determined by peptide mapping and isoelectric point determinations of the radiolabelled proteins that are specific substrates for cholera toxin. The regulatory proteins specifically labelled by cholera toxin will be partially purified, and after elution from an SDS-acrylamide gel used for the production of antisera against adenylate cyclase regulatory proteins.
| Zawistowski, Jon S; Nakamura, Kazuhiro; Parker, Joel S et al. (2013) MicroRNA 9-3p targets ?1 integrin to sensitize claudin-low breast cancer cells to MEK inhibition. Mol Cell Biol 33:2260-74 |
| Duncan, James S; Whittle, Martin C; Nakamura, Kazuhiro et al. (2012) Dynamic reprogramming of the kinome in response to targeted MEK inhibition in triple-negative breast cancer. Cell 149:307-21 |
| Cronan, M R; Nakamura, K; Johnson, N L et al. (2012) Defining MAP3 kinases required for MDA-MB-231 cell tumor growth and metastasis. Oncogene 31:3889-900 |
| Johnson, Gary L (2011) Defining MAPK interactomes. ACS Chem Biol 6:18-20 |
| Huang, Weichun; Umbach, David M; Vincent Jordan, Nicole et al. (2011) Efficiently identifying genome-wide changes with next-generation sequencing data. Nucleic Acids Res 39:e130 |
| Abell, Amy N; Jordan, Nicole Vincent; Huang, Weichun et al. (2011) MAP3K4/CBP-regulated H2B acetylation controls epithelial-mesenchymal transition in trophoblast stem cells. Cell Stem Cell 8:525-37 |
| Jordan, Nicole Vincent; Johnson, Gary L; Abell, Amy N (2011) Tracking the intermediate stages of epithelial-mesenchymal transition in epithelial stem cells and cancer. Cell Cycle 10:2865-73 |
| Nakamura, Kazuhiro; Kimple, Adam J; Siderovski, David P et al. (2010) PB1 domain interaction of p62/sequestosome 1 and MEKK3 regulates NF-kappaB activation. J Biol Chem 285:2077-89 |
| Konhilas, John P; Boucek, Dana M; Horn, Todd R et al. (2010) The role of MEKK1 in hypertrophic cardiomyopathy. Int Heart J 51:277-84 |
| Nakamura, Kazuhiro; Johnson, Gary L (2010) Activity assays for extracellular signal-regulated kinase 5. Methods Mol Biol 661:91-106 |
Showing the most recent 10 out of 14 publications
