1985 — 1987 |
Wolfman, Alan |
F32Activity Code Description: To provide postdoctoral research training to individuals to broaden their scientific background and extend their potential for research in specified health-related areas. |
Control of the Phosphotidylinositol Response by P21ras @ University of Rochester |
0.948 |
1989 — 1993 |
Wolfman, Alan |
R29Activity Code Description: Undocumented code - click on the grant title for more information. |
Role of Multiple G-Proteins in Signal Transduction @ Cleveland Clinic Foundation
I wish to test the hypothesis that individual signal transduction systems are mediated by unique plasma membrane GDP-binding proteins. Previous reports, using anti-ras monoclonal Y13-259 have implied this oncogenic protein may be involved in coupling receptor-mediated phosphatidylinositol turnover and the generation of a proliferative responses to either oncogenic tyrosine kinase transformation or normal cellular response to serum addition. Coupling of cell surface receptors to the generation of intracellular second messengers theoretically occurs through the accelerated exchange of bound GDP for GTP by receptor specific G-proteins. Utilizing high resolution anion exchange chromatography, we have discovered at least ten plasma membrane GOP-binding proteins, five of which react with Y13-259. Growth factor dependent changes in the GDP off-rates for each individual GDP-binding species will be examined. Positive coupling between a specific growth factor receptor and a unique GDP-binding protein will be monitored by accelerated rates of quanine nucleotide exchange. Practically, this will be apparent by the disappearance of a single GDP-binding protein in the elution profile from a high resolution anion exchange column. Possible ATP dependent and tyrosine kinase dependent changes in GDP off-rates will be examined. Tyrosine kinase dependent guanine nucleotide exchange will also be examined in membranes containing a temperature sensitive src protein. Requirements for soluble guanine nucleotide exchange factors will also be analyzed. GDP-binding proteins purified from brain tissue will be used in the development of monoclonal antibodies against proven coupling factors. These antibodies will be used in microinjection experiments to determine if single coupling events are sufficient to generate a proliferative signal. Reconstitution experiments will also be done using purified unique plasma membrane G-proteins and GDP-binding protein deficient solubilized membranes. The native molecular weights, possible subunit interactions, pertussis/cholera toxin catalyzed ADP-ribosylation, and similarities to genetically defined ras-related oncogene families will also be evaluated.
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0.948 |
1993 — 1997 |
Wolfman, Alan |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R55Activity Code Description: Undocumented code - click on the grant title for more information. |
Association of a Map Kinase Activator With P21ras-Gtp @ Cleveland Clinic Foundation
Several groups have been successful in the in vitro p21ras. GTP specific activation of MEK and MAPK, mimicking the biological responses observed in whole cells. Stable signaling complexes of p21ras. GTP with Raf-1 and MEK have been identified. In addition, data suggest that the signaling complexes formed between Raf-1 and MEK with p21ras. GTP are unique and independent of one another. Aim 1: Characterize the signaling complexes formed between Raf-1 and members of the MAP kinase cascade with p21ras. We will examine the requirements for Raf-1 and MEK/MEK kinase association and activation by p21ras.GTP. We will also examine the isoform specific activation of both MEK and MAPK by p21ras.GTP. Aim 2: Determination of proteins which directly interact with p21ras. (a) We will examine the ability of purified recombinant proteins, Raf-1, MEK and MEK kinase to directly associate with p21ras.GDP and GMP-PNP. (b) We will examine whether the mammalian homologues to the yeast STE5 and STE20 proteins, which are both upstream of STE11 (the yeast homolog to MEK kinase) associate with p21ras.GDP and/or GMP-PNP. (c) Identification and purification of proteins required for signaling complexes between Raf-1 and MEK/MEK kinase with p21ras. Aim 3: Examine (a) the existence and nature of p21ras dependent signaling complexes in quiescent and serum-stimulated fibroblasts and (b) the role of p21ras.GMP-PNP associated proteins in p21ras-mediated entry of quiescent cell into S phase. We will examine the presence of preformed signal complexes in cultured fibroblasts. We will also test, by microinjection, the ability of signaling proteins isolated from immobilized-p21ras.GMP-PNP to drive quiescent cells or ras-blocked serum-stimulated cells into S phase. Aim 4: Examine the potential activation of signaling proteins which interact with [21ras.GMP-PNP in a transient fashion. We have reported that the transforming p21Val12 protein appears to interact with Raf-1 more transiently than p21c-ras. We will remove the immobilized-21ras and test the activity of the Raf-1 which does not form stabile complexes with p21ras. In similar experiments, we will remove immobilized-p21ras and test the remaining protein extracts for their ability to activate recombinant MAP kinase, MEK and MEK kinase. Active samples will be separated and tested for identity with previously described MEK and MEK kinases.
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0.93 |
2000 — 2003 |
Wolfman, Alan |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Regulation of Apoptosis by Cellular Ras Isoforms @ Cleveland Clinic Foundation
The Ras family of GTPases are known to be critical in the transient activation of proliferative signals by extracellular mitogens. Oncogenic Ras proteins provide a constitutive, rather than transient signal, thereby resulting in cell transformation. Oncogenic Ras proteins can, in some instances, protect cells from apoptosis, primarily through the constitutive activation of PI-3 kinase and its downstream target, Akt. In other cases, oncogenic Ras can activate the apoptotic pathway, resulting in cell death. Using N-Ras and K(i)-Ras knockout cell lines, we have demonstrated that these proteins play a critical role in regulating apoptotic sensitivity in normal cells. In the absence of c-N-Ras expression, cells become hypersensitive to apoptotic agents, inclusive of TNFalpha, anti-Fas and serum-withdrawal. Ectopic expression of c-N-Ras in the N-Ras deficient cell lines restored their resistance to each of the apoptotic treatments. Ectopic expression of c-K(A)-Ras, however, failed to rescue the apoptotic sensitivity of the N-Ras knockout cell lines. In fact, expression of the K(A)-Ras protein enhanced even further the apoptotic sensitivity of the N-Ras knockout cell lines. Our preliminary data suggest that c-N-Ras deficiency results in a higher than normal level of mitochondrial instability. Raf-1, MEK1 and c-N-Ras are all found in a purified mitochondrial preparation, supporting our hypothesis that c-N-Ras plays a vital role in maintaining mitochondrial integrity. While N-Ras negative cells are hypersensitive to the induction of apoptosis, K(i)-Ras negative cells were more resistant than their cognate control cells (K+/+) to apoptotic agents, suggesting that cellular K(i)-Ras proteins provide a steady-state pro-apoptotic signal. We will test the hypothesis that endogenous cellular Ras isoforms (N, K(A), and K(B)-Ras) regulate the threshold that determines whether or not extracellular stimuli promote programmed cell death. We will test this hypothesis through the execution of the following specific aims: 1. We will determine which well-defined apoptotic components are altered by the lack o c-N-Ras expression. 2. We will characterize the requirement for cellular K-Ras proteins in sensitizing fibroblasts towards apoptotic signals. 3. We will perform structure/function analysis on the variable regions between N-Ras K(A)-Ras and K(B)-Ras to determine specific regions within these proteins which define their opposing functions in regulating cellular sensitivity towards apoptotic agents.
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0.93 |