Faculty Bibliography

The activation of Ras proteins is a key step in the signal transduction pathways triggered by ligand-bound cell surface receptors. The GTPase activating proteins (GAPs) p120-GAP and neurofibromin, the neurofibromatosis-type 1 (NF1) gene product, are thought to play an essential role in the regulation of Ras activity by increasing the GTPase activity of wild type, but not activated Ras in vitro. Both GAPs are widely expressed in mammalian tissues thus raising the question of whether or not they have different regulatory functions. In this study, we have analysed the distribution of p120-GAP and neurofibromin in splenic B lymphocytes by immunofluorescent staining. Crosslinking of surface immunoglobulin (slg), the B-lymphocyte antigen receptor, induced the redistribution of neurofibromin. In contrast, no apparent change in the cellular localization of p120-GAP occurred followed the cross-linking of slg. The redistribution of neurofibromin coincided both spatially and temporally with the relocalization of crosslinked slg and was inhibited by the cytoskeletal disrupting agents colchicine and cytochalasin D. These findings indicated that neurofibromin and p120-GAP can be differentially regulated in vivo and suggest that neurofibromin is a component of the signaling pathway initiated by crosslinking of B lymphocyte slg. Furthermore, our observations that cocapping neurofibromin with slg is independent of the p21ras redistribution suggests that the role of neurofibromin in B cells is not solely related to its ability to act as a Ras regulator

Widespread experience indicates that application of suboptimal concentrations of stimulating ligands (secretagogues) to secretory cells elicits submaximal extents of secretion. Similarly, for permeabilized secretory cells, the extent of secretion is related to the concentration of applied intracellular effectors. We investigated the relationship between the extent of secretion from mast cells (assessed as the release of hexosaminidase) and the degranulation (exocytosis) responses of individual cells. For permeabilized mast cells stimulated by the effector combination Ca2+ plus GTP-gamma-S and for intact cells stimulated by the Ca2+ ionophore ionomycin, we found that exocytosis has the characteristics of an all-or-none process at the level of the individual cells. With a suboptimal stimulus, the population comprised only totally degranulated cells and fully replete cells. In contrast, a suboptimal concentration of compound 48/80 applied to intact cells induced a partial degree of degranulation. This was determined by observing the morphological changes accompanying degranulation by light and electron microscopy and also as a reduction in the intensity of light scattered at 90 degrees, indicative of a change in the cell-refractive index. These results may be explained by the existence of a threshold sensitivity to the combined effectors that is set at the level of individual cells and not at the granule level. We used flow cytometry to establish the relationship between the extent of degranulation in individual rat peritoneal mast cells and the extent of secretion in the population (measured as the percentage release of total hexosaminidase). For comparison, secretion was also elicited by applying the Ca2+ ionophore ionomycin or compound 48/80 to intact cells. For permeabilized cells and also for intact cells stimulated with the ionophore, levels of stimulation that generate partial secretion gave rise to bimodal frequency distributions of 90 degrees light scatter. In contrast, a partial stimulus to secretion by compound 48/80 resulted in a single population of partially degranulated cells, the degree of degranulation varying across the cell population. The difference between the all-or-none responses of the permeabilized or ionophore-treated cells and the graded responses of cells activated by compound 48/80 is likely to stem from differences in the effective calcium stimulus. Whereas cell stimulated with receptor-directed agonists can undergo transient and localized Ca2+ changes, a homogeneous and persistent stimulus is sensed at every potential exocytotic site in the permeabilized cells

Mammalian D-type cyclins are growth factor-regulated, delayed early response genes that are presumed to control progression through the G1 phase of the cell cycle by governing the activity of cyclin-dependent kinases (cdks). Overexpression of mouse cyclin D1 in serum-stimulated mouse NIH-3T3 and rat-2 fibroblasts increased their rates of G0 to S- and G1- to S-phase transit by several hours, leading to an equivalent contraction of their mean cell generation times. Although such cells remained contact inhibited and anchorage dependent, they manifested a reduced serum requirement for growth and were smaller in size than their normal counterparts. Ectopic expression of cyclin D2 in rodent fibroblasts, either alone or together with exogenous cdk4, shortened their G0- to S-phase interval and reduced their serum dependency, but cyclin D2 alone did not alter cell size significantly. When cells were microinjected during the G1 interval with a monoclonal antibody specifically reactive to cyclin D1, parental rodent fibroblasts and derivatives overexpressing this cyclin were inhibited from entering S phase, but cells injected near the G1/S phase transition were refractory to antibody-induced growth suppression. Thus, cyclin D1, and most likely D2, are rate limiting for G1 progression

In this study we describe the cellular distribution of the SH2 and SH3 domains of phospholipase C-gamma (PLC-gamma) and of the adaptor protein GRB2 following their microinjection into living rat embryo fibroblasts. Using immunofluorescence microscopy, we show that a truncated protein composed of the SH2 and SH3 domains of PLC-gamma was localized to the actin cytoskeleton. A similar localization pattern was observed when only the SH3 domain of PLC-gamma was microinjected. In contrast, a truncated protein composed of only the SH2 domains of PLC-gamma exhibited diffuse cytoplasmic distribution. Microinjected GRB2 protein was localized primarily to membrane ruffles, as was GRB2 protein containing SH2 loss-of-function point mutations. Hence, the localization of GRB2 to membrane ruffles does not require interaction with tyrosine-phosphorylated moieties. However, GRB2 proteins with SH3 loss-of-function point mutations exhibited diffuse cytoplasmic distribution. These results indicate that SH3 domains are responsible for the targeting of signaling molecules to specific subcellular locations

A human complementary DNA was isolated that encodes a widely expressed protein, hSos1, that is closely related to Sos, the product of the Drosophila son of sevenless gene. The hSos1 protein contains a region of significant sequence similarity to CDC25, a guanine nucleotide exchange factor for Ras from yeast. A fragment of hSos1 encoding the CDC25-related domain complemented loss of CDC25 function in yeast. This hSos1 domain specifically stimulated guanine nucleotide exchange on mammalian Ras proteins in vitro. Mammalian cells overexpressing full-length hSos1 had increased guanine nucleotide exchange activity. Thus hSos1 is a guanine nucleotide exchange factor for Ras. The hSos1 interacted with growth factor receptor-bound protein 2 (GRB2) in vivo and in vitro. This interaction was mediated by the carboxyl-terminal domain of hSos1 and the Src homology 3 (SH3) domains of GRB2. These results suggest that the coupling of receptor tyrosine kinases to Ras signaling is mediated by a molecular complex consisting of GRB2 and hSos1

Many of the actions of receptor tyrosine kinases are mediated by the protein Ras, including the activation of various downstream serine/threonine kinases and the stimulation of growth and differentiation. The human protein Grb2 binds to ligand-activated growth factor receptors and downstream effector proteins through its Src-homology (SH) domains SH2 and SH3, respectively, and like its homologue from Caenorhabditis elegans, Sem-5, apparently forms part of a highly conserved pathway by which these receptors can control Ras activity. Here we show that the SH3 domains of Grb2 bind to the carboxy-terminal part of hSos1, the human homologue of the Drosophila guanine-nucleotide-releasing factor for Ras, which is essential for control of Ras activity by epidermal growth factor receptor and sevenless. Moreover, a synthetic 10-amino-acid peptide containing the sequence PPVPPR specifically blocks the interaction. These results indicate that the Grb2/hSos1 complex couples activated EGF receptor to Ras signalling

Activation of receptor tyrosine kinases such as those for epidermal growth factor (EGF), platelet-derived growth factor, or nerve growth factor converts the inactive, GDP-bound form of Ras to the active, GTP-bound form, and a dominant negative mutant of Ras interferes with signalling from such receptors. The mechanisms by which receptor tyrosine kinases and Ras are coupled, however, are not well understood. Many cytoplasmic proteins regulated by such receptors contain Src-homology (SH) 2 and 3 domains, and the SH2- and SH3-containing protein Grb2, like its homologue from Caenorhabditis elegans, Sem-5, appears to play an important role in the control of Ras by receptor tyrosine kinases. Here we show that overexpression of Grb2 potentiates the EGF-induced activation of Ras and mitogen-activated protein kinase by enhancing the rate of guanine nucleotide exchange on Ras. Cellular Grb2 appears to form a complex with a guanine-nucleotide-exchange factor for Ras, which binds to the ligand-activated EGF receptor, allowing the tyrosine kinase to modulate Ras activity

A cDNA clone encoding a novel, widely expressed protein (called growth factor receptor-bound protein 2 or GRB2) containing one src homology 2 (SH2) domain and two SH3 domains was isolated. Immunoblotting experiments indicate that GRB2 associates with tyrosine-phosphorylated epidermal growth factor receptors (EGFRs) and platelet-derived growth factor receptors (PDGFRs) via its SH2 domain. Interestingly, GRB2 exhibits striking structural and functional homology to the C. elegans protein sem-5. It has been shown that sem-5 and two other genes called let-23 (EGFR like) and let-60 (ras like) lie along the same signal transduction pathway controlling C. elegans vulval induction. To examine whether GRB2 is also a component of ras signaling in mammalian cells, microinjection studies were performed. While injection of GRB2 or H-ras proteins alone into quiescent rat fibroblasts did not have mitogenic effect, microinjection of GRB2 together with H-ras protein stimulated DNA synthesis. These results suggest that GRB2/sem-5 plays a crucial role in a highly conserved mechanism for growth factor control of ras signaling

The role of Ras in the transduction of signals that control cell growth is undisputed. However, the identity of the Ras signalling pathway remains unknown. Evidence is mounting that Ras can receive signals from different cell surface receptors most likely via a common intermediate, GAP. A new insight into the possible function of Ras is provided by the recent findings that certain ligands can induce the coordinated redistribution of Ras and cell surface receptors. The next challenge is to identify the specific targets for the action of Ras