Faculty Bibliography

The RAS guanine nucleotide binding proteins activate multiple signaling events that regulate cell growth and differentiation. In quiescent fibroblasts, ectopic expression of activated H-RAS (H-RASV12, where V12 indicates valine-12) induces membrane ruffling, mitogen-activated protein (MAP) kinase activation, and stimulation of DNA synthesis. A mutant of activated H-RAS, H-RASV12C40 (where C40 indicates cysteine-40), was identified that was defective for MAP kinase activation and stimulation of DNA synthesis, but retained the ability to induce membrane ruffling. Another mutant of activated H-RAS, H-RASV12S35 (where S35 indicates serine-35), which activates MAP kinase, was defective for stimulation of membrane ruffling and induction of DNA synthesis. Expression of both mutants resulted in a stimulation of DNA synthesis that was comparable to that induced by H-RASV12. These results indicate that membrane ruffling and activation of MAP kinase represent distinct RAS effector pathways and that input from both pathways is required for the mitogenic activity of RAS

The guanine nucleotide exchange factor Son of sevenless (Sos) performs a crucial step in the coupling of receptor tyrosine kinases to Ras activation. Mammalian cells contain two related but distinct Sos proteins, Sos1 and Sos2. Although they share a high degree of overall similarity, it is not known to what extent their biological and biochemical properties overlap. In the present study, we have compared the interactions of the two human homologues of Sos, hSos1 and hSos2, with the adaptor protein Grb2. We show that hSos2 interacts with Grb2 via its proline-rich COOH-terminal domain and that this interaction is dependent on the SH3 domains of Grb2. In general, these characteristics are similar to the ones reported previously for the interaction of hSos1 with Grb2. However, the apparent binding affinity of hSos2 for Grb2 is significantly higher relative to that of hSos1 both in vitro and in vivo. The region conferring this higher binding affinity has been mapped to residues 1126-1242 of the hSos2 COOH-terminal domain. These results suggest that Sos1 and Sos2 may differentially contribute to receptor-mediated Ras activation

The cyclin-dependent kinase 4 (CDK4) regulates progression through the G1 phase of the cell cycle. The activity of CDK4 is controlled by the opposing effects of the D-type cyclin, an activating subunit, and p16INK4, an inhibitory subunit. Ectopic expression of p16INK4 blocked entry into S phase of the cell cycle induced by oncogenic Ha-Ras, and this block was relieved by coexpression of a catalytically inactive CDK4 mutant. Expression of p16INK4 suppressed cellular transformation of primary rat embryo fibroblasts by oncogenic Ha-Ras and Myc, but not by Ha-Ras and E1a. Together, these observations provide direct evidence that p16INK4 can inhibit cell growth

Mitogen-activated protein kinases (MAP kinases) are common components of signaling pathways induced by diverse growth stimuli. Although the guanidine nucleotide-binding Ras proteins are known to be upstream activators of MAP kinases, the extent to which MAP kinases directly contribute to the mitogenic effect of Ras is as yet undefined. In this study, inhibition of MAP kinases by the MAP kinase phosphatase MKP-1 blocked the induction of DNA synthesis in quiescent rat embryonic fibroblast REF-52 cells by an activated mutant of Ras, V12Ras. These results suggest an essential role for activation of MAP kinases in the transition from the quiescent to the DNA replication phase of the eukaryotic cell cycle

The amiloride-sensitive Na+ channel constitutes the rate-limiting step for Na+ transport in epithelia. Immunolocalization and electrophysiological studies have demonstrated that this channel is localized at the apical membrane of polarized epithelial cells. This localization is essential for proper channel function in Na+ transporting epithelia. In addition, the channel has been shown to associate with the cytoskeletal proteins ankyrin and alpha-spectrin in renal epithelia. However, the molecular mechanisms underlying the cytoskeletal interactions and apical membrane localization of this channel are largely unknown. In this study we show that the putative pore forming subunit of the rat epithelial (amiloride-sensitive) Na+ channel (alpha ENaC) binds to alpha-spectrin in vivo, as determined by co-immunoprecipitation. This binding is mediated by the SH3 domain of alpha-spectrin which binds to a unique proline-rich sequence within the C-terminal region of alpha rENaC. Accordingly, the C-terminal region is sufficient to mediate binding to intact alpha-spectrin from alveolar epithelial cell lysate. When microinjected into the cytoplasm of polarized primary rat alveolar epithelial cells, a recombinant fusion protein containing the C-terminal proline-rich region of alpha rENaC localized exclusively to the apical area of the plasma membrane, as determined by confocal microscopy. This localization paralleled that of alpha-spectrin. In contrast, microinjected fusion protein containing the N-terminal (control) protein of alpha rENaC remained diffuse within the cytoplasm. These results suggest that an SH3 binding region in alpha rENaC mediates the apical localization of the Na+ channel. Thus, cytoskeletal interactions via SH3 domains may provide a novel mechanism for retaining proteins in specific membranes of polarized epithelial cells

The Drosophila Son of sevenless (Sos) gene functions in the signaling pathway initiated by the Sevenless receptor tyrosine kinase. It encodes the Drosophila homologue of CDC25, an activator of Ras in the yeast Saccharomyces cerevisiae. Two widely expressed mammalian homologues of Sos (mSos) have now been identified and characterized. They encode for 150-kD proteins that are Ras-specific guanine nucleotide exchange factors. Genetic and biochemical studies indicate that Sos proteins bind directly to the SH2- and SH3-domain-containing adaptor protein GRB2/Drk. This interaction defines a pathway by which receptor tyrosine kinases can communicate with Ras

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