Faculty Bibliography

RAS interacts with multiple targets in the cell and controls at least two signaling pathways, one regulating extracellular signal-regulated kinase (ERK) activation and the other controlling membrane ruffling formation. These two pathways appear to act synergistically to cause transformation. SCH 51344 is a pyrazolo-quinoline derivative identified based on its ability to derepress transformation sensitive alpha-actin promoter in RAS-transformed cells. Previous studies have shown that SCH 51344 is a potent inhibitor of RAS-transformation. However, SCH 51344 had very little effect on the activities of proteins in the ERK pathway, suggesting that it inhibits RAS-transformation by a novel mechanism. In this study, we show that SCH 51344 specifically blocks membrane ruffling induced by activated forms of H-RAS, K-RAS, N-RAS and RAC. Treatment of fibroblast cells with this compound had very little effect on RAS-mediated activation of ERK and JUN kinase activities. SCH 51344 was effective in inhibiting the anchorage-independent growth of Rat-2 fibroblast cells transformed by the three forms of oncogenic RAS and RAC V12. These results indicate that SCH 51344 inhibits a critical component of the membrane ruffling pathway downstream from RAC and suggest that targeting this pathway may be an effective approach to inhibit transformation by RAS and other oncogenes

Ras proteins are membrane-bound GTP-binding proteins that play a critical role in the control of cell growth. Through a large number of genetic and biochemical studies it is becoming increasingly evident that the biological activity of Ras proteins is mediated by multiple signaling pathways. This review provides an account of the target proteins that interact with Ras and the functional consequences of these interactions. The relative contribution of the different Ras effector pathways to the mitogenic and oncogenic effects of Ras are discussed

Activation of the Ras/MAPK signaling cascade is essential for growth factor-induced cell proliferation and differentiation. In this report, we describe the purification, cloning, and characterization of a novel protein, designated FRS2, that is tyrosine phosphorylated and binds to Grb2/Sos in response to FGF or NGF stimulation. We find that FRS2 is myristylated and that this modification is essential for membrane localization, tyrosine phosphorylation, Grb2/Sos recruitment, and MAPK activation. FRS2 functions as a lipid-anchored docking protein that targets signaling molecules to the plasma membrane in response to FGF stimulation to link receptor activation with the MAPK and other signaling pathways essential for cell growth and differentiation. Finally, we demonstrate that FRS2 is closely related and probably indentical to SNT, the long-sought target of FGF and NGF receptors

The pleckstrin homology (PH) domain is a conserved protein module present in diverse signal transducing proteins. To investigate the function of the PH domain of the Ras exchanger Sos, we have generated a recombinant (His)6-tagged PH domain from human Sos1 (PH-Sos). Here we show that PH-Sos binds with high affinity(1.5 microM) to lipid vesicles containing the negatively charged phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2). When microinjected into serum-deprived rat embryo fibroblasts or COS cells, PH-Sos displays a homogenous subcellular distribution. However, PH-Sos rapidly accumulates in the plasma membrane following serum stimulation and, under these conditions, is localized preferentially to the leading edge of motile cells. Surprisingly, the membrane localization of PH-Sos is not dependent on its ability to bind PIP2. Overexpression of the PH domain of Sos has a pronounced dominant-negative effect on serum-induced activation of the Ras signaling pathway. These results suggest that the PH domain of Sos participates in regulating the inducible association of Sos with the membrane, and indicate the presence of specific ligands that interact with this domain to bring about the activation of Ras

BACKGROUND: Activation of phospholipase D (PLD) is an important but poorly understood component of receptor-mediated signal transduction responses and regulated secretion. We recently reported the cloning of the human gene encoding PLD1; this enzyme has low basal activity and is activated by protein kinase C and the small GTP-binding proteins, ADP-ribosylation factor (ARF), Rho, Rac and Cdc42. Biochemical and cell biological studies suggest, however, that additional and distinct PLD activities exist in cells, so a search was carried out for novel mammalian genes related to PLD1. RESULTS: We have cloned the gene for a second PLD family member and characterized the protein product, which appears to be regulated differently from PLD1: PLD2 is constitutively active and may be modulated in vivo by inhibition. Unexpectedly, PLD2 localizes primarily to the plasma membrane, in contrast to PLD1 which localizes solely to peri-nuclear regions (the endoplasmic reticulum, Golgi apparatus and late endosomes), where PLD activity has been shown to promote ARF-mediated coated-vesicle formation. PLD2 provokes cortical reorganization and undergoes redistribution in serum-stimulated cells, suggesting that it may have a role in signal-induced cytoskeletal regulation and/or endocytosis. CONCLUSIONS: PLD2 is a newly identified mammalian PLD isoform with novel regulatory properties. Our findings suggest that regulated secretion and morphological reorganization, the two most frequently proposed biological roles for PLD, are likely to be effected separately by PLD1 and PLD2

The RAC guanine nucleotide binding proteins regulate multiple biological activities, including actin polymerization, activation of the Jun kinase (JNK) cascade, and cell proliferation. RAC effector loop mutants were identified that separate the ability of RAC to interact with different downstream effectors. One mutant of activated human RAC protein, RACV12H40 (with valine and histidine substituted at position 12 and 40, respectively), was defective in binding to PAK3, a Ste20-related p21-activated kinase (PAK), but bound to POR1, a RAC-binding protein. This mutant failed to stimulate PAK and JNK activity but still induced membrane ruffling and mediated transformation. A second mutant, RACV12L37 (with leucine substituted at position 37), which bound PAK but not POR1, induced JNK activation but was defective in inducing membrane ruffling and transformation. These results indicate that the effects of RAC on the JNK cascade and on actin polymerization and cell proliferation are mediated by distinct effector pathways that diverge at the level of RAC itself

The Son of sevenless proteins (Sos) are guanine nucleotide exchange factors involved in the activation of Ras by cytoplasmic and receptor tyrosine kinases. Growth factor stimulation rapidly induces the phosphorylation of Sos on multiple serine and threonine sites. Previous studies have demonstrated that growth factor-induced Sos phosphorylation occurs at the C-terminal region of the protein and is mediated, in part, by mitogen-activated protein (MAP) kinase. In this report, we describe the identification of five MAP kinase sites (S-1137, S-1167, S-1178, S-1193, and S-1197) on hSos1. We demonstrate that four of these sites, S-1132, S-1167, S-1178, and S-1193, become phosphorylated following growth factor stimulation. The MAP kinase phosphorylation sites are clustered within a region encompassing three proline-rich SH3-binding sites in the C-terminal domain of hSos1. Replacing the MAP kinase phosphorylation sites with alanine residues results in an increase in the binding affinity of Grb2 to hSos1. Interestingly, hSos2 contains only one MAP kinase phosphorylation site and, as demonstrated previously, has an increased affinity toward Grb2 compared with hSos1. These results suggest a role for MAP kinase in the regulation of Grb2-Sos interactions. Since the binding of Grb2 is important for Sos function, the phosphorylation-dependent modulation of Grb2-Sos association may provide a means of controlling Ras activation

The Rac GTP binding proteins are implicated in actin cytoskeleton-membrane interaction in mammalian cells. In fibroblast cells, Rac has been shown to mediate growth factor-induced polymerization of actin to form membrane ruffles and lamellipodia. We report here the isolation of a noval Rac1-interacting protein, POR1. POR1 binds directly to Rac1, and the interaction of POR1 with Rac1 is GTP dependent. A mutation in the Rac1 effector binding loop shown to abolish membrane ruffling also abolishes interaction with POR1. Truncated versions of POR1 inhibit the induction of membrane ruffling by an activated mutant of Rac1, V12Rac1, in quiescent rat embryonic fibroblast REF52 cells. Furthermore, POR1 synergizes with an activated mutant of Ras, V12Ras, in the induction of membrane ruffling. These results suggest a potential role for POR1 in Rac1-mediated signaling pathways

The Ras superfamily of small GTPases comprises a group of molecular switches that regulate an astonishing diversity of cellular functions. A deep understanding of mitogenesis, cytoskeletal organization, vesicle traffic, and nuclear transport now requires the inclusion of the small GTPases as essential components of the molecular machines that drive these processes. The rich complexity of the control mechanisms involved is evidenced by the recent discoveries of GTPase cascades, multiple downstream effectors, and interconnected networks of GTPase-regulated protein kinase cascades. The 1995 FASEB Summer Conference at Snowmass Village, Colorado, on the Ras GTPase superfamily provided testimony to the broad impact that the study of these proteins continues to exert on cell biology

Activation of Ras by a number of receptor tyrosine kinases is mediated by the guanine nucleotide exchange factor Sos. This activation is thought to occur as a result of the recruitment to the plasma membrane of a complex consisting of Sos and the adaptor molecule Grb2. Growth factor stimulation has been shown to induce the rapid phosphorylation of Sos on serine and threonine residues. In rat L6 cells, insulin-induced Sos phosphorylation is accompanied by a partial dissociation of the Grb2-Sos complex. In this study we have investigated the relationship between Sos phosphorylation and Grb2 association. To this end, we have utilized cAMP because it has been demonstrated that elevation of cytoplasmic levels of cAMP inhibits growth factor-induced Sos phosphorylation. We show that in rat L6 cells, cAMP treatment prevents both the insulin-stimulated Sos phosphorylation and Grb2 dissociation. However, cAMP treatment has no effect on the duration of insulin-induced Ras activation. These results suggest that the kinetics of Ras activation are independent of the phosphorylation-induced dissociation of Sos from Grb2