Faculty Bibliography

The Ras-specific guanine nucleotide-exchange factors Son of sevenless (Sos) and Ras guanine nucleotide-releasing factor 1 (RasGRF1) transduce extracellular stimuli into Ras activation by catalyzing the exchange of Ras-bound GDP for GTP. A truncated form of RasGRF1 containing only the core catalytic Cdc25 domain is sufficient for stimulating Ras nucleotide exchange, whereas the isolated Cdc25 domain of Sos is inactive. At a site distal to the catalytic site, nucleotide-bound Ras binds to Sos, making contacts with the Cdc25 domain and with a Ras exchanger motif (Rem) domain. This allosteric Ras binding stimulates nucleotide exchange by Sos, but the mechanism by which this stimulation occurs has not been defined. We present a crystal structure of the Rem and Cdc25 domains of Sos determined at 2.0-A resolution in the absence of Ras. Differences between this structure and that of Sos bound to two Ras molecules show that allosteric activation of Sos by Ras occurs through a rotation of the Rem domain that is coupled to a rotation of a helical hairpin at the Sos catalytic site. This motion relieves steric occlusion of the catalytic site, allowing substrate Ras binding and nucleotide exchange. A structure of the isolated RasGRF1 Cdc25 domain determined at 2.2-A resolution, combined with computational analyses, suggests that the Cdc25 domain of RasGRF1 is able to maintain an active conformation in isolation because the helical hairpin has strengthened interactions with the Cdc25 domain core. These results indicate that RasGRF1 lacks the allosteric activation switch that is crucial for Sos activity

The RTK-Ras-ERK cascade is a central signaling module implicated in the control of diverse biological processes including cell proliferation, differentiation, and survival. The coupling of RTK to Ras is mediated by the Ras-specific nucleotide-exchange factor Son of Sevenless (Sos), which activates Ras by inducing the exchange of GDP for GTP . Considerable evidence indicates that the duration and amplitude of Ras signals are important determinants in controlling the biological outcome . However, the mechanisms that regulate the quantitative output of Ras signaling remain poorly understood. We define a previously unrecognized regulatory component of the machinery that specifies the kinetic properties of signals propagated through the RTK-Ras-ERK cascade. We demonstrate that the establishment of a positive feedback loop involving Ras.GTP and Sos leads to an increase in the amplitude and duration of Ras activation in response to EGF stimulation. This effect is propagated to downstream elements of the pathway as reflected by sustained EGF-induced ERK phosphorylation and enhanced SRE-dependent transcription. As a consequence, the physiological endpoint of EGF action is switched from proliferation to differentiation. We propose that the engagement of Ras/Sos positive feedback loop may contribute to the mechanism by which ligand stimulation is coupled to discrete biological responses

Pancreatic ductal adenocarcinoma (PDA) is generally considered to have originated from pancreatic duct epithelial cells (PDEC). The ability to manipulate the growth properties of PDEC is, therefore, critical for understanding the molecular events involved in the initiation of PDA. Here, we describe methods that we have established for the isolation and maintenance of PDEC in two-dimensional and three-dimensional culture systems. The availability of these culture systems should be particularly useful for studying their relationships between specific genetic lesions and the morphogenic changes that accompany pancreatic ductal tumorigenesis

BACKGROUND & AIMS: Missense mutations in human cationic trypsinogen PRSS1 are frequently detected in patients with hereditary pancreatitis, a rare genetic disease of the pancreas characterized by autodigestive necrosis, chronic inflammation, and fibrosis. To examine the link between PRSS1 mutations and the initiation and progression of hereditary pancreatitis, we have sought to generate a transgenic mouse that carries a missense mutation in the PRSS1 that is most frequently observed in patients. METHODS: A transgenic mouse was generated in which the expression of the mouse PRSS1 mutant R122H (R122H_mPRSS1) is targeted to pancreatic acinar cells by fusion to the elastase promoter. The expression of the mutant trypsinogen was assessed by immunohistochemical staining and real-time reverse transcription polymerase chain reaction analysis. The relationship between transgene expression and inflammation was analyzed by morphologic assessment of H&E-stained tissue sections, responsiveness to cerulein-induced pancreatitis, and immunohistochemical identification of cellular and biochemical components of the inflammatory response. RESULTS: Pancreata from transgenic mice display early-onset acinar cell injury and inflammatory cell infiltration. With progressing age, the transgenic mice develop pancreatic fibrosis and display acinar cell dedifferentiation. Moreover, the expression of R122H_mPRSS1 transgene is associated with enhanced response to cerulein-induced pancreatitis. Finally, cell-specific activation of the inflammation-associated signaling pathways, c-jun-N-terminal kinase and extracellular signal-regulated kinase, was observed in response to expression of R122H_mPRSS1. CONCLUSIONS: These results underscore the importance of PRSS1 mutations as pathogenic mediators of hereditary pancreatitis and indicate that persistent pancreatic injury might be causally linked to chronic pancreatitis

The three mammalian Ras isoforms: HRas, NRas and KRas have been widely implicated in the control of cell proliferation, survival, motility and transformation. Although nearly identical with respect to their catalytic and effector-binding properties, HRas, NRas and KRas lead to different biological outcomes in development, cell growth and cancer. This functional distinction is believed to result at least in part from the differential membrane compartmentalization of Ras isoforms. The different distribution of Ras proteins in cellular membranes dictates unique spatio-temporal patterns of activation of effector pathways. This perspective focuses on the factors that control membrane compartmentalization of Ras with an emphasis on a recently discovered novel posttranslational modification of Ras--ubiquitination. The properties of Ras ubiquitination, its contribution to the regulation of Ras intracellular trafficking and finally the influence of Ras ubiquitination on its signaling potential are discussed

Molecular switches such as small GTPases of the Ras family cycle between inactive GDP-bound and active GTP-bound states. Their essential role in controlling development and cell homeostasis requires mechanisms which determine amplitude and timing of activation. This is achieved in part by the action of guanine nucleotide exchange factors, which function as highly controlled enzymes whose activity relies on spatial segregation and intra- and intermolecular regulation. Here, we describe two experimental methodologies that permit the identification and characterization of GTPase binding sites on activators by assaying complex formation within a broad range of affinities. In the first assay system, proteins presented on the surface of filamentous phage are used to probe affinity determinants of protein-protein interactions. In this application, a protein-displayed phage library is generated by random mutagenesis and a plate-based selection is performed to identify mutations that confer higher binding affinity to an immobilized target. The second method uses light scattering as a tool for measuring the molecular weight, stoichiometry, and polydispersity of protein complexes in solution. In this application, conventional gel filtration chromatography provides initial fractionation, and in-line light scattering measurements allow accurate determination of molar masses of the eluent. This technique also provides information about conformational homogeneity which can be used as a quality

The Ras-specific nucleotide exchange factor son of sevenless (SOS) is a large, multidomain protein with complex regulation, including a Ras-dependent allosteric mechanism. The N-terminal segment of SOS, the histone domain, contains two histone folds, which is highly unusual for a cytoplasmic protein. Using a combination of computational docking, small-angle x-ray scattering, mutagenesis, and calorimetry, we show that the histone domain folds into the rest of SOS and docks onto a helical linker that connects the pleckstrin-homology (PH) and Dbl-homology (DH) domains of SOS to the catalytic domain. In this model, a positively charged surface region on the histone domain is positioned so as to provide a fourth potential anchorage site on the membrane for SOS in addition to the PH domain, the allosteric Ras molecule, and the C-terminal adapter-binding site. The histone domain in SOS interacts with the helical linker, using a region of the surface that in nucleosomes is involved in histone tetramerization. Adjacent surface elements on the histone domain that correspond to the DNA-binding surface of nucleosomes form the predicted interaction site with the membrane. The orientation and position of the histone domain in the SOS model implicates it as a potential mediator of membrane-dependent activation signals

It is well established that Ras oncogenes facilitate neoplastic conversion by stimulating tumor cell growth, survival and motility. However, current studies have indicated that the role of Ras in malignant transformation extends beyond these cell-intrinsic effects to include the establishment of a pro-tumorigenic host environment. We have recently demonstrated that Ras-induced secretion of the chemokine Interleukin-8 (CXCL-8/IL-8) elicits a local inflammatory reaction that is critical for neo-vascularization and sustained tumor growth. Our data identify a novel mechanism by which the Ras oncogene promotes tumor-host interactions that are essential for cancer progression, and suggest that CXCL-8 could serve as a surrogate marker for in-vivo Ras activity

Substituting alanine for glycine at position 60 in v-H-Ras generated a dominant negative mutant that completely abolished the ability of v-H-Ras to transform NIH 3T3 cells and to induce germinal vesicle breakdown in Xenopus oocytes. The crystal structure of the GppNp-bound form of RasG60A unexpectedly shows that the switch regions adopt an open conformation reminiscent of the structure of the nucleotide-free form of Ras in complex with Sos. Critical residues that normally stabilize the guanine nucleotide and the Mg(2+) ion have moved considerably. Sos binds to RasG60A but is unable to catalyze nucleotide exchange. Our data suggest that the dominant negative effect observed for RasG60A.GTP could result from the sequestering of Sos in a non-productive Ras-GTP-guanine nucleotide exchange factor ternary complex

Pancreatic cancer is a challenging disease for patients, doctors and researchers who for decades have searched for a cure for this deadly malignancy. Although existing mouse models of pancreatic cancer have shed light on the mechanistic basis of the neoplastic conversion of the pancreas, their impact in terms of offering new diagnostics and therapeutic modalities remains limited. Chronic pancreatitis is an inflammatory disease of the pancreas that is associated with a gradual damage of the organ and an increased risk of developing neoplastic lesions. In this review, we propose that detailed studies of chronic inflammatory processes in the pancreas will provide insights into the evolution of pancreatic cancer. This information may prove useful in the design of effective therapeutic strategies to battle the disease