Role of the pleckstrin homology domain in intersectin-L Dbl homology domain activation of Cdc42 and signaling.

Intersectin-long (ITSN-L) contains the invariant Dbl homology (DH) and pleckstrin homology (PH) domain structure characteristic of the majority of Dbl family proteins. This strict domain topography suggests that the PH domain serves an essential, conserved function in the regulation of the intrinsic guanine nucleotide exchange activity of the DH domain. We evaluated the role of the PH domain in regulating the DH domain function of ITSN-L.

Ras family signaling: therapeutic targeting.

Mutationally activated and oncogenic versions of the ras genes were first identified in human tumors in 1982. This discovery prompted great interest in the development of anti-Ras strategies as novel, target-based approaches for cancer treatment. The three human ras genes represent the most frequently mutated oncogenes in human cancers.

Critical role of the pleckstrin homology domain in Dbs signaling and growth regulation.

Dbl family proteins act as guanine nucleotide exchange factors and positive regulators of Rho GTPase function by stimulating formation of the active, GTP-bound state. All Dbl family Rho guanine nucleotide exchange factors possess an invariant tandem domain structure consisting of a Dbl homology (DH) catalytic domain followed by a pleckstrin homology (PH) regulatory domain. We determined previously that the PH domain of Dbs was critical for the intrinsic catalytic activity of the DH domain in vitro and for Dbs transformation in vivo.

Selective deficiency in protein kinase C isoenzyme expression and inadequacy in mitogen-activated protein kinase activation in cord blood T cells.

The biochemical basis for the reduced lymphokine production by neonatal T cells compared with adult T cells remains poorly defined. Previous studies have raised the possibility that neonatal T cells could be deficient in their ability to transmit signals via protein kinase (PK) C. We now report that while PKC-dependent activation of the mitogen-activated protein (MAP) kinases, c-Jun N-terminal protein kinase and the extracellular signal-regulated protein kinase (ERK)1/ERK2, was deficient in cord blood T cells compared with adult blood T cells, marked activation of the MAP kinases in cord blood T cells was achieved via PKC-independent means.

RhoG signals in parallel with Rac1 and Cdc42.

RhoG is a member of the Rho family of small GTPases and shares high sequence identity with Rac1 and Cdc42. Previous studies suggested that RhoG mediates its effects through activation of Rac1 and Cdc42. To further understand the mechanism of RhoG signaling, we studied its potential activation pathways, downstream signaling properties, and functional relationship to Rac1 and Cdc42 in vivo.

XPLN, a guanine nucleotide exchange factor for RhoA and RhoB, but not RhoC.

Rho proteins cycle between an inactive, GDP-bound state and an active, GTP-bound state. Activation of these GTPases is mediated by guanine nucleotide exchange factors (GEFs), which promote GDP to GTP exchange. In this study we have characterized XPLN, a Rho family GEF.

RhoA biological activity is dependent on prenylation but independent of specific isoprenoid modification.

Recent studies showed that specific isoprenoid modification may be critical for RhoB subcellular location and function. Therefore, we determined whether the function of the highly related RhoA protein is also critically dependent on specific isoprenoid modification: (a) in contrast to observations with RhoB or Ras proteins, where farnesylated and geranylgeranylated versions showed differences in subcellular location, both prenylated versions of RhoA showed the same plasma membrane and cytosolic location; (b) a farnesylated version of activated RhoA(63L) retained the same diverse functions as the normally geranylgeranylated RhoA(63L) protein, and both proteins show indistinguishable abilities to stimulate gene expression, cause growth transformation of NIH 3T3 mouse fibroblasts, to stimulate the motility of T47D human breast epithelial cells, and to block HIV-1 viral replication and gene expression; and (c) cells expressing farnesylated RhoA retained sensitivity to the growth inhibition caused by inhibition of geranylgeranyltransferase I, indicating that other proteins are critical targets for inhibitors of geranylgeranylation.

Distinct requirements for Ras oncogenesis in human versus mouse cells.

The spectrum of tumors associated with oncogenic Ras in humans often differs from those in mice either treated with carcinogens or engineered to sporadically express oncogenic Ras, suggesting that the mechanism of Ras transformation may be different in humans. Ras stimulates primarily three main classes of effector proteins, Rafs, PI3-kinase, and RalGEFs, with Raf generally being the most potent at transforming murine cells. Using oncogenic Ras mutants that activate single effectors as well as constitutively active effectors, we find that the RalGEF, and not the Raf or PI3-kinase pathway, is sufficient for Ras transformation in human cells.

Critical role of the pleckstrin homology and cysteine-rich domains in Vav signaling and transforming activity.

Vav family proteins are members of the Dbl family of guanine nucleotide exchange factors and activators of Rho family small GTPases. In addition to the Dbl homology (DH) domain important for guanine nucleotide exchange factor catalytic function, all Dbl family proteins contain an adjacent pleckstrin homology (PH) domain that serves to regulate DH domain activity. Although the role of the PH domain in Vav function has been evaluated extensively, its precise role and whether it serves a distinct role in different Vav proteins remain unresolved.

Tiam1 mediates Ras activation of Rac by a PI(3)K-independent mechanism.

Rac is a member of the Ras superfamily of GTPases and functions as a GDP/GTP-regulated switch. Formation of active Rac-GTP is stimulated by Dbl family guanine nucleotide exchange factors (GEFs), such as Tiam1 (ref. 2).

Farnesyltransferase inhibitors: promises and realities.

Farnesyltransferase inhibitors have recently shown clinical efficacy against leukemias, gliomas and even non-small-cell lung cancers, especially when administered in combination with taxanes. It is possible that the critical target downstream of farnesyltransferase responsible for these effects is not either Ras or RhoB, the two most cited possibilities - but the hunt is on.

Raf-independent deregulation of p38 and JNK mitogen-activated protein kinases are critical for Ras transformation.

Activated Ras, but not Raf, causes transformation of RIE-1 epithelial cells, supporting the importance of Raf-independent pathways in mediating Ras transformation. The p38 and JNK mitogen-activated protein kinase cascades are activated by Ras via Raf-independent effector function. Therefore, we determined whether p38 and JNK activation are involved in Ras transformation of RIE-1 epithelial cells.

Role of MAP kinases in the 1,25-dihydroxyvitamin D3-induced transactivation of the rat cytochrome P450C24 (CYP24) promoter. Specific functions for ERK1/ERK2 and ERK5.

The current study investigated the action of 1,25-dihydroxyvitamin D(3) (1,25D) at the genomic and signal transduction levels to induce rat cytochrome P450C24 (CYP24) gene expression. A rat CYP24 promoter containing two vitamin D response elements and an Ets-1 binding site was used to characterize the mechanism of actions for the 1,25D secosteroid hormone. The Ets-1 binding site was determined to function cooperatively with the most proximal vitamin D response element in a hormone-dependent fashion.

Leukemia-associated Rho guanine nucleotide exchange factor promotes G alpha q-coupled activation of RhoA.

Leukemia-associated Rho guanine-nucleotide exchange factor (LARG) belongs to the subfamily of Dbl homology RhoGEF proteins (including p115 RhoGEF and PDZ-RhoGEF) that possess amino-terminal regulator of G protein signaling (RGS) boxes also found within GTPase-accelerating proteins (GAPs) for heterotrimeric G protein alpha subunits. p115 RhoGEF stimulates the intrinsic GTP hydrolysis activity of G alpha 12/13 subunits and acts as an effector for G13-coupled receptors by linking receptor activation to RhoA activation. The presence of RGS box and Dbl homology domains within LARG suggests this protein may also function as a GAP toward specific G alpha subunits and couple G alpha activation to RhoA-mediating signaling pathways.

Structural basis for the selective activation of Rho GTPases by Dbl exchange factors.

Activation of Rho-family GTPases involves the removal of bound GDP and the subsequent loading of GTP, all catalyzed by guanine nucleotide exchange factors (GEFs) of the Dbl-family. Despite high sequence conservation among Rho GTPases, Dbl proteins possess a wide spectrum of discriminatory potentials for Rho-family members. To rationalize this specificity, we have determined crystal structures of the conserved, catalytic fragments (Dbl and pleckstrin homology domains) of the exchange factors intersectin and Dbs in complex with their cognate GTPases, Cdc42 and RhoA, respectively.

Critical but distinct roles for the pleckstrin homology and cysteine-rich domains as positive modulators of Vav2 signaling and transformation.

Vav2, like all Dbl family proteins, possesses tandem Dbl homology (DH) and pleckstrin homology (PH) domains and functions as a guanine nucleotide exchange factor for Rho family GTPases. Whereas the PH domain is a critical positive regulator of DH domain function for a majority of Dbl family proteins, the PH domains of the related Vav and Vav3 proteins are dispensable for DH domain activity. Instead, Vav proteins contain a cysteine-rich domain (CRD) critical for DH domain function.

RasGRP4 is a novel Ras activator isolated from acute myeloid leukemia.

Although a number of genetic defects are commonly associated with acute myeloid leukemia (AML), a large percentage of AML cases are cytogenetically normal. This suggests a functional screen for transforming genes is required to identify genetic mutations that are missed by cytogenetic analyses. We utilized a retrovirus-based cDNA expression system to identify transforming genes expressed in cytogenetically normal AML patients.

Rho family GTPases regulate mammary epithelium cell growth and metastasis through distinguishable pathways.

Background: Relatively few genes have been shown to directly affect the metastatic phenotype of breast cancer epithelial cells in vivo. The Rho family of proteins, incluing the Rho, Rac and Cdc42 subfamilies, are related to the small GTP binding protein Ras and regulated diverse biological processes including gene transcription, cytoskeletal organization, cell proliferation and transformation. The effects of Cdc42, Rac and Rho on the actin cytoskeleton suggested a possible role for Rho proteins in cellular motility and metastasis; however, a formal analysis of the role of Rho proteins in breast cancer cellular growth and metastasis in vivo had not previously been performed.

Cellular N-Ras promotes cell survival by downregulation of Jun N-terminal protein kinase and p38.

Cellular N-Ras provides a steady-state antiapoptotic signal, at least partially through the regulation of phosphorylated Akt and Bad levels. Fibroblasts lacking c-N-Ras expression are highly sensitive to the induction of apoptosis by a variety of agents. Reduction of pBad and pAkt levels using a phosphatidylinositol 3-kinase inhibitor was not sufficient to sensitize the control cell population to the high level of apoptosis observed in the N-Ras knockout cell lines, suggesting that c-N-Ras provides at least one other antiapoptotic signal.

Involvement of phosphatidylinositol 3-kinase, but not RalGDS, in TC21/R-Ras2-mediated transformation.

Oncogenic Ras and activated forms of the Ras-related protein TC21/R-Ras2 share similar abilities to alter cell proliferation. However, in contrast to Ras, we found previously that TC21 fails to activate the Raf-1 serine/threonine kinase. Thus, TC21 must utilize non-Raf effectors to regulate cell function.

Loss of transgelin in breast and colon tumors and in RIE-1 cells by Ras deregulation of gene expression through Raf-independent pathways.

Activated Ras but not Raf can transform RIE-1 and other epithelial cells, indicating the critical importance of Raf-independent effector function in Ras transformation of epithelial cells. To elucidate the nature of these Raf-independent activities, we utilized representational difference analysis to identify genes aberrantly expressed by Ras through Raf-independent mechanisms in RIE-1 cells. We identified a total of 22 genes, both known and novel, whose expression was either activated or abolished by Ras but not Raf.

Role of MLK3-mediated activation of p70 S6 kinase in Rac1 transformation.

The signaling pathways that mediate the transforming activity of the Rac1 GTPase remain to be determined. In the present study, we used effector domain mutants of the constitutively activated Rac(61L) mutant that display differential transforming activities and differential activation of downstream effector pathways to investigate the contribution of p70 S6 kinase (p70(S6K)) to Rac1 transformation and to decipher the signaling pathways leading from Rac1 to p70(S6K). First, we found that Rac1 transforming activity could be dissociated from Rac1 activation of p70(S6K).

The Nf2 tumor suppressor, merlin, functions in Rac-dependent signaling.

Mutations in the neurofibromatosis type II (NF2) tumor suppressor predispose humans and mice to tumor development. The study of Nf2+/- mice has demonstrated an additional effect of Nf2 loss on tumor metastasis. The NF2-encoded protein, merlin, belongs to the ERM (ezrin, radixin, and moesin) family of cytoskeleton:membrane linkers.