Biomedical subjects

Jeffrey Settleman

Publications and source records attributed to Jeffrey Settleman.

7 recordsLinked to original sources

PubMed2003

Specific biochemical inactivation of oncogenic Ras proteins by nucleoside diphosphate kinase.

Activating mutations of Ras have been implicated in approximately 30% of human cancers. In every case, the biochemical consequence of such mutations is to disrupt the GTPase activity of Ras and to render Ras resistant to the actions of GTPase activating proteins. Consequently, oncogenic Ras mutants are "locked" in a GTP-bound active state. We detected a potent activity in Escherichia coli extract that can efficiently convert mutationally activated GTP-bound Ras to the inactive GDP-bound form. Purification of the protein responsible for this activity led to the identification of the enzyme nucleoside diphosphate kinase (Ndk). The human orthologue of Ndk is the NM23 metastasis suppressor, which we found to exhibit a similar activity. Purified Ndk effectively inactivates several of the oncogenic forms of Ras that are seen frequently in human cancers, including RasD12, the most commonly detected Ras mutation. Significantly, Ndk does not detectably affect wild-type Ras or an activated form of the Ras-related Rho GTPase. These results demonstrate that it is possible, through biochemical means, to specifically inactivate oncogenic Ras as a potential therapeutic approach to tumors that harbor Ras mutations. Moreover, the results suggest that the loss of NM23 expression that is commonly observed during tumor progression could lead to increased potency of oncogenic Ras proteins.

Escherichia coli↗

PubMed2003

Rnd proteins function as RhoA antagonists by activating p190 RhoGAP.

BACKGROUND: The Rnd proteins Rnd1, Rnd2, and Rnd3 (RhoE) comprise a unique branch of Rho-family G-proteins that lack intrinsic GTPase activity and consequently remain constitutively "active." Prior studies have suggested that Rnd proteins play pivotal roles in cell regulation by counteracting the biological functions of the RhoA GTPase, but the molecular basis for this antagonism is unknown. Possible mechanisms by which Rnd proteins could function as RhoA antagonists include sequestration of RhoA effector molecules, inhibition of guanine nucleotide exchange factors, and activation of GTPase-activating proteins (GAPs) for RhoA. However, effector molecules of Rnd proteins with such properties have not been identified. RESULTS: Here we identify p190 RhoGAP (p190), the most abundant GAP for RhoA in cells, as an interactor with Rnd proteins and show that this interaction is mediated by a p190 region that is distinct from the GAP domain. Using Rnd3-RhoA chimeras and Rnd3 mutants defective in p190 binding, as well as p190-deficient cells, we demonstrate that the cellular effects of Rnd expression are mediated by p190. We moreover show that Rnd proteins increase the GAP activity of p190 toward GTP bound RhoA and, finally, demonstrate that expression of Rnd3 leads to reduced cellular levels of RhoA-GTP by a p190-dependent mechanism. CONCLUSIONS: Our results identify p190 RhoGAPs as effectors of Rnd proteins and demonstrate a novel mechanism by which Rnd proteins function as antagonists of RhoA.

3T3 Cells↗

PubMed2003

Modulation of Rho GTPase signaling regulates a switch between adipogenesis and myogenesis.

Mature adipocytes and myocytes are derived from a common mesenchymal precursor. While IGF-1 promotes the differentiation of both cell types, the signaling pathways that specify the distinct cell fates are largely unknown. Here, we show that the Rho GTPase and its regulator, p190-B RhoGAP, are components of a critical switch in the adipogenesis-myogenesis "decision." Cells derived from embryos lacking p190-B RhoGAP exhibit excessive Rho activity, are defective for adipogenesis, but undergo myogenesis in response to IGF-1 exposure. In vitro, activation of Rho-kinase by Rho inhibits adipogenesis and is required for myogenesis. The activation state of Rho following IGF-1 signaling is determined by the tyrosine-phosphorylation status of p190-B RhoGAP and its resulting subcellular relocalization. Moreover, adjusting Rho activity is sufficient to alter the differentiation program of adipocyte and myocyte precursors. Together, these results identify the Rho GTPase as an essential modulator of IGF-1 signals that direct the adipogenesis-myogenesis cell fate decision.

Adipose Tissue↗

PubMed2003

p190-B RhoGAP regulates mammary ductal morphogenesis.

Previous studies from our laboratory have demonstrated that p190-B RhoGAP (p190-B) is differentially expressed in the Cap cells of terminal end buds (TEBs) and poorly differentiated rodent mammary tumors. Based on these observations we hypothesized that p190-B might play an essential role in invasion of the TEBs into the surrounding fat pad during ductal morphogenesis. To test this hypothesis, mammary development was studied in p190-B-deficient mice. A haploinsufficiency phenotype was observed in p190-B heterozygous mice as indicated by decreased number and rate of ductal outgrowth(s) at 3, 4, and 5 wk of age when compared with their wild-type littermates. This appeared to result from decreased proliferation in the Cap cells of the TEBs, a phenotype remarkably similar to that observed previously in IGF-I receptor null mammary epithelium. Furthermore, decreased expression of insulin receptor substrates 1 and 2 were observed in TEBs of p190-B heterozygous mice. These findings are consistent with decreased IGF signaling observed previously in p190-B-/- mouse embryo fibroblasts. To further assess if this defect was cell autonomous or due to systemic endocrine effects, the mammary anlagen from p190-B+/+, p190-B+/-, and p190-B-/- mice was rescued by transplantation into the cleared fat pad of recipient Rag1-/- mice. Surprisingly, as opposed to 75-80% outgrowths observed using wild-type donor epithelium, only 40% of the heterozygous and none of the p190-B-/- epithelial transplants displayed any outgrowths. Together, these results suggest that p190-B regulates ductal morphogenesis, at least in part, by modulating the IGF signaling axis.

Analysis of Variance↗

PubMed2003

A memory GAP.

Pavlovian conditioning of the response to fear has provided an important model for memory formation in mammals. Several studies have begun to identify the key signal-transduction components that provide the molecular basis for this complex neural process. A recent report has revealed that the Rho GTPase and its regulator p190 Rho-GTPase-activating protein (p190 RhoGAP) also play an important regulatory role in fear memory formation.

Animals↗

PubMed2003

A nuclear MAL-function links Rho to SRF.

The activation of SRF-mediated transcription in response to Rho GTPase-induced actin assembly involves the ability of SRF to sense changes in the cellular level of actin monomers. An article in the May 2 issue of Cell identifies the transcription factor MAL as an actin binding protein that functions as an SRF coactivator and whose translocation from cytoplasm to nucleus depends on its dissociation from actin monomers (Miralles et al., 2003).

Actins↗

PubMed2002

Modulation of CREB activity by the Rho GTPase regulates cell and organism size during mouse embryonic development.

Rho GTPases regulate several aspects of tissue morphogenesis during animal development. We found that mice lacking the Rho-inhibitory protein, p190-B RhoGAP, are 30% reduced in size and exhibit developmental defects strikingly similar to those seen in mice lacking the CREB transcription factor. In p190-B RhoGAP-deficient mice, CREB phosphorylation is substantially reduced in embryonic tissues. Embryo-derived cells contain abnormally high levels of active Rho protein, are reduced in size, and exhibit defects in CREB activation upon exposure to insulin or IGF-1. The cell size defect is rescued by expression of constitutively activated CREB, and in wild-type cells, expression of activated Rho or dominant-negative CREB results in reduced cell size. Together, these results suggest that activity of the Rho GTPase modulates a signal from insulin/IGFs to CREB that determines cell size and animal size during embryogenesis.

Animals↗