IkappaBalpha and IkappaBalpha /NF-kappa B complexes are retained in the cytoplasm through interaction with a novel partner, RasGAP SH3-binding protein 2.

IkappaBalpha inhibits the transcriptional activity of NF-kappaB both in the cytoplasm by preventing the nuclear translocation of NF-kappaB and in the nucleus where it dissociates NF-kappaB from DNA and transports it back to the cytoplasm. Cytoplasmic localization of inactive NF-kappaB/IkappaBalpha complexes is controlled by mutual masking of nuclear import sequences of NF-kappaB p65 and IkappaBalpha and active CRM1-mediated nuclear export. Here, we describe an additional mechanism accounting for the cytoplasmic anchoring of IkappaBalpha or NF-kappaB/IkappaBalpha complexes. The N-terminal domain of IkappaBalpha contains a sequence responsible for the cytoplasmic retention of IkappaBalpha that is specifically recognized by G3BP2, a cytoplasmic protein that interacts with both IkappaBalpha and IkappaBalpha/NF-kappaB complexes. G3BP2 is composed of an N-terminal domain homologous to the NTF2 protein, followed by an acidic domain sufficient for the interaction with the IkappaBalpha cytoplasmic retention sequence, a region containing five PXXP motifs and a C-terminal domain containing RNA-binding motifs. Overexpression of G3BP2 directly promotes retention of IkappaBalpha in the cytoplasm, indicating that subcellular distribution of IkappaBalpha and NF-kappaB/IkappaBalpha complexes likely results from a equilibrium between nuclear import, nuclear export, and cytoplasmic retention. The molecular organization of G3BP2 suggests that this putative scaffold protein might connect the NF-kappaB signal transduction cascade with cellular functions such as nuclear transport or RNA metabolism.

A novel phosphorylation-dependent RNase activity of GAP-SH3 binding protein: a potential link between signal transduction and RNA stability.

A potential p120 GTPase-activating protein (RasGAP) effector, G3BP (RasGAP Src homology 3 [SH3] binding protein), was previously identified based on its ability to bind the SH3 domain of RasGAP. Here we show that G3BP colocalizes and physically interacts with RasGAP at the plasma membrane of serum-stimulated but not quiescent Chinese hamster lung fibroblasts. In quiescent cells, G3BP was hyperphosphorylated on serine residues, and this modification was essential for its activity. Indeed, G3BP harbors a phosphorylation-dependent RNase activity which specifically cleaves the 3'-untranslated region of human c-myc mRNA. The endoribonuclease activity of G3BP can initiate mRNA degradation and therefore represents a link between a RasGAP-mediated signaling pathway and RNA turnover.

Sam68 is a Ras-GAP-associated protein in mitosis.

Sam68 is the major tyrosine-phosphorylated and Src-associated protein in mitotic cells. Sam68 stimulates G1/S transition and this effect is dependent on the integrity of its KH domain (hnRNPK Homology) which confers nucleic acid binding properties. During mitosis, Sam68 undergoes tyrosine phosphorylation, which negatively regulates its nucleic acid binding properties and mediates the interaction of Sam68 with critical SH2-containing signaling proteins such as Grb2, PLC gamma 1 and Ras-GAP. However, the interaction of Ras-GAP with Sam68 has been brought into question, based on the lack of co-immunoprecipitation between Sam68 and Ras-GAP in interphase cells. Here we show that the choice of anti-Ras-GAP antibodies is critical for the detection of Ras-GAP/Sam68 complex formation, and that this interaction is specific for G2/M transition in both NIH3T3 and Src-transformed cells. Such data reinforce the importance of the interaction of Ras-GAP with RNA binding proteins during cell proliferation through its SH2 and SH3 domains.

A role for Sam68 in cell cycle progression antagonized by a spliced variant within the KH domain.

Sam68 is the main tyrosine-phosphorylated and Src-associated protein in mitotic cells. Sam68 exhibits a conserved functional KH (hnRNPK homology) RNA binding domain and binds single strand nucleic acids. Tyrosine phosphorylation mediates the interaction of Sam68 with many SH3- and SH2-containing proteins and negatively regulates its nucleic acid binding properties. But the function and the impact of Sam68 on cell signaling and cell proliferation remains elusive. We report here the identification of a natural isoform of Sam68 with a deletion within the KH domain. This isoform, called Sam68DeltaKH, is specifically expressed at growth arrest upon confluency in normal cells. In cells that do not enter quiescence at confluency such as Src-transformed cells, no recruitment of Sam68DeltaKH is observed. Transfected Sam68DeltaKH inhibits serum-induced DNA synthesis and cyclin D1 expression. Sam68 overcomes these effects, suggesting that isoforms of Sam68 are involved, through KH domain signaling, in cell proliferation, and more precisely in G1/S transition.

TGF-beta 1 and cAMP attenuate cyclin A gene transcription via a cAMP responsive element through independent pathways.

Transforming growth factor beta (TGF-beta) is a potent inhibitor of the proliferation of many cell lines. The expression of Cyclin A is down-regulated by TGF-beta 1 in Chinese hamster lung fibroblasts and most of this effect is mediated at the transcriptional level through a cAMP-responsive element (CRE), but does not require a functional cAMP-dependent protein kinase. However, activation of the cAMP pathway in these cells gives rise to a strong inhibition of proliferation, paralleled by a down-regulation of Cyclin A promoter activity. This effect requires the integrity of the CRE, suggesting a role for CRE-binding proteins in late G1/S controls.

Nuclear import of serum response factor (SRF) requires a short amino-terminal nuclear localization sequence and is independent of the casein kinase II phosphorylation site.

Serum stimulation of resting cells is mediated at least in part at the transcriptional level by the activation of numerous genes among which c-fos constitutes a model. Serum response factor (SRF) forms a ternary complex at the c-fos serum response element (SRE) with an accessory protein p62TCF/Elk-1. Both proteins are the targets of multiple phosphorylation events and their role is still unknown in the amino terminus of SRF. While the transcriptional activation domain has been mapped between amino acids 339 and 508, the DNA-binding and the dimerization domains have been mapped to between amino acids 133-235 and 168-235, respectively, no role has been proposed for the amino-terminal portion of the molecule. We demonstrate in the present work that amino acids 95 to 100 contain a stretch of basic amino acids that are sufficient to target a reporter protein to the nucleus. Moreover, this sequence appears to be the only nuclear localization signal operating in SRF. Finally, whereas the global structure around this putative nuclear location signal is reminiscent of what is found in the SV40 T antigen, the casein kinase II phosphorylation site does not determine the rate of cyto-nuclear protein transport of this protein.

Saccharomyces cerevisiae CDC25 (1028-1589) is a guanine nucleotide releasing factor for mammalian ras proteins and is oncogenic in NIH3T3 cells.

The best characterized yeast guanine nucleotide releasing factor is CDC25, which acts on RAS and thereby stimulates cAMP production in Saccharomyces cerevisiae. In order to determine if CDC25 could be a specific GDP-GTP releasing factor for the mammalian proteins Ha-ras, Ki-ras, and N-ras, its functions were studied both in vitro and in NIH3T3 cells. The 561 amino acid composing the C-terminal domain of CDC25 (CDC25 C-domain) released guanine nucleotides (both GDP and GTP) from Ha-, Ki-, and N-ras but not from Rap1A, Rab5, and Rab11. CDC25 acted on oncogenically activated Ha-ras even if the last 23 amino acids (167-189) of the Ras proteins were not present. CDC25 transformed NIH3T3 cells; its transforming capacity was enhanced by overexpression of wild-type Ha-ras. CDC25 C-domain probably exerts its effects through the activation of cellular Ras proteins. These data suggest that the CDC25 C-domain can function as an upstream activator of Ras proteins in a heterologous system and therefore could be a useful tool to study the regulation of Ras activation by growth factor receptors.

Loss of the G1-S control of cyclin A expression during tumoral progression of Chinese hamster lung fibroblasts.

The expression of cyclin A, one of the key regulators of cell cycle progression in association with cdc2/cdk2 protein kinases and which undergoes cyclic accumulation during the cell cycle, has been investigated in CCL39 Chinese hamster lung fibroblasts and in two transformed variants, A71 and 39Py. Whereas A71 (selected after tumor induction in nude mice) is subject to growth arrest (less than 5% of labeled nuclei after 24 h of serum starvation), 39Py (obtained after transformation by polyoma virus) is not (more than 50% of labeled nuclei). In both cells, cyclin A expression was correlated with establishment of S phase, with a progressive deregulation of its G1 controls. This deregulation was not detected with the two early response genes c-fos and c-myc. The kinetics of accumulation of cyclin A lagged behind that of [3H]thymidine incorporation, thereby questioning a direct role for cyclin A in S phase triggering. Moreover, transforming growth factor beta 1, which is known to inhibit alpha-thrombin or fibroblast growth factor-induced mitogenicity in G0-arrested CCL39 cells, is shown here to down-regulate cyclin A expression in both CCL39 and A71 cells but has no effect on 39Py cells. These data establish cyclin A as a sensitive marker for the loss of growth factor requirement.

The Saccharomyces cerevisiae gene product SDC25 C-domain functions as an oncoprotein in NIH3T3 cells.

Ras proteins in mammalian cells cycle between a GTP-bound 'on' state and a GDP-bound 'off' state. Activation of Ras p21 results from the dissociation of tightly bound GDP and the exchange of bound GDP for GTP. A guanine nucleotide exchange factor is required for this activation. Activation promotes interaction with effector molecules and allows the signal to be transduced. In Saccharomyces cerevisiae, the function of guanine nucleotide exchange has been ascribed to the product of the CDC25 gene. The C-terminus domain of SDC25, a homologue of CDC25, can substitute for the CDC25 protein in yeast. We have demonstrated that the SDC25 C-terminus domain promotes GTP binding to Ras p21 in CHO cells. In the present study, we found that the stable expression of the SDC25 C-terminus domain induced transformation of NIH3T3 cells. Ras proteins in these tumorigenic cells were GTP bound. In addition, the coexpression of wild-type Ha-Ras protein with the SDC25 C-terminus was found to enhance the tumorigenic properties of the NIH3T3 cells. These results imply that, in subsets of human tumours, cellular Ras p21 might be found in its GTP-bound active form as a consequence of an oncogenic activation of a mammalian Ras guanine nucleotide exchange factor.

Implication of GAP in Ras-dependent transactivation of a polyoma enhancer sequence.

Controversy exists as to whether the interaction of a guanosine triphosphatase activating protein (GAP) with Ras proteins functions both to initiate and to terminate Ras-dependent signaling events or only to terminate them. GAP-C, a carboxyl-terminal fragment of GAP that is sufficient to stimulate GTPase activity, inhibited the stimulation of transcription produced by some oncoproteins (v-Src, polyoma middle T, wild-type Ras, and oncogenic Ras) but not that produced by v-Mos. Wild-type GAP did not affect transcription induced by oncogenic Ras but reversed the inhibitory effect of GAP-C on transcription induced by oncogenic Ras. These results indicate that GAP is a negative regulator of wild-type Ras and elicits a downstream signal by interacting with Ras-GTP (guanosine triphosphate).

The COOH-domain of the product of the Saccharomyces cerevisiae SCD25 gene elicits activation of p21-ras proteins in mammalian cells.

The evidence presented here indicates that the domain containing the COOH-terminal part of the Saccharomyces cerevisiae SCD25 gene product (C-domain), which is homologous to the COOH-terminal part of CDC25 protein, can elicit activation of mammalian ras proteins in CHO cells. Transfection of expression vectors carrying the C-domain of SCD25, but not of CDC25, promotes the GTP-bound form of ras proteins as determined by analysis of the guanine nucleotides bound to ras proteins immunoprecipitated by Y13-259 mAb, and enhances transcription of a HIV-LTR-CAT construct. This is the first demonstration of the activation of ras proteins by transfection of a single heterologous gene.