No abstract

Activation of Ras induces a variety of cellular responses depending on the specific effector activated and the intensity and amplitude of this activation. We have previously shown that calmodulin is an essential molecule in the down-regulation of the Ras/Raf/MEK/extracellularly regulated kinase (ERK) pathway in cultured fibroblasts and that this is due at least in part to an inhibitory effect of calmodulin on Ras activation. Here we show that inhibition of calmodulin synergizes with diverse stimuli (epidermal growth factor, platelet-derived growth factor, bombesin, or fetal bovine serum) to induce ERK activation. Moreover, even in the absence of any added stimuli, activation of Ras by calmodulin inhibition was observed. To identify the calmodulin-binding protein involved in this process, calmodulin affinity chromatography was performed. We show that Ras and Raf from cellular lysates were able to bind to calmodulin. Furthermore, Ras binding to calmodulin was favored in lysates with large amounts of GTP-bound Ras, and it was Raf independent. Interestingly, only one of the Ras isoforms, K-RasB, was able to bind to calmodulin. Furthermore, calmodulin inhibition preferentially activated K-Ras. Interaction between calmodulin and K-RasB is direct and is inhibited by the calmodulin kinase II calmodulin-binding domain. Thus, GTP-bound K-RasB is a calmodulin-binding protein, and we suggest that this binding may be a key element in the modulation of Ras signaling.

The SET protein and the cell cycle inhibitor p21 Cip1 interact in vivo and in vitro. We identified here the domain 157 LIF 159 of p21 Cip1 as essential for the binding of SET. We also found that SET contains at least two domains of interaction with p21Cip1 , one located in the fragment amino acids 81-180 and the other one in the fragment including amino acids 181-277. SET and p21Cip1 co-localize in the cell nucleus in a temporal manner. Overexpression of SET blocks the cell cycle at the G 2 /M transition in COS and HCT116 cells. Moreover, SET inhibits cyclin B-CDK1 activity both in vivo and in vitro in both cell types. This effect is specific for these complexes since SET did not inhibit either cyclin A-CDK2 or cyclin E-CDK2 complexes. SET and p21Cip1 cooperate in the inhibition of cyclin B-CDK1 activity. The inhibitory effect of SET resides in its acidic C terminus, as demonstrated by the ability of this domain to inhibit cyclin B-CDK1 activity and by the lack of blocking G 2 /M transition when a mutated form of SET lacking this C terminus domain was overexpressed in COS cells. These results indicate that SET might regulate G 2 /M transition by modulating cyclin B-CDK1 activity.

Resistance to TGF-b1 occurred in pancreatic cancer cells suggesting that inactivation of TGF-b inhibitory signaling pathways may play an important role in human pancreatic cancer. The aim of our study was to determine the presence of alterations in the main putative components of the TGF-b inhibitory signaling pathways (p15, Smad4, Smad2, TGFb-RII, CDC25A). A panel of human carcinomas of the exocrine pancreas orthotopically implanted and perpetuated in nude mice and pancreatic cancer cell lines were studied. p15 gene alterations, mainly homozygous deletions that involved exons 1 and/or 2, were found in the 62.5% (5 of 8) of pancreatic xenografts whereas Smad4 gene aberrations were found in one of eight xenografts and in two of seven cell lines. Additional aberrations in these genes were acquired during in vivo perpetuation and distal dissemination. Paradoxically, TGFb-RII overexpression and a decrease in CDC25A protein levels were found in all tumors and cell lines. In one cell line, resistance to TGFb1 occurred in the absence of alterations in the genes analysed so far. We conclude that all human pancreatic tumor cells analysed herein have non-functional TGF-b pathways. The majority of cells harbor alterations in at least one of the putative components of TGF-b pathways, mainly in p15 and Smad4 genes. These results suggest that inactivation of TGF-b signaling pathways plays an important role in human pancreatic tumorigenesis.

We report here that different cell stresses regulate the stability of cyclin D1 protein. Exposition of Granta 519 cells to osmotic shock, oxidative stress, and arsenite induced the post-transcriptional down-regulation of cyclin D1. In the case of osmotic shock, this effect was completely reversed by the addition of p38 SAPK2 -specific inhibitors (SB203580 or SB220025), indicating that this effect is dependent on p38 SAPK2 activity. Moreover, the use of proteasome inhibitors prevented this down-regulation. Thus, osmotic shock induces proteasomal degradation of cyclin D1 protein by a p38 SAPK2 -dependent pathway. The effect of p38 SAPK2 on cyclin D1 stability might be mediated by direct phosphorylation at specific sites. We found that p38 SAPK2 phosphorylates cyclin D1 in vitro at Thr 286 and that this phosphorylation triggers the ubiquitination of cyclin D1. These results link for the first time a stress-induced MAP kinase pathway to cyclin D1 protein stability, and they will help to understand the molecular mechanisms by which stress transduction pathways regulate the cell cycle machinery and take control over cell proliferation.Mammalian cell cycle progression depends on the sequential activation of different members of a family of serine-threonine kinases named cyclin-dependent kinases (CDKs). 1 The activity of these kinases is positively regulated by cyclin binding, and phosphorylation by the CDK-activating kinase. The activity is also modulated negatively by phosphorylation at specific residues of the CDKs and by the binding of CDK inhibitors (1-3). Progression through G 1 phase is controlled first by CDK4 and CDK6, which bind combinatorially to cyclins D1, D2, and D3; and later on by CDK2, which associates with cyclin E (4). During G 1 these complexes are responsible for the phosphorylation of different members of the pocket proteins family, which includes the retinoblastoma protein, p107, and p130 (5-8). The hyperphosphorylation of the pocket proteins leads to the transactivation of genes that are necessary for the onset and progression of DNA replication (9 -11).Quiescent cells contain low levels of D-type cyclins. After growth factor stimulation, their synthesis is induced, and then cyclin D1-CDK4/6 complexes can be formed during G 1 (12, 13). Cyclin D1 expression, assembly of cyclin D-CDK complexes, and their nuclear translocation require the activation of Ras, Raf1, MAP kinase kinase 1/2, ERKs, and the transcription factor c-Ets-2 (14 -17). The maintenance of active cyclin D1-CDK4/6 complexes requires persistent mitogenic signaling, and mitogen withdrawal cancels cyclin D1 synthesis and cyclin D1-CDK4 complexes rapidly dissipate (18). Cyclin D1 turnover is regulated by degradation, mediated by phosphorylation of a specific threonine residue (Thr 286 ) located near the carboxyl terminus. This phosphorylation promotes its polyubiquitination and subsequent degradation by the 26 S proteasome (19). Recently, an alternative mechanism of cyclin D1 ubiquitination, independent of Thr 286 phosphorylation, has be...

The fermented extract of wheat germ, trade name Avemar, is a complex mixture of biologically active molecules with potent anti-metastatic activities in various human malignancies. Here we report the effect of Avemar on Jurkat leukemia cell viability, proliferation, cell cycle distribution, apoptosis, and the activity of key glycolytic/pentose cycle enzymes that control carbon flow for nucleic acid synthesis. The cytotoxic IC 50 concentration of Avemar for Jurkat tumor cells is 0.2 mg/ml, and increasing doses of the crude powder inhibit Jurkat cell proliferation in a dose-dependent fashion. At concentrations higher than 0.2 mg/ml, Avemar inhibits cell growth by more than 50% (72 h of incubation), which is preceded by the appearance of a sub-G 1 peak on flow histograms at 48 h. Laser scanning cytometry of propidium iodideand annexin V-stained cells indicated that the growthinhibiting effect of Avemar was consistent with a strong induction of apoptosis. Inhibition by benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone of apoptosis but increased proteolysis of poly(ADP-ribose) indicate caspases mediate the cellular effects of Avemar. Activities of glucose-6-phosphate dehydrogenase and transketolase were inhibited in a dose-dependent fashion, which correlated with decreased 13 C incorporation and pentose cycle substrate flow into RNA ribose. This decrease in pentose cycle enzyme activities and carbon flow toward nucleic acid precursor synthesis provide the mechanistic understanding of the cell growth-controlling and apoptosis-inducing effects of fermented wheat germ. Avemar exhibits about a 50-fold higher IC 50 (10.02 mg/ml) for peripheral blood lymphocytes to induce a biological response, which provides the broad therapeutic window for this supplemental cancer treatment modality with no toxic effects.The preventive and therapeutic potential of two natural wheat products, wheat bran and fermented wheat germ (Avemar), in experimental carcinogenesis has recently been described (1, 2). Although no chemical constituents are yet isolated and tested experimentally, it is likely that benzoquinones and wheat germ agglutinin in wheat germ and fiber and lipids and phytic acid in wheat bran play a significant role in exerting anti-carcinogenic effects. In a recent report utilizing intracellular carbon flow studies with a 13 C-labeled isotope of glucose and biological mass spectrometry (GC/MS), 1 it was demonstrated that the crude powder of fermented wheat germ dosedependently inhibits nucleic acid ribose synthesis primarily through the nonoxidative steps of the pentose cycle while increasing direct glucose carbon oxidation and acetyl-CoA utilization toward fatty acid synthesis in pancreatic adenocarcinoma cells (3). These metabolic changes indicate that fermented wheat germ exerts its anti-proliferative action through altering metabolic enzyme activities, which primarily control glucose carbon flow toward nucleic acid synthesis.In vivo, Avemar has a marked inhibitory effect on metastasis formation in tumor-bearing animals (4), an...

We previously showed that K-Ras is a calmodulin-binding protein. Involvement of this interaction in anterograde and retrograde transport of K-Ras was then suggested. To test this we have analyzed here the domains of K-Ras essential for the interaction with calmodulin. At least three different regions in the K-Ras molecule were important; they are the hypervariable region, the ␣-helix between amino acids 151 and 166, and the Switch II. Within the hypervariable region, both the hydrophobic farnesyl group and the positive-charged amino acids were essential for the interaction between K-Ras and calmodulin in cellular extracts. Consistently, K-Ras S181D, which mimics phosphorylation of Ser-181 of K-Ras, also completely abolished binding to calmodulin. K-Ras mutants correctly farnesylated that did not bind calmodulin were all located at plasma membrane, showing that calmodulin interaction was not required for the transport of K-Ras to plasma membrane. In NIH3T3 cells, K-Ras and calmodulin colocalized mainly in the plasma membrane even after the addition of Ca 2؉ ionophore, indicating that interaction did not directly lead to K-Ras internalization. Furthermore, using a K-Ras with impaired binding to calmodulin but with membrane localization, we could demonstrate in striatal neurones that interaction between K-Ras and calmodulin was not required for Golgi K-Ras translocation induced by Ca 2؉

Ras proteins are small guanosine triphosphatases involved in the regulation of important cellular functions such as proliferation, differentiation, and apoptosis. Understanding the intracellular trafficking of Ras proteins is crucial to identify novel Ras signaling platforms. In this study, we report that epidermal growth factor triggers Kirsten Ras (KRas) translocation onto endosomal membranes (independently of calmodulin and protein kinase C phosphorylation) through a clathrin-dependent pathway. From early endosomes, KRas but not Harvey Ras or neuroblastoma Ras is sorted and transported to late endosomes (LEs) and lysosomes. Using yellow fluorescent protein–Raf1 and the Raichu-KRas probe, we identified for the first time in vivo–active KRas on Rab7 LEs, eliciting a signal output through Raf1. On these LEs, we also identified the p14–MP1 scaffolding complex and activated extracellular signal-regulated kinase 1/2. Abrogation of lysosomal function leads to a sustained late endosomal mitogen-activated protein kinase signal output. Altogether, this study reveals novel aspects about KRas intracellular trafficking and signaling, shedding new light on the mechanisms controlling Ras regulation in the cell.