Role Of Ras Proteins Research Articles

The Role of the Guanosine Nucleotide-Binding Protein in the Corpus Luteum.

Simple SummaryThis review aims to discuss the role of the guanosine nucleotide-binding protein (RAS) family in the biological events that occur during the formation and regression of the corpus luteum in the ovary. RAS proteins mediate extracellular signals, transduce through their receptors via multiple signaling pathways, and regulate a wide array of cellular processes. RAS exhibits a notable function in the regulation of vascular endothelial growth factor, fibroblast growth factor, insulin-like growth factor, angiopoietins (ANPT), and hypoxia-inducible factor (HIF). RAS proteins appear to be involved in several factors that are notably associated with the regulation of the corpus luteum. Further research is necessary to enhance our understanding of the role of the RAS family in the ovarian corpus luteum.The corpus luteum is a temporary endocrine gland in the ovary. In the ovarian cycle, repeated patterns of specific cellular proliferation, differentiation, and transformation occur that accompany the formation and regression of the corpus luteum. Molecular mechanism events in the ovarian microenvironment, such as angiogenesis and apoptosis, are complex. Recently, we focused on the role of RAS protein in the ovarian corpus luteum. RAS protein plays a vital role in the modulation of cell survival, proliferation, and differentiation by molecular pathway signaling. Additionally, reproductive hormones regulate RAS activity in the cellular physiological function of ovarian follicles during pre-ovulatory maturation and ovulation. Thus, we have reviewed the role of RAS protein related to the biological events of the corpus luteum in the ovary.

Direct physical interaction of active Ras with mSIN1 regulates mTORC2 signaling

BackgroundThe mechanistic (or mammalian) target of rapamycin (mTOR), a Ser/Thr kinase, associates with different subunits forming two functionally distinct complexes, mTORC1 and mTORC2, regulating a diverse set of cellular functions in response to growth factors, cellular energy levels, and nutrients. The mechanisms regulating mTORC1 activity are well characterized; regulation of mTORC2 activity, however, remains obscure. While studies conducted in Dictyostelium suggest a possible role of Ras protein as a potential upstream regulator of mTORC2, definitive studies delineating the underlying molecular mechanisms, particularly in mammalian cells, are still lacking.MethodsProtein levels were measured by Western blotting and kinase activity of mTORC2 was analyzed by in vitro kinase assay. In situ Proximity ligation assay (PLA) and co-immunoprecipitation assay was performed to detect protein-protein interaction. Protein localization was investigated by immunofluorescence and subcellular fractionation while cellular function of mTORC2 was assessed by assaying extent of cell migration and invasion.ResultsHere, we present experimental evidence in support of the role of Ras activation as an upstream regulatory switch governing mTORC2 signaling in mammalian cancer cells. We report that active Ras through its interaction with mSIN1 accounts for mTORC2 activation, while disruption of this interaction by genetic means or via peptide-based competitive hindrance, impedes mTORC2 signaling.ConclusionsOur study defines the regulatory role played by Ras during mTORC2 signaling in mammalian cells and highlights the importance of Ras-mSIN1 interaction in the assembly of functionally intact mTORC2.

Genetic analysis of KRAS mutation status in metastatic colorectal cancer patients

Colorectal carcinoma (CRC) represents a serious problem worldwide: in the Slovak republic are diagnosed about 2600 new CRC cases annually and its incidence is increasing. Colorectal cancer patients may succumb to the disease because of local recurrence or local formation of metastasis. Therefore, it is necessary to modulate therapeutic algorithm with new methods, leading to early diagnostic of CRC or changing the existing therapeutic procedures. Recent progresses have been made in understanding of EGFR pathway involved in CRC carcinogenesis, especially the role of Ras protein. Mutations in KRAS oncogene are frequently found in human cancers, particularly colorectal, pancreatic, billiary tract and lung tumors. The presence of the KRAS mutations in metastatic colorectal cancer patients correlates with lack of response to the certain epidemal growth factor receptor (EGFR) inhibitor therapies, such as Panitumumab and Cetuximab. Consequently, screening for KRAS mutations status may be used as a prognostic marker, because the CRC patients with KRAS positive tumors have a worse prognosis. The aim of our study was to establish the methods for rapid and sensitive detection of KRAS mutation status in formalin fixed paraffin embedded (FFPE) tissues DNA. We applied Real Time PCR analysis (TheraScreen KRAS Mutation Test Kit) and sequencing analysis (optimised for the analysis of FFPE tissues) to detect somatic mutations in codon 12 and 13 of KRAS gene. Both methods were used concurrently in the panel of DNA isolated from 25 colorectal FFPE tissues tumor. The positive or negative results from all 25 samples were identified by both methods independently. The KRAS mutations were presented in 8 of 25 patients (32%). Our results demonstrate that the Real Time PCR analysis can be used for detection of somatic KRAS mutations in FFPE clinical samples. However, we also recognize that the sequencing analysis of approximately 200bp amplicons may be used for mutations status screening, but with care of method sensitivity.

Detection of N-RAS and K-RAS in their active GTP-bound form in acute myeloid leukemia without activating RAS mutations

RAS genes, predominantly N-RAS and K-RAS, have been implicated in the pathogenesis of acute myeloid leukemia (AML), due to activating RAS mutations detectable in ∼20% of AML patients. In the present study, RAS proteins were detected in their activated, GTP-bound form, in AML patients (n = 10) not expressing mutated forms of H-RAS, K-RAS and N-RAS. Further analysis revealed the simultaneous presence of N-RAS and K-RAS proteins in the GTP-bound state in seven out of 10 AML samples. In four out of 10 samples the levels of RAS-GTP were comparable to an AML cell line (TF-1) with an activating N-RAS mutation (Q61P). The detection of RAS-GTP in AML patients without RAS mutations further supports a functional role of RAS proteins in the pathogenesis of AML and may explain the observed effects of RAS inhibitors in some AML patients in the absence of activating RAS mutations.

Ras is involved in the negative control of autophagy through the class I PI3-kinase.

Ras proteins exert a pivotal regulatory function in signal transduction involved in cell proliferation and their activation mutation leads to malignant cell transformation. However, the role of Ras proteins in autophagy, an intracellular protein degradation process in cell growth control is unknown. In the present study, we demonstrate that the degradation of long-lived proteins in NIH3T3 cells in response to nutrient starvation was significantly suppressed by oncogenic RasVal12 transformation in a rapamycin (mTOR inhibitor)-sensitive manner. Morphologic observations also show the decrease in the formation of autophagic vacuoles upon the Ras transformation. Furthermore, epidermal growth factor or serum downregulated the protein degradation induced by serum starvation and the dominant-negative RasAsn17 mutant counteracted this suppressive effect, indicating that Ras mediates the growth factor downregulation of autophagy. The suppression of protein degradation by the activated RasVal12 was mediated by the class I phosphatidyl inositol 3-kinase (PI3-kinase), but not either or Raf Ral GDS. Consistent with this, RasVal12 and class I PI3-kinase inhibited the rate of autophagic sequestration of LDH. These data suggest that Ras plays a critical role as a negative regulator for nutrient deprivation-induced autophagy through the class I PI3-kinase signaling pathway.

Ablation of Goalpha overrides G1 restriction point control through Ras/ERK/cyclin D1-CDK activities.

We have generated stable IIC9 cell lines, Goa1 and Goa2, that overexpress full-length antisense Goalpha RNA. As shown previously, expression of antisense Goalpha RNA ablated the alpha subunit of the heterotrimeric G protein, Go, resulting in growth in the absence of mitogen. To better understand this change in IIC9 phenotype, we have characterized the signaling pathway and cell cycle events previously shown to be important in control of IIC9 G1/S phase progression. In this paper we clearly demonstrate that ablation of Goalpha results in growth, constitutively active Ras/ERK, elevated expression of cyclin D1, and constitutively active cyclin D1-CDK complexes, all in the absence of mitogen. Furthermore, these characteristics were abolished by the transient overexpression of the transducin heterotrimeric G protein alpha subunit strongly suggesting the transformation of Goalpha-ablated cells involves Gobetagamma subunits. This is the first study to implicate a heterotrimeric G protein in tumor suppression.

Ras proteins mediate induction of uncoupling protein, IGF-I, and IGF-I receptor in rat fetal brown adipocyte cell lines.

Since Ras proteins are essential intermediates of some insulin-like growth factor I (IGF-I)/insulin signaling pathways, we examined whether Ras proteins mediate the IGF-I-induced uncoupling protein expression. Additionally, the role of Ras proteins on IGF-I and IGF-I receptor expression was studied. IGF-I treatment of fetal brown adipocytes cotransfected with inducible gene constructs of SV40 large T antigen (SV40LTag) and a transforming ras gene induced uncoupling protein expression (UCP) in the absence of expression of the transfected genes. The expression of the dexamethasone-inducible transforming ras gene alone or in combination with the Zn-inducible SV40LTag mimicked the IGF-I effect inducing UCP expression and IGF-I did not induce it further. However, the expression of the Zn-inducible SV40LTag did not increase UCP expression in the absence of IGF-I. Expression of the transfected ras oncogene also induced IGF-I and IGF-I receptor mRNAs, whereas expression of SV40LTag did not increase them. Specific IGF-I binding was also specifically increased by expression of the transfected ras oncogene but was not affected by expression of the SV40LTag construct. These results indicate that Ras proteins mediate the IGF-I-induced effect on UCP expression and play a role in the expression of IGF-I and IGF-I receptor. Therefore, an IGF-I autocrine/paracrine loop might be implicated in the process of thermogenic differentiation of brown adipose tissue by a new mechanism unlike that induced by norepinephrine.

The role of ras proteins in insulin signal transduction.

Ras-proteins are guanine nucleotide binding proteins, which, in the GTP bound state emit a strong mitogenic signal. In the GDP bound state, the protein appears inactive. We have found that stimulation by insulin of cells expressing elevated levels of insulin receptors results in a rapid conversion of Ras-GDP into Ras-GTP. This process is part of the signalling pathway leading to immediate-early gene expression and a mitogenic response. There seems to be no involvement of Ras-GTP formation in the process of insulin stimulated glucose transport. Though the precise mechanism by which Ras is converted to the GTP bound state remains to be established, a tight correlation exists between receptor autophosphorylation and Ras-GTP formation.

Malignant transformation by ras and other oncogenes produces common alterations in inositol phospholipid signaling pathways.

The role of ras proteins in signal transduction was assessed by studying inositol phospholipid metabolism and inositol phospholipid-mediated cellular responsiveness to agonists in cells transformed by ras and other oncogenes. Specific alterations were observed in the inositol phospholipid cycle of ras-transformed fibroblasts, but similar changes were also produced by spontaneous transformation or transformation mediated by either membrane-associated oncogenes, such as src, met, or trk, or cytoplasmic oncogenes, mos and raf; the nuclear oncogenes fos and myc did not produce these changes. The alterations included (i) stimulation of phospholipase A2 activity as indicated by elevated levels of glycerophosphoinositol and nonesterified arachidonic acid and (ii) specific uncoupling between surface receptor-mediated stimulation by platelet-derived growth factor, bombesin, or serum and activation of intracellular phospholipase C. These findings suggest the existence of common biochemical pathways for transformation by cytoplasmic and membrane-associated oncogenes and are not consistent with the hypothesis that 21-kDa ras proteins (p21) are direct or distinct regulatory elements of phospholipase C or phospholipase A2 in inositol phospholipid signal transduction pathways.

Injection of an antibody against a p21 c-Ha-ras protein inhibits cleavage in axolotl eggs.

The presence of a ras protein was demonstrated in cleaving axolotl eggs by selective immunoprecipitation with a polyclonal antibody against a peptide encoded by the c-Ha-ras oncogene, cellular homolog of the v-Ha-ras oncogene of Harvey rat sarcoma virus. Injection of this antibody into axolotl oocytes subjected to progesterone treatment does not prevent meiotic maturation. Injection of the same antibody into a blastomere of axolotl eggs at the 2- or 4-cell stage causes cleavage arrest in the descendants of the injected blastomere. Cytological observations of the injected eggs show, in the arrested blastomeres, enlarged nuclei always surrounded by an intact nuclear envelope and containing uncondensed chromatin. The possible role of ras protein in meiosis and mitosis is discussed.

Microinjection of the ras oncogene protein into PC12 cells induces morphological differentiation

To investigate the possible role of ras proteins in the differentiation process signaled by nerve growth factor, we have microinjected the proto-oncogenic and oncogenic (T24) forms of the human H- ras protein into living rat pheochromocytoma cells (PC12). PC12 cells, which have the phenotype of replicating chromaffin-like cells under normal growth conditions, respond to nerve growth factor by differentiating into nonreplicating sympathetic neuron-like cells. Microinjection of the ras oncogene protein promoted the morphological differentiation of PC12 cells into neuron-like cells. In contrast, microinjection of similar amounts of the proto-oncogene form of the ras protein had no apparent effect on PC12 cells. The induction of morphological differentiation by the ras oncogene protein occurred in the absence of nerve growth factor, was dependent on protein synthesis, and was accompanied by cessation of cell division. Treatment of PC12 cells with nerve growth factor or cAMP analogue prior to injection did not alter the phenotypic changes induced by the ras oncogene protein.