PTEN is a recently identified tumor suppressor inactivated in a variety of cancers such as glioblastoma and endometrial and prostate carcinoma. It contains an amino-terminal phosphatase domain and acts as a phosphatidylinositol 3,4,5-trisphosphate phosphatase antagonizing the activity of the phosphatidylinositol 3-OH kinase. PTEN also contains a carboxyl-terminal domain, and we addressed the role of this region that, analogous to the amino-terminal phosphatase domain, is the target of many mutations identified in tumors. Expression of carboxyl-terminal mutants in PTEN-deficient glioblastoma cells permitted the anchorageindependent growth of the cells that otherwise was suppressed by wild-type PTEN. The stability of these mutants in cells was reduced because of rapid degradation. Although the carboxylterminal region contains regulatory PEST sequences and a PDZ-binding motif, these specific elements were dispensable for the tumor-suppressor function. The study of carboxylterminal point mutations affecting the stability of PTEN revealed that these were located in strongly predicted -strands. Surprisingly, the phosphatase activity of these mutants was affected in correlation with the degree of disruption of these structural elements. We conclude that the carboxyl-terminal region is essential for regulating PTEN stability and enzymatic activity and that mutations in this region are responsible for the reversion of the tumorsuppressor phenotype. We also propose that the molecular conformational changes induced by these mutations constitute the mechanism for PTEN inactivation.PTEN was identified recently as a tumor-suppressor gene located on human chromosome 10q23.3 (1, 2). Deletions or somatic mutations of PTEN occur with high frequency in malignant gliomas (3, 4) and endometrial cancer (5) and with a lower rate in other malignancies such as prostate (6) or small-cell lung cancer (7). Germ-line mutations of PTEN are the cause of Cowden disease, an autosomal-dominant hamartoma syndrome with increased risk for development of tumors in a variety of tissues (8).The PTEN gene contains 9 exons and encodes a 403-aa protein that displays high homology in its N-terminal region to dual-specificity protein phosphatases and also to tensin, a cytoskeleton protein (1,2,9). A recent study demonstrated that PTEN acts as a phospholipid phosphatase dephosphorylating the position D3 of the phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ), which is the direct product of the phosphatidylinositol 3-OH kinase (PI-3 kinase) (10). Subsequent studies have confirmed this finding and have shown that cells lacking wild-type PTEN from PTEN-deficient mice (11), from gliomas (12), or from patients with Cowden disease (13) have elevated levels of PIP 3 . As a result, the activity of protein kinase B (PKB͞Akt) was also elevated in these cells, indicating that PTEN exerts its tumor-suppressor function by negatively regulating the antiapoptotic PI-3 kinase-PKB-signaling pathway.Biological evidence that PTEN acts as a tumor suppressor came from...
By using a plasmid-based transient protein expression system in cultured cels and an in vitro transcription/translation system, we analyzed the proteolytic processing of the putative nonstructural protein region of the precursor polyprotein from a Japanese type of hepatitis C virus. In addition to the previously reported viral proteins, p21 and p70, we identified products of 4 kDa (p4), 27 kDa (p27), 56 kDa (p56), 58 kDa (p58), and 66 kDa (p66). These products were produced in a viral serine proteinase (proteinase 2)-dependent manner from the region downstream of p70 in the precursor polyprotein and were arranged as NH2-p70-p4-p27-p58(p56)-p66-COOH as determined with region-specific antibodies. We showed that p56 was an N-terminally truncated form of p58, which suggested that a small polypeptide of 2 kDa (p2) was produced from the N-terminal part of p58. Cleavage between p4 and p27 was inefficient in vitro and we saw the 31-kDa precursor polypeptide (p31) accumulate. Furthermore, efficient cleavage at this site in vivo required the presence of p58/p56. Immunoprecipitation analysis in vitro also suggested
Gene products of hepatitis C virus (HCV), a possible major causative agent of posttransfusion non-A, non-B hepatitis, are considered to be produced from a precursor polyprotein via proteolytic processing mediated by either host cell or viral proteinases. The presence of HCV serine proteinase has been proposed from analyses of amino acid sequence homology. To examine the processing mechanism of the HCV precursor polyprotein, the amino-terminal region of the putative nonstructural protein region of the HCV genome, containing the serine proteinase motif, was expressed and analyzed by using an in vitro transcription/translation system and a transient expression system in cultured cells. Two distinct proteinase activities which function in the production of a 70-kDa protein (p70) from the precursor polyprotein were detected. One of these proteinase activities, which cleaved the carboxyl (C)-terminal side of p70, required the presence of the serine proteinase motif, which is located in the amino (N)-terminal region of p70. That suggested that the predicted HCV serine proteinase was functional. The other activity, which was responsible for the cleavage of the N-terminal side of p70, required the expression of the region upstream and downstream of that cleavage site, including the p70 serine proteinase domain. From the results of pulse-chase analysis, using proteinase inhibitors coupled with a point mutation analysis, the latter activity was proposed to be a novel zinc-dependent metalloproteinase.
Human cells are known to be more refractory than rodent cells against oncogenic transformation in vitro. To date, the molecular mechanisms underlying such resistance remain largely unknown. The combination of simian virus 40 early region and H-Ras V12 has been effective for transformation of rat embryo fibroblasts, but not for human cells. However, the additional ectopic expression of the telomerase catalytic subunit (hTERT) was reported to be capable of causing transformation of normal human cells. In this study, however, we demonstrate that the combined expression of the above-mentioned three genetic elements is not always sufficient to transform normal human diploid fibroblasts (HDF). Although the expression and function of these introduced genetic elements were essentially the same, among four HDF, TIG-1 and TIG-3 were resistant to transformation. The other two (BJ and IMR-90) showed transformed phenotypes, but they were much restricted compared with rat embryo fibroblasts in expressing simian virus 40 early region and H-Ras V12. In correlation with these phenotypes, TIG-1 and TIG-3 remained diploid after the introduction of these genetic elements, whereas BJ and IMR-90 became highly aneuploid. These results strongly suggest that the lack of telomerase is not the sole reason for the refractory nature of HDF against transformation and that normal human cells have still undefined intrinsic mechanisms rendering them resistant to oncogenic transformation.
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