To investigate the molecular basis of PTENmediated tumor suppression, we introduced a null mutation into the mouse Pten gene by homologous recombination in embryonic stem (ES) cells. Pten ؊/؊ ES cells exhibited an increased growth rate and proliferated even in the absence of serum. ES cells lacking PTEN function also displayed advanced entry into S phase. This accelerated G 1 ͞S transition was accompanied by down-regulation of p27 KIP1 , a major inhibitor for G 1 cyclindependent kinases. Inactivation of PTEN in ES cells and in embryonic fibroblasts resulted in elevated levels of phosphatidylinositol 3,4,5,-trisphosphate, a product of phosphatidylinositol 3 kinase. Consequently, PTEN deficiency led to dosagedependent increases in phosphorylation and activation of Akt͞ protein kinase B, a well-characterized target of the phosphatidylinositol 3 kinase signaling pathway. Akt activation increased Bad phosphorylation and promoted Pten ؊/؊ cell survival. Our studies suggest that PTEN regulates the phosphatidylinositol 3,4,5,-trisphosphate and Akt signaling pathway and consequently modulates two critical cellular processes: cell cycle progression and cell survival.The tumor susceptibility gene encoding PTEN͞MMAC1͞TEP1 (1-3) is mutated at high frequency in many primary human cancers and several familial cancer predisposition disorders (4). PTEN contains the sequence motif that is highly conserved in the members of the protein tyrosine phosphatase family. PTEN has been shown in vitro to possess phosphatase activity on phosphotyrosyl, phosphothreonyl-containing substrates (3, 5) and more recently, on phosphatidylinositol 3,4,5-trisphosphate (PIP3), a product of phosphatidylinositol 3 (PI3) kinase (6). Many cancerrelated mutations have been mapped within the conserved catalytic domain of PTEN, suggesting that the phosphatase activity of PTEN is required for tumor suppressor function. In addition, wild-type PTEN, but not mutant derivatives lacking phosphatase activity, suppresses the growth of glioblastoma cells and their tumorigenecity in nude mice (7-9), confirming the functional relevance of the PTEN phosphatase domain for tumor suppression. Very recently, inactivation of PTEN in a mouse model has confirmed the role of PTEN as a bona fide tumor suppressor (10). However, the exact function of PTEN in regulation of cell growth and tumorigenesis remains unclear.In this study, we have investigated the molecular basis underlying the tumor suppression function of PTEN by using a combination of molecular genetic, cell biological, and biochemical approaches. We have identified PIP3, a product of PI3 kinase, as an intracellular target of PTEN. Our studies suggest that PTEN acts as a negative regulator for the PI3-kinase͞Akt signaling pathway, which controls and coordinates two major cellular processes: cell cycle progression and cell death.
PTEN͞MMAC1͞TEP1 is a tumor suppressor that possesses intrinsic phosphatase activity. Deletions or mutations of its encoding gene are associated with a variety of human cancers. However, very little is known about the molecular mechanisms by which this important tumor suppressor regulates cell growth. Here, we show that PTEN expression potently suppressed the growth and tumorigenicity of human glioblastoma U87MG cells. The growth suppression activity of PTEN was mediated by its ability to block cell cycle progression in the G 1 phase. Such an arrest correlated with a significant increase of the cell cycle kinase inhibitor p27 KIP1 and a concomitant decrease in the activities of the G 1 cyclindependent kinases. PTEN expression also led to the inhibition of Akt͞protein kinase B, a serine-threonine kinase activated by the phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathway. In addition, the effect of PTEN on p27 KIP1 and the cell cycle can be mimicked by treatment of U87MG cells with LY294002, a selective inhibitor of PI 3-kinase. Taken together, our studies suggest that the PTEN tumor suppressor modulates G 1 cell cycle progression through negatively regulating the PI 3-kinase͞Akt signaling pathway, and one critical target of this signaling process is the cyclin-dependent kinase inhibitor p27 KIP1 .
Pten (Phosphatase and tensin homolog deleted on chromosome 10) is a recently identified tumor suppressor gene which is deleted or mutated in a variety of primary human cancers and in three cancer predisposition syndromes [1]. Pten regulates apoptosis and cell cycle progression through its phosphatase activity on phosphatidylinositol (PI) 3,4,5-trisphosphate (PI(3,4,5)P(3)), a product of PI 3-kinase [2-5]. Pten has also been implicated in controlling cell migration [6], but the exact mechanism is not very clear. Using the isogenic Pten(+/+) and Pten(-/-) mouse fibroblast lines, here we show that Pten deficiency led to increased cell motility. Reintroducing the wild-type Pten, but not the catalytically inactive Pten C124S or lipid-phosphatase-deficient Pten G129E mutant, reduced the enhanced cell motility of Pten-deficient cells. Moreover, phosphorylation of the focal adhesion kinase p125(FAK) was not changed in Pten(-/-) cells. Instead, significant increases in the endogenous activities of Rac1 and Cdc42, two small GTPases involved in regulating the actin cytoskeleton [7], were observed in Pten(-/-) cells. Overexpression of dominant-negative mutant forms of Rac1 and Cdc42 reversed the cell migration phenotype of Pten(-/-) cells. Thus, our studies suggest that Pten negatively controls cell motility through its lipid phosphatase activity by down-regulating Rac1 and Cdc42.
Microorganisms play an important role in soil phosphorus (P) cycling and regulation of P availability in agroecosystems. However, the responses of the functional and ecological traits of P-transformation microorganisms to long-term nutrient inputs are largely unknown. This study used metagenomics to investigate changes in the relative abundance of microbial Ptransformation genes at four long-term experimental sites that received various inputs of N and P nutrients (up to 39 years). Long-term P input increased microbial P immobilization by decreasing the relative abundance of the P-starvation response gene (phoR) and increasing that of the low-affinity inorganic phosphate transporter gene (pit). This contrasts with previous findings that low-P conditions facilitate P immobilization in culturable microorganisms in short-term studies. In comparison, long-term nitrogen (N) input significantly decreased soil pH, and consequently decreased the relative abundances of total microbial P-solubilizing genes and the abundances of Actinobacteria, Gammaproteobacteria, and Alphaproteobacteria containing genes coding for alkaline phosphatase, and weakened the connection of relevant key genes. This challenges the concept that microbial P-solubilization capacity is mainly regulated by N:P stoichiometry. It is concluded that long-term N inputs decreased microbial P-solubilizing and mineralizing capacity while P inputs favored microbial immobilization via altering the microbial functional profiles, providing a novel insight into the regulation of P cycling in sustainable agroecosystems from a microbial perspective.
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