It has long been postulated that protein tyrosine phosphatases may act as tumor suppressors because of their ability to counteract the oncogenic actions of protein tyrosine kinases. The cloning and characterization of a novel human protein tyrosine phosphatase, TEP1, is reported. TEP1 contains the protein tyrosine phosphatase signature motif, and it possesses an intrinsic protein tyrosine phosphatase activity. TEP1 also shares extensive homology with tensin, a cytoskeletal protein localized to focal adhesions, and with auxilin (see Drosophila auxilin), a protein involved in synaptic vesicle transport. Immunofluorescence studies show that TEP1 is a cytoplasmic protein. The abundance of TEP1 transcription is altered in many transformed cells. In the transforming growth factor beta-sensitive cells, TEP1 expression is rapidly down-regulated by transforming growth factor beta, a cytokine shown to be involved in regulating cell adhesion and cell motility. The gene encoding TEP1 maps to chromosome 10q23, a locus that is frequently deleted in a variety of human cancers. TEP1 protein is identical to the protein encoded by the candidate tumor suppressor gene PTEN/MMAC1. Functional studies of the TEP1 protein suggest that its tumor suppressor function may associate with its intrinsic protein tyrosine phosphatase activity and its cytoplasmic localization (D. Li, 1997).
In Saccharomyces cerevisiae the CDC14 gene is essential for cell cycle progression. Strains carrying the cdc14-1(ts) allele enter the cell cycle and arrest at restrictive temperatures. Two human cDNAs have been identified encoding proteins which share sequence identity to the yeast CDC14p. The cell cycle arrest in cdc14-1(ts) can be specifically complemented by the human cDNAs suggesting that they are functionally equivalent to the yeast CDC14 gene. Another clone identified in the search for human CDC14-like proteins corresponds to the putative tumor suppressor gene PTEN/MMAC1. Analysis of the PTEN/MMAC1 shows that it does not complement the cdc14-1(ts) allele and that it is more closely related to the yeast open reading frame YNL128W. Human CDC14p and PTEN/MMAC1 were expressed as recombinant proteins, and both were shown to have kinetic properties characteristic of dual specific phosphatases. The human CDC14p is localized in the nucleus while PTEN/MMAC1 has been reported to be localized in the cytoplasm. These results suggest that CDC14 and YNL128W/PTEN/MMAC1 represent two related, but distinct, families of human and yeast phosphatases (L. Li, 1997).
Shallow gradients of chemoattractants, sensed by G protein-linked signaling pathways, elicit localized binding of PH domains specific for PI(3,4,5)P3 at sites on the membrane where rearrangements of the cytoskeleton and pseudopod extension occur. Disruption of the PI 3-phosphatase, PTEN, in Dictyostelium discoideum dramatically prolongs and broadens the PH domain relocation and actin polymerization responses, causing the cells lacking PTEN to follow a circuitous route toward the attractant. Exogenously expressed PTEN-GFP localizes to the surface membrane at the rear of the cell. Membrane localization requires a putative PI(4,5)P2 binding motif and is required for chemotaxis. These results suggest that specific phosphoinositides direct actin polymerization to the cell's leading edge and regulation of PTEN through a feedback loop plays a critical role in gradient sensing and directional migration (Iijima, 2002).
Studies in Dictyostelium and in mammalian leukocytes have shown that chemoattractant receptors and G protein subunits remain distributed uniformly on the perimeter of a chemotaxing cell and therefore, the signaling pathways must be selectively activated on the cell's leading edge. In an experiment that first visualized localized activation, the PH domain of cytosolic regulator of adenylyl cyclase (Crac) has been shown to selectively translocate to the membrane at the front of a chemotaxing D. discoideum cell. It is likely that Crac is recruited to the membrane by binding to specific phosphoinositides. Other PH domains with the same phosphoinositide binding specificity as Crac have subsequently been shown to respond similarly to chemoattractants in D. discoideum and leukocytes. The profile of PH domains that reports the directional response most effectively suggests changes occur in the local concentrations of phosphatidylinositol 3,4,5 trisphosphate (PI(3,4,5)P3) and phosphatidylinositol 3,4 bisphosphate (PI(3,4)P2). These observations have led to the proposal that the determination of the directional response occurs within the signaling pathway and that the activity of the cytoskeleton is locally controlled by these phosphoinositides (Iijima, 2002 and references therein).
Exposure of cells to a uniform increment in chemoattractant results in transient increases in phosphoinositides, polymerized actin, cAMP, cGMP, IP3, and Ca+2 influx, phosphorylation of myosins as well as rapid changes in cell shape. Evidence suggests that the G protein ßgamma complex propagates the signal to these responses, although the identities of its direct and relevant effectors are unknown. It is possible that the G protein couples to only a few effector enzymes and that an increase in a single messenger regulates multiple downstream responses. In fact, many responses display similar kinetics and several are known to occur at the leading edge of the cell, suggesting a level of coordination. It has been proposed that specific phosphoinositides act as a 'node' in the chemoattractant signaling pathway and that downstream components bearing PH domains and involved in pseudopodia extension are directed to the site on the membrane marked by highest concentration of these lipids (Iijima, 2002).
Thus far, a causal relationship between the elicited appearance of specific phosphoinositides, actin polymerization, other physiological responses, and directional pseudopod extension has not been established. Evidence suggesting a role of phosphatidylinositol 3-kinases (PI3K) in directed migration has been confusing. D. discoideum cells lacking multiple PI3Ks have alternately been described as displaying either faster or impaired chemotaxis. Neutrophils from mice lacking PI3Kgamma show defective chemotaxis, while lymphocytes do not show strong defects. Similarly, inconsistent results have been obtained with the PI3K inhibitors, LY294002 and wortmannin, which have been reported to either inhibit or have little effect on chemotaxis. Furthermore, actin polymerization, the salient response underlying pseudopod extension, is unaffected by mutations or inhibitors of PI3K. Disruption of the PI 3-phophatase, PTEN, and the PI 5-phosphatase, SHIP, in mice, D. melanogaster, and C. elegans has been reported to cause dramatic pleiotropic phenotypes, including reports of enhanced cytoskeletal activity (Iijima, 2002).
The cells lacking PTEN activity are defective in both polarization and directional sensing, suggesting that similar biochemical mechanisms underlie both processes. The pten- cells are able to weakly sense the direction of a chemotactic gradient since membrane-associated phosphoinositides, reflected as PHCrac-GFP binding, are broadly distributed to the front half of the cell. In addition, the large activation of physiological responses in pten- cells induced by uniform stimulation eventually subsides. This implies that the phosphoinositide synthetic activity (i.e., PI3K activity) must still be regulated in the pten- cells and that other lipid-degrading enzymes (such as PI 4- or PI 5-phosphatase) operate to remove phosphoinositides. This regulation of PI3K activity is apparently sufficient to mediate rudimentary directional sensing in the absence of PTEN function. In polarized cells, chemoattractant receptors are uniformly distributed on the membrane. However, G protein ß subunits are localized in a shallow anterior-posterior gradient, and there is a faster frequency of receptor-chemoattractant interaction at the front of a polarized cell. The accumulation of PTEN-GFP at the rear of a cell is expected to influence the distribution of phosphoinositides on the cell membranes and may therefore also strongly contribute to polarization. Indeed, PTEN shares homology with tensin, suggesting it might associate with cytoskeletal components. Thus, polarization may result from a spontaneous redistribution of the same signal transduction components that are involved in directional sensing (Iijima, 2002).
The mechanisms of leading edge formation in chemotaxing Dictyostelium cells have been investigated. While phosphatidylinositol 3-kinase (PI3K) transiently translocates to the plasma membrane in response to chemoattractant stimulation and to the leading edge in chemotaxing cells, PTEN, a negative regulator of PI3K pathways, exhibits a reciprocal pattern of localization. By uniformly localizing PI3K along the plasma membrane, it has been shown that chemotaxis pathways are activated along the lateral sides of cells and PI3K can initiate pseudopod formation, providing evidence for a direct instructional role of PI3K in leading edge formation. These findings provide evidence that differential subcellular localization and activation of PI3K and PTEN is required for proper chemotaxis (Funamoto, 2002).
The phosphatidylinositol 3-kinase (PI3K) signaling pathway is a conserved signal transduction cascade that is fundamental for the correct development of the nervous system. The major negative regulator of PI3K signaling is the lipid phosphatase DAF-18/PTEN, which can modulate PI3K pathway activity during neurodevelopment. This study identified a novel role for DAF-18 in promoting neurite outgrowth during development in C. elegans. DAF-18 modulates the PI3K signaling pathway to activate DAF-16/FOXO and promote developmental neurite outgrowth. This activity of DAF-16 in promoting outgrowth is isoform-specific, being effected by the daf-16b isoform but not the daf-16a or daf-16d/f isoform. It was also demonstrated that the capacity of DAF-16/FOXO in regulating neuron morphology is conserved in mammalian neurons. These data provide a novel mechanism by which the conserved PI3K signaling pathway regulates neuronal cell morphology during development through FOXO (Christensen, 2011).
The ability of injured axons to regenerate declines with age, yet the mechanisms that regulate axon regeneration in response to age are not known. This study shows that axon regeneration in aging C. elegans motor neurons is inhibited by the conserved insulin/IGF1 receptor DAF-2. DAF-2's function in regeneration is mediated by intrinsic neuronal activity of the forkhead transcription factor DAF-16/FOXO. DAF-16 regulates regeneration independently of lifespan, indicating that neuronal aging is an intrinsic, neuron-specific, and genetically regulated process. In addition, DAF-18/PTEN was found to inhibit regeneration independently of age and FOXO signaling via the TOR pathway. Finally, DLK-1, a conserved regulator of regeneration, is downregulated by insulin/IGF1 signaling, bound by DAF-16 in neurons, and required for both DAF-16- and DAF-18-mediated regeneration. Together, these data establish that insulin signaling specifically inhibits regeneration in aging adult neurons and that this mechanism is independent of PTEN and TOR (Byrne, 2004).
Phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) is a key molecule involved in cell growth signaling. Overexpression of PTEN, a putative tumor suppressor, reduces insulin-induced PtdIns(3,4,5)P3 production in human 293 cells without effecting insulin-induced phosphoinositide 3-kinase activation. Further, transfection of the catalytically inactive mutant of PTEN (C124S) caused PtdIns(3,4,5)P3 accumulation in the absence of insulin stimulation. Purified recombinant PTEN catalyzes dephosphorylation of PtdIns(3,4,5)P3, specifically at position 3 on the inositol ring. PTEN also exhibites 3-phosphatase activity toward inositol 1,3,4,5-tetrakisphosphate. These results raise the possibility that PTEN acts in vivo as a phosphoinositide 3-phosphatase by regulating PtdIns(3,4,5)P3 levels. As expected, the C124S mutant of PTEN is incapable of catalyzing dephosphorylation of PtdIns(3,4,5)P3 consistent with the mechanism observed in protein-tyrosine phosphatase-catalyzed reactions (Maehama, 1998).
The PTEN structure, revealed by X-ray crystallography, reveals a phosphatase domain that is similar to protein phosphatases but has an enlarged active site important for the accommodation of the phosphoinositide substrate. The structure also reveals that PTEN has a C2 domain. The PTEN C2 domain binds phospholipid membranes in vitro, and mutation of basic residues that could mediate this reduces PTEN's membrane affinity and its ability to suppress the growth of glioblastoma tumor cells. The phosphatase and C2 domains associate across an extensive interface, suggesting that the C2 domain may serve to productively position the catalytic domain on the membrane (Lee, 1999).
PTEN is a tumor suppressor gene mutated in human cancers. Although many mutations target the phosphatase domain, others create a truncated protein lacking the C-terminal PDZ-binding motif or a protein that extends beyond the PDZ-binding motif. Using the yeast two-hybrid system, a membrane-associated guanylate kinase family protein was isolated with multiple PDZ domains [AIP-1 (atrophin interacting protein 1), renamed MAGI-2 (membrane associated guanylate kinase inverted-2)]. MAGI-2 contains eight potential protein-protein interaction domains and is localized to tight junctions in the membrane of epithelial cells. PTEN binds to MAGI-2 through an interaction between the PDZ-binding motif of PTEN and the second PDZ domain of MAGI-2. MAGI-2 enhances the ability of PTEN to suppress Akt activation. Furthermore, certain PTEN mutants have reduced stability, which is restored by adding the minimal PDZ-binding motif back to the truncated protein. It is proposed that MAGI-2 improves the efficiency of PTEN signaling through assembly of a multiprotein complex at the cell membrane (Wu, 2000).
The PTEN tumor suppressor is frequently affected in cancer cells, and inherited PTEN mutation causes cancer-susceptibility conditions such as Cowden syndrome. PTEN acts as a plasma-membrane lipid-phosphatase antagonizing the phosphoinositide 3-kinase/AKT cell survival pathway. However, PTEN is also found in cell nuclei, but mechanism, function, and relevance of nuclear localization remain unclear. This study shows that nuclear PTEN is essential for tumor suppression and that PTEN nuclear import is mediated by its monoubiquitination. A lysine mutant of PTEN, K289E associated with Cowden syndrome, retains catalytic activity but fails to accumulate in nuclei of patient tissue due to an import defect. This and another lysine residue have been identified as major monoubiquitination sites essential for PTEN import. While nuclear PTEN is stable, polyubiquitination leads to its degradation in the cytoplasm. Thus, cancer-associated mutations of PTEN have been identified that target its posttranslational modification, and it has been demonstrated how a discrete molecular mechanism dictates tumor progression by differentiating between degradation and protection of PTEN (Trotman, 2007).
The tumor suppressor PTEN, a critical regulator for multiple cellular processes, is mutated or deleted frequently in various human cancers. Subtle reductions in PTEN expression levels have profound impacts on carcinogenesis. This study shows that PTEN level is regulated by ubiquitin-mediated proteasomal degradation, and its ubiquitin ligase has been purified as HECT-domain protein NEDD4-1. In cells NEDD4-1 negatively regulates PTEN stability by catalyzing PTEN polyubiquitination. Consistent with the tumor-suppressive role of PTEN, overexpression of NEDD4-1 potentiates cellular transformation. Strikingly, in a mouse cancer model and multiple human cancer samples where the genetic background of PTEN was normal but its protein levels were low, NEDD4-1 is highly expressed, suggesting that aberrant upregulation of NEDD4-1 can posttranslationally suppress PTEN in cancers. Elimination of NEDD4-1 expression inhibits xenotransplanted tumor growth in a PTEN-dependent manner. Therefore, NEDD4-1 is a potential proto-oncogene that negatively regulates PTEN via ubiquitination, a paradigm analogous to that of Mdm2 and p53 (Wang, 2007).
PTEN is a tumor suppressor whose function is frequently lost in human cancer. It possesses a lipid phosphatase activity that represses the activation of PI3 kinase/Akt signaling, leading to decreased cell growth, proliferation, and survival. The potential for PTEN to regulate transcription of the large rRNAs by RNA polymerase I (RNA Pol I) was investigated. Since increased synthesis of rRNAs is a hallmark of neoplastic transformation, the ability of PTEN to control the transcription of rRNAs might be crucial for its tumor suppressor function. The expression of PTEN in PTEN-deficient cells represses RNA Pol I transcription, while decreasing PTEN expression enhances transcription. PTEN-mediated repression requires its lipid phosphatase activity and is independent of the p53 status of the cell. This event can be uncoupled from PTEN's ability to regulate the cell cycle. RNA Pol I is regulated through PI3 kinase/Akt/mammalian target of rapamycin/S6 kinase, and the expression of constitutively activated S6 kinase is able to abrogate transcription repression by PTEN. No change in the expression of the RNA Pol I transcription components, upstream binding factor or SL1 (which consists of TATA-binding protein (TBP) and three associated factors (TAFIs) was observed upon PTEN expression. However, chromatin immunoprecipitation assays demonstrate that PTEN differentially reduces the occupancy of the SL1 subunits on the rRNA gene promoter. Furthermore, PTEN induces dissociation of the SL1 subunits. Together, these results demonstrate that PTEN represses RNA Pol I transcription through a novel mechanism that involves disruption of the SL1 complex (Zhang, 2005).
No apparent change was found in the expression of SL1 or UBF proteins upon PTEN induction. Therefore, potential alterations in the RNA Pol I transcription components and SL1/UBF interactions were examined at the rRNA gene promoter responsible for the reduction in transcription. At 6 h following PTEN induction, a selective reduction was observed in the occupancy of TBP on the endogenous rRNA gene promoter. These results are consistent with a previous study showing that induction of RNA Pol I transcription by insulin-like growth factor-dependent activation of PI3 kinase signaling is associated with enhanced occupancy of TBP on rRNA gene promoters. Surprisingly, however, the decrease in promoter occupancy of TBP is not correlated with a decrease in binding of the other SL1 components, suggesting that PTEN expression induces the dissociation of the SL1 complex. Coimmunoprecipitation assays further support this idea, indicating that PTEN leads to a decreased association of TBP with the SL1 TAFs. This initial event is independent of the phosphorylation state of TBP or TAFI110. Thus, these studies provide new evidence that the formation or stability of the SL1 complex itself can limit the rate of rRNA gene synthesis (Zhang, 2005).
Chromatin organization and dynamics are integral to global gene transcription. Histone modification influences chromatin status and gene expression. PTEN plays multiple roles in tumor suppression, development, and metabolism. This study, performed with HeLa cells, reports on the interplay of PTEN, histone H1, and chromatin. Loss of PTEN leads to dissociation of histone H1 from chromatin and decondensation of chromatin. PTEN deletion also results in elevation of histone H4 acetylation at lysine 16, an epigenetic marker for chromatin activation. PTEN and histone H1 physically interact through their C-terminal domains. Disruption of the PTEN C terminus promotes the chromatin association of MOF acetyltransferase and induces H4K16 acetylation. Hyperacetylation of H4K16 impairs the association of PTEN with histone H1, which constitutes regulatory feedback that may reduce chromatin stability. These results demonstrate that PTEN controls chromatin condensation, thus influencing gene expression. It is proposed that PTEN regulates global gene transcription profiling through histones and chromatin remodeling (Chen, 2014: PubMed).
The PTEN/PI3K signaling pathway regulates a vast array of fundamental cellular responses. Cardiomyocyte-specific inactivation of tumor suppressor PTEN results in hypertrophy, and unexpectedly, a dramatic decrease in cardiac contractility. Analysis of double-mutant mice has revealed that the cardiac hypertrophy and the contractility defects can be genetically uncoupled. PI3Kalpha mediates the alteration in cell size while PI3Kgamma acts as a negative regulator of cardiac contractility. Mechanistically, PI3Kgamma inhibits cAMP production and hypercontractility can be reverted by blocking cAMP function. These data show that PTEN has an important in vivo role in cardiomyocyte hypertrophy and GPCR signaling and identify a function for the PTEN-PI3Kgamma pathway in the modulation of heart muscle contractility (Crackower, 2002).
The tumor suppressor gene PTEN, which is frequently mutated in human cancers, encodes a lipid phosphatase for phosphatidylinositol 3,4,5-triphosphate and antagonizes phosphatidylinositol 3 kinase. Primordial germ cells (PGCs), which are the embryonic precursors of gametes, are the source of testicular teratoma. To elucidate the intracellular signaling mechanisms that underlie germ cell differentiation and proliferation, mice with a PGC-specific deletion of the Pten gene were generated. Male mice that lacked PTEN exhibit bilateral testicular teratoma, which results from impaired mitotic arrest and outgrowth of cells with immature characters. Experiments with PTEN-null PGCs in culture have revealed that these cells have greater proliferative capacity and enhance pluripotent embryonic germ (EG) cell colony formation. PTEN appears to be essential for germ cell differentiation and an important factor in testicular germ cell tumor formation (Kimura, 2003).
PTEN is a direct antagonist of phosphatidylinositol 3 kinase. Pten is a well recognized tumor suppressor and is one of the most commonly mutated genes in human malignancies. More recent studies of development and stem cell behavior have shown that PTEN regulates the growth and differentiation of progenitor cells. Significantly, PTEN is found in osteoprogenitor cells that give rise to bone-forming osteoblasts; however, the role of PTEN in bone development is incompletely understood. To define how PTEN functions in osteoprogenitors during bone development, Pten was conditionally deleted in mice using the cre-deleter strain Dermo1cre, which targets undifferentiated mesenchyme destined to form bone. Deletion of Pten in osteoprogenitor cells led to increased numbers of osteoblasts and expanded bone matrix. Significantly, osteoblast development and synthesis of osteoid in the nascent bone collar was uncoupled from the usual tight linkage to chondrocyte differentiation in the epiphyseal growth plate. The expansion of osteoblasts and osteoprogenitors was found to be due to augmented FGF signaling as evidenced by (1) increased expression of FGF18, a potent osteoblast mitogen, and (2) decreased expression of SPRY2, a repressor of FGF signaling. The differentiation of osteoblasts was autonomous from the growth plate chondrocytes and was correlated with an increase in the protein levels of GLI2, a transcription factor that is a major mediator of hedgehog signaling. Evidence is provided that increased GLI2 activity is also a consequence of increased FGF signaling through downstream events requiring mitogen-activated protein kinases. To test whether FGF signaling is required for the effects of Pten deletion, one allele of fibroblast growth factor receptor 2 (FGFR2) was deleted. Significantly, deletion of FGFR2 caused a partial rescue of the Pten-null phenotype. This study identifies activated FGF signaling as the major mediator of Pten deletion in osteoprogenitors (Guntur, 2011).
The PTEN tumor suppressor gene modulates several cellular functions, including cell migration, survival, and proliferation by antagonizing phosphatidylinositol 3-kinase (PI 3-kinase)-mediated signaling cascades. Mechanisms by which the expression of PTEN is regulated are, however, unclear. The ligand-activated nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) has been shown to regulate differentiation and/or cell growth in a number of cell types, which has led to the suggestion that PPARgamma, like PTEN, could act as a tumor suppressor. PPARgamma has also been implicated in anti-inflammatory responses, although downstream mediators of these effects are not well defined. The activation of PPARgamma by its selective ligand, rosiglitazone, upregulates PTEN expression in human macrophages, Caco2 colorectal cancer cells, and MCF7 breast cancer cells. This upregulation correlates with decreased PI 3-kinase activity as measured by reduced phosphorylation of protein kinase B. One consequence of this was that rosiglitazone treatment reduces the proliferation rate of Caco2 and MCF7 cells. Antisense-mediated disruption of PPARgamma expression prevents the upregulation of PTEN that normally accompanies monocyte differentiation and reduces the proportion of macrophages undergoing apoptosis, while electrophoretic mobility shift assays show that PPARgamma is able to bind two response elements in the genomic sequence upstream of PTEN. These results demonstrate a role for PPARgamma in regulating PI 3-kinase signaling by modulating PTEN expression in inflammatory and tumor-derived cells.
The dauer formation gene daf-18 is the C. elegans homolog of PTEN. DAF-18 is a component of the insulin-like signalling pathway controlling entry into diapause and adult longevity that is regulated by the DAF-2 receptor tyrosine kinase and the AGE-1 PI 3-kinase. Others have shown that mutation of daf-18 suppresses the life extension and constitutive dauer formation associated with daf-2 or age-1 mutants. Similarly, it is shown that inactivation of daf-18 by RNA-mediated interference mimics this suppression, and that a wild-type daf-18 transgene rescues the dauer defect. These results indicate that PTEN/daf-18 antagonizes the DAF-2-AGE-1 pathway, perhaps by catalyzing dephosphorylation of the PIP3 generated by AGE-1. These data further support the notion that mutations of PTEN contribute to the development of human neoplasia through an aberrant activation of the PI 3-kinase signaling cascade (Rouault, 1999).
An insulin-like signaling pathway, from the DAF-2 receptor, the AGE-1 phosphoinositide 3-kinase, and the AKT-1/AKT-2 serine/threonine kinases to the DAF-16 Fork head transcription factor, regulates the metabolism, development, and life span of C. elegans. Inhibition of daf-18 gene activity bypasses the normal requirement for AGE-1 and partially bypasses the need for DAF-2 signaling. The suppression of age-1 mutations by a daf-18 mutation depends on AKT-1/AKT-2 signaling, showing that DAF-18 acts between AGE-1 and the AKT input to DAF-16 transcriptional regulation. daf-18 encodes a homolog of the human tumor suppressor PTEN (MMAC1/TEP1), which has 3-phosphatase activity toward phosphatidylinositol 3,4,5-trisphosphate (PIP3). DAF-18 PTEN may normally limit AKT-1 and AKT-2 activation by decreasing PIP3 levels. The action of daf-18 in this metabolic control pathway suggests that mammalian PTEN may modulate insulin signaling and may be variant in diabetic pedigrees (Ogg, 1998).
To determine the function of PTEN in vivo a PTEN homolog in Caenorhabditis elegans has been studied. A strong loss-of-function allele of the PTEN homolog was generated and the deficient strain has been shown to be unable to enter dauer diapause. An insulin-like phosphatidylinositol 3-OH kinase (PI3'K) signaling pathway regulates dauer-stage entry. Mutations in either the daf-2 insulin receptor-like (IRL) gene or the age-1 encoded PI3'K catalytic subunit homolog cause constitutive dauer formation and also affect the life span, brood size, and metabolism of nondauer animals. Strikingly, loss-of-function mutations in the age-1 PI3'K and daf-2 IRL genes are suppressed by loss-of-function mutations in the PTEN homolog. The PTEN homolog is encoded by daf-18, a previously uncloned gene that has been shown to interact genetically with the DAF-2 IRL AGE-1 PI3'K signaling pathway. This interaction provides clear genetic evidence that PTEN acts to antagonize PI3'K function in vivo. Given the conservation of the PI3'K signaling pathway between C. elegans and mammals, the analysis of daf-18 PTEN mutant nematodes should shed light on the role of human PTEN in the etiology of metabolic disease, aging, and cancer (Gil, 1999).
A homolog of PTEN has been found in Caenorhabditis elegans and it has been found to correspond to the daf-18 gene, which had been defined by a single, phenotypically weak allele, daf-18(e1375). By analyzing an allele, daf-18(nr2037) , which bears a deletion of the catalytic portion of CePTEN/DAF-18, it has been shown that mutation in daf-18 can completely suppress the dauer-constitutive phenotype caused by inactivation of daf-2 or age-1, which encode an insulin receptor-like molecule and the catalytic subunit of phosphatidylinositol 3-kinase, respectively. In addition, daf-18(nr2037) dramatically shortens lifespan, both in a wild-type background and in a daf-2 mutant background that normally prolongs lifespan. The lifespan in a daf-18(nr2037) mutant can be restored to essentially that of wild type when combined with a daf-2 mutation. These studies provide genetic evidence that, in C. elegans, the PTEN homolog DAF-18 functions as a negative regulator of the DAF-2 and AGE-1 signaling pathway, consistent with the notion that DAF-18 acts a phosphatidylinositol 3,4,5-trisphosphate phosphatase in vivo. Furthermore, these studies have uncovered a longevity-promoting activity of the PTEN homolog in C. elegans (Mihaylova, 1999).
In C. elegans, reduced insulin-like signalling induces developmental quiescence, reproductive delay and lifespan extension. The C. elegans orthologues of LKB1 (Drosophila homolog: lkb1) and AMPK cooperate during conditions of reduced insulin-like signalling to establish cell cycle quiescence in the germline stem cell population, in addition to prolonging lifespan. The inactivation of either protein causes aberrant germline proliferation during diapause-like 'dauer' development, whereas the loss of AMPK uncouples developmental arrest from lifespan extension. Reduced TGF-beta activity also triggers developmental quiescence independent of the insulin-like pathway. These data suggest that these two signalling pathways converge on the C. elegans PTEN orthologue to coordinate germline proliferation with somatic development during dauer formation, via the regulation of AMPK and its upstream activator LKB1, rather than through the canonical insulin-like signalling cascade. In humans, germline mutations in TGF-beta family members, PTEN or LKB1 result in related tumour-predisposing syndromes. These findings establish a developmental relationship that may underscore their shared, characteristic aetiology (Narbonne, 2006).
PTEN is one of the most commonly lost tumor suppressors in human cancer and is known to inhibit insulin signaling. Eph receptor tyrosine kinases (RTKs) have also been implicated in cancer formation and progression, and they have diverse functions, including nervous and vascular system development. This study shows that in C. elegans, the VAB-1 Eph kinase domain physically interacts with and phosphorylates PTEN (DAF-18), diminishing its protein levels and function. vab-1 mutants show increased longevity and sensitivity to dauer conditions, consistent with increased DAF-18/PTEN activity and decreased insulin-like signaling. Moreover, daf-18 mutations suppress vab-1 oocyte maturation phenotypes independent of PI3K signaling. Evidence is presented that DAF-18 has protein phosphatase activity to antagonize VAB-1 action. Possible implications for human cancers are discussed, based on the idea that mutually inhibitory interactions between PTEN and Eph RTKs may be conserved (Brisbin, 2009).
A novel tumor suppressor gene, PTEN, is inactivated in a number of different tumor types including breast cancer. An investigation of the functional role suggests that PTEN transcriptionally represses both exogenous and endogenous c-myc expressions in human breast carcinoma cells. PTEN, when ectopically expressed in human breast carcinoma cells, exhibits an inhibition of phosphorylation of both activating residues of protein kinase B (PKB)/AKT at Ser-473 and Thr-308 without any significant alteration of AKT expression. Furthermore, introduction of PTEN into human breast carcinoma cells induces apoptotic cell death and inhibited cell growth and tumor formation in nude mice. Taken together, these data suggest that PTEN acts as a transcriptional repressor, inhibits the AKT-mediated cell survival signaling pathway, and negatively regulates human breast carcinoma cell growth. These results further emphasize the potential of PTEN as a gene therapeutic agent (Ghosh, 1999).
mPTEN-mutant mouse embryos display regions of increased proliferation. In contrast, mPTEN-deficient immortalized mouse embryonic fibroblasts exhibit decreased sensitivity to cell death in response to a number of apoptotic stimuli, accompanied by constitutively elevated activity and phosphorylation of protein kinase B/Akt, a crucial regulator of cell survival. Expression of exogenous PTEN in mutant cells restores both their sensitivity to agonist-induced apoptosis and normal pattern of PKB/Akt phosphorylation. Furthermore, PTEN negatively regulates intracellular levels of phosphatidylinositol (3,4,5) trisphosphate in cells and dephosphorylates it in vitro. These results show that PTEN may exert its role as a tumor suppressor by negatively regulating the PI3'K/PKB/Akt signaling pathway (Stambolic, 1998).
The molecular pathways that link nutritional cues to developmental programs are poorly understood. C. elegans hatchlings arrest in a dormant state termed 'L1 diapause' until food is supplied. However, little is known about what signal transduction pathways mediate nutritional status to control arrest and initiation of postembryonic development. This study reports that C. elegans embryonic germline precursors undergo G2 arrest with condensed chromosomes and remain arrested throughout L1 diapause. Loss of the DAF-18/PTEN tumor suppressor bypasses this arrest, resulting in inappropriate germline growth dependent on the AGE-1/PI-3 and AKT-1/PKB kinases. DAF-18 also regulates an insulin/IGF-like pathway essential for longevity and dauer larva formation. However, DAF-16/FoxO, which is repressed by this pathway, is not required for germline arrest in L1 diapause. Thus, these findings indicate that quiescence of germline development during L1 diapause is not a passive consequence of nutrient deprivation, but rather is actively maintained by DAF-18 through a pathway distinct from that which regulates longevity and dauer formation (Fukuyama, 2006).
The pathway controlling germline quiescence during L1 diapause overlaps with, but is distinct from, those involved in longevity and dauer formation, because there was no requirement of daf-16 for arresting germline growth during L1 diapause. Whether daf-2 regulates initiation of postembryonic germline growth by directly activating developmental programs, or simply by promoting feeding, remains to be investigated. In contrast to the germline, no somatic cell divisions were observed when newly hatched daf-18 and daf-16 mutants are starved in M9 buffer. However, it was recently found that during L1 diapause in the presence of a carbon source, DAF-18, as well as DAF-16, also acts to restrict developmental events, including cell division, in somatic tissue. Further identification and characterization of genes required for maintenance of L1 diapause and initiation of larval development should provide additional insights into the relationship between the nutrient-derived signaling pathway and developmental programs (Fukuyama, 2006).
The tumor suppressor PTEN is a phosphatase with sequence similarity to the cytoskeletal protein tensin. Here the cellular roles of PTEN were investigated. Overexpression of PTEN inhibits cell migration, whereas antisense PTEN enhances migration. Integrin-mediated cell spreading and the formation of focal adhesions are down-regulated by wild-type PTEN but not by PTEN with an inactive phosphatase domain. PTEN interacts with the focal adhesion kinase FAK and reduces its tyrosine phosphorylation. Overexpression of FAK partially antagonizes the effects of PTEN. Thus, PTEN phosphatase may function as a tumor suppressor by negatively regulating cell interactions with the extracellular matrix (Tamura, 1998).
The tumor suppressor PTEN dephosphorylates focal adhesion kinase (FAK) and inhibits integrin-mediated cell spreading and cell migration. Expression of PTEN selectively inhibits activation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. PTEN expression in glioblastoma cells lacking the protein results in inhibition of integrin-mediated MAP kinase activation. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)- induced MAPK activation are also blocked. To determine the specific point of inhibition in the Ras/Raf/ MEK/ERK pathway, these components were examined after stimulation by fibronectin or growth factors. Shc phosphorylation and Ras activity are inhibited by expression of PTEN, whereas EGF receptor autophosphorylation is unaffected. The ability of cells to spread at normal rates is partially rescued by coexpression of constitutively activated MEK1, a downstream component of the pathway. In addition, focal contact formation is enhanced as indicated by paxillin staining. The phosphatase domain of PTEN is essential for all of these functions, because PTEN with an inactive phosphatase domain does not suppress MAP kinase or Ras activity. In contrast to its effects on ERK, PTEN expression dies not affect c-Jun NH2-terminal kinase (JNK) or PDGF-stimulated Akt. These data suggest that a general function of PTEN is to down-regulate FAK and Shc phosphorylation, Ras activity, downstream MAP kinase activation, and associated focal contact formation and cell spreading (Gu, 1998).
PTEN/MMAC1 is a major new tumor suppressor gene that encodes a dual-specificity phosphatase with sequence similarity to the cytoskeletal protein tensin. PTEN dephosphorylates focal adhesion kinase (FAK) and inhibits cell migration, spreading, and focal adhesion formation. Effects of PTEN on cell invasion, migration, and growth as well as the involvement of FAK and p130 Crk-associated substrate (p130Cas; see CAS/CSE1 segregation protein) were investigated in U87MG glioblastoma cells missing PTEN. Cell invasion, migration, and growth are down-regulated by expression of phosphatase-active forms of PTEN but not by PTEN with an inactive phosphatase domain; these effects are correlated with decreased tyrosine phosphorylation levels of FAK and p130Cas. Overexpression of FAK concomitant with PTEN results in increased total tyrosine phosphorylation levels of FAK and p130Cas and effectively antagonizes the effects of PTEN on cell invasion and migration and partially on cell growth. Overexpression of p130Cas increases total tyrosine phosphorylation levels of p130Cas without affecting those of FAK; however, although p130Cas can reverse PTEN inhibition of cell invasion and migration, it does not rescue cell growth in U87MG cells. In contrast to FAK, p130Cas cannot be shown to interact with PTEN in cells, and it is not dephosphorylated directly by PTEN in vitro. These results suggest important roles of PTEN in the phenotype of tumor progression, and that the effects of PTEN on cell invasion, migration, and growth are mediated by distinct downstream pathways that diverge at the level of FAK (Tamura, 1999a).
The tumor suppressor PTEN is a phosphatase with sequence homology to tensin. PTEN dephosphorylates phosphatidylinositol 3,4, 5-trisphosphate (PIP3) and focal adhesion kinase (FAK), and it can inhibit cell growth, invasion, migration, and focal adhesions. Molecular interactions of PTEN and FAK were investigated in glioblastoma and breast cancer cells lacking PTEN. The PTEN trapping mutant D92A binds wild-type FAK, requiring FAK autophosphorylation site Tyr397. In PTEN-mutated cancer cells, FAK phosphorylation is retained even in suspension after detachment from extracellular matrix, accompanied by enhanced PI 3-K association with FAK and sustained PI 3-K activity, PIP3 levels, and Akt phosphorylation; expression of exogenous PTEN suppressed all five properties. PTEN-mutated cells are resistant to apoptosis in suspension, but most of the cells enter apoptosis after expression of exogenous PTEN or wortmannin treatment. Moreover, overexpression of FAK in PTEN-transfected cells reverses the decreased FAK phosphorylation and PI 3-K activity, and it partially rescues PIP3 levels, Akt phosphorylation, and PTEN-induced apoptosis. These results show that FAK Tyr397 is important in PTEN interactions with FAK, that PTEN regulates FAK phosphorylation and molecular associations after detachment from matrix, and that PTEN negatively regulates the extracellular matrix-dependent PI 3-K/Akt cell survival pathway in a process that can include FAK (Tamura, 1999b).
Cell migration is modulated by regulatory molecules such as growth factors, oncogenes, and the tumor suppressor PTEN. PTEN inhibits of cell migration and focal adhesion kinase (FAK) and p130 Crk-associated substrate (p130Cas) restores this PTEN inhibited migration. A second novel pathway is describe regulating random cell motility involving Shc and mitogen-activated protein (MAP) kinase, which is downmodulated by PTEN and additive to a FAK pathway regulating directional migration. Overexpression of Shc or constitutively activated MEK1 in PTEN- reconstituted U87-MG cells stimulates integrin-mediated MAP kinase activation and cell migration. Conversely, overexpression of dominant negative Shc inhibits cell migration; Akt appears to be uninvolved. PTEN directly dephosphorylates Shc. The migration induced by FAK or p130(Cas) is directionally persistent and involves extensive organization of actin microfilaments and focal adhesions. In contrast, Shc or MEK1 induces a random type of motility associated with less actin cytoskeletal and focal adhesion organization. These results identify two distinct, additive pathways regulating cell migration that are downregulated by tumor suppressor PTEN: one involves Shc, a MAP kinase pathway, and random migration, whereas the other involves FAK, p130(Cas), more extensive actin cytoskeletal organization, focal contacts, and directionally persistent cell motility. Integration of these pathways provides an intracellular mechanism for regulating the speed and the directionality of cell migration (Gu, 1999).
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. Pten regulates apoptosis and cell cycle progression through its phosphatase activity on phosphatidylinositol (PI) 3,4,5-trisphosphate (PI(3,4,5)P3), a product of PI 3-kinase. Pten has also been implicated in controlling cell migration, but the exact mechanism is not very clear. Using isogenic Pten+/+ and Pten-/- mouse fibroblast lines, it is shown that Pten deficiency leads to increased cell motility. Reintroducing the wild-type Pten, but not the catalytically inactive Pten C124S or lipid-phosphatase-deficient Pten G129E mutant, reduces the enhanced cell motility of Pten-deficient cells. Moreover, phosphorylation of the focal adhesion kinase p125FAK is 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, are observed in Pten-/- cells. Overexpression of dominant-negative mutant forms of Rac1 and Cdc42 reverses the cell migration phenotype of Pten-/- cells. Thus, these studies suggest that Pten negatively controls cell motility through its lipid phosphatase activity by down-regulating Rac1 and Cdc42. It is suggested that Pten exerts its tumor suppressor function not only at the stage of tumor initiation, but also in tumor progression and metastasis (Liliental, 2000).
Growth factors promote cell survival and cell motility, presumably through the activation of Akt and the Rac and Cdc42 GTPases, respectively. Because Akt is dispensable for Rac/Cdc42 regulation of actin reorganization, it has been assumed that Rac and Cdc42 stimulate cell motility independent of Akt in mammalian cells. However, this study demonstrates that Akt is essential for Rac/Cdc42-regulated cell motility in mammalian fibroblasts. A dominant-negative Akt inhibits cell motility stimulated by Rac/Cdc42 or by PDGF treatment, without affecting ruffling membrane-type actin reorganization. Akt is activated by expression of Rac and Cdc42; colocalization of endogenous phosphorylated Akt with Rac and Cdc42 is observed at the leading edge of fibroblasts. Importantly, expression of active Akt but not the closely related kinase SGK is sufficient for increasing cell motility. This effect of Akt is cell autonomous and not mediated by inhibition of GSK3. Dominant-negative Akt but not SGK reverses the increased cell motility phenotype of fibroblasts lacking the PTEN tumor suppressor gene. Taken together, these results suggest that Akt promotes cell motility downstream of Rac/Cdc42 in growth factor-stimulated cells and in invasive PTEN-deficient cells (Higuchi, 2002).
PTEN is a tumor suppressor gene involved in cell cycle control, apoptosis and mediation of adhesion and migration signaling. Germline mutations of PTEN in humans are associated with familial tumor syndromes, among them Cowden disease. Glioblastomas, highly malignant glial tumours of the central nervous system frequently show loss of PTEN. Recent reports have outlined some aspects of PTEN function in central nervous system development. Using a conditional gene disruption approach, Pten was inactivated locally in mice early during embryogenesis, in a region specific fashion, and later during postnatal development in a cell-specific manner, to study the role of PTEN in differentiation, migration and neoplastic transformation. PTEN is shown to be required for the realization of normal cerebellar architecture, for regulation of cell and organ size, and for proper neuronal and glial migration. However, PTEN is not required for cell differentiation and lack of PTEN is not sufficient to induce neoplastic transformation of neuronal or glial cells (Marino, 2002).
Phosphatase and tensin homolog (PTEN) mediates many of its effects on proliferation, growth, survival, and migration through its PtdIns(3,4,5)P3 lipid phosphatase activity, suppressing phosphoinositide 3-kinase (PI3K)-dependent signaling pathways. PTEN also possesses a protein phosphatase activity, the role of which is less well characterized. This study investigated the role of PTEN in the control of cell migration of mesoderm cells ingressing through the primitive streak in the chick embryo. Overexpression of PTEN strongly inhibits the epithelial-to-mesenchymal transition (EMT) of mesoderm cells ingressing through the anterior and middle primitive streak, but it does not affect EMT of cells located in the posterior streak. The inhibitory activity on EMT is completely dependent on targeting PTEN through its C-terminal PDZ binding site, but can be achieved by a PTEN mutant (PTEN G129E) with only protein phosphatase activity. Expression either of PTEN lacking the PDZ binding site or of the PTEN C2 domain, or inhibition of PI3K through specific inhibitors, does not inhibit EMT, but results in a loss of both cell polarity and directional migration of mesoderm cells. The PTEN-related protein TPTE, which normally lacks any detectable lipid and protein phosphatase activity, can be reactivated through mutation, and only this reactivated mutant leads to nondirectional migration of these cells in vivo. It is concluded that PTEN modulates cell migration of mesoderm cells in the chick embryo through at least two distinct mechanisms: controlling EMT, which involves its protein phosphatase activity; and controlling the directional motility of mesoderm cells, through its lipid phosphatase activity (Leslie, 2007; full text of article).
Axon-dendrite polarity is a cardinal feature of neuronal morphology essential for information flow. A differential distribution of GSK-3ß activity is found in the axon versus the dendrites. A constitutively active GSK-3ß mutant inhibits axon formation, whereas multiple axons formed from a single neuron when GSK-3ß activity is reduced by pharmacological inhibitors, a peptide inhibitor, or siRNAs. An active mechanism for maintaining neuronal polarity was revealed by the conversion of preexisting dendrites into axons upon GSK-3 inhibition. Biochemical and functional data show that the Akt kinase and the PTEN phosphatase are upstream of GSK-3ß in determining neuronal polarity. These results demonstrate that there are active mechanisms for maintaining as well as establishing neuronal polarity, indicate that GSK-3ß relays signaling from Akt and PTEN to play critical roles in neuronal polarity, and suggest that application of GSK-3ß inhibitors can be a novel approach to promote generation of new axons after neural injuries (Jiang, 2005).
Serine (Ser) 9 in GSK-3ß can be regulated by multiple kinases, among which Akt is differentially localized in axons. To test for GSK-3ß regulation by the candidate upstream molecules, the same inhibitors for PI3K as those used previously for studies of neuronal polarity were used. LY294002 significantly inhibits Akt phosphorylation at Ser 473 and GSK-3ß phosphorylation at Ser 9. LY294002 does not affect GSK-3ß phosphorylation at Tyr216. In addition to the biochemical evidences, the distribution of GSK-3ß and pGSK-3ß was examined. LY294002 reduces the ratio of pGSK-3ß over GSK-3ß. aPKC is another candidate GSK-3ß regulator, but GSK-3ß phosphorylation is not affected by the aPKC inhibitor bisindolylmaleimide I (Bis), or by the MAPK inhibitor U0126 or the PKA inhibitor KT5720 (Jiang, 2005).
To test for the functional significance of Akt, attempts were made to examine the effect of inhibiting Akt by using short hairpin RNAs (shRNAs), but they caused neuronal death. The effect of increasing Akt activity on neuronal polarity was examined by using Myr-Akt, a constitutively active Akt derived by fusion with the myristoylation signal of Src. The wild-type Akt does not affect neuronal polarity, whereas Myr-Akt causes the formation of multiple axons (Jiang, 2005).
Because GSK-3ß S9A should not be phosphorylated by Akt after the substation of Ser 9, it was possible to test for the relationship between Akt and GSK-3ß by cotransfection of GSK-3ß S9A and Myr-Akt. GSK-3ß S9A could partially, but not completely, reverse the effect of Myr-Akt on the formation of multiple axons. These results are consistent with the idea of Akt being upstream of GSK-3ß. The partial reversal could result from either incomplete overlap in GSK-3ß S9A and Myr-Akt transfection or the possibility that GSK-3ß constitutes a part, but not all, of the output for Akt (Jiang, 2005).
Detailed examination indicates that Akt plays multiple roles in axon and dendrite development. Myr-Akt increases the number of neurites per neuron both in neurons forming multiple axons, and in neurons with single axons. Myr-Akt increases both the numbers of axons and dendrites from the normal total of five neurites to eight. When the number of axons is increased in Myr-Akt neurons, axon length is shorter than normal, perhaps due to limited materials for an increased number of axons. When the number of axons is not increased in Myr-Akt neurons, the single axon is longer than normal, indicating that Akt promotes axon outgrowth. The length of dendrites is not affected by Myr-Akt (Jiang, 2005).
Only the effect of increasing axon number is shared between Akt activation and GSK-3ß inactivation. GSK-3ß is therefore only one downstream component of Akt, specifically mediating the function of Akt in axon-dendrite polarity, but not in neurite number or axon length (Jiang, 2005).
PTEN is a lipid and protein phosphatase that functions in opposition to PI3K by dephosphorylating the lipid product of PI3K, phosphatidylinositol 3,4,5-trisphosphate (PIP3) . The role of PTEN was examined further and the functional relationship of PTEN and GSK-3ß was characterized. Neuronal polarity is lost upon PTEN overexpression. Detailed analysis indicates that PTEN transfection inhibits axon formation without affecting dendrite formation. The inactive PTEN control (PTEN G129R) does not affect neuronal polarity (Jiang, 2005).
To investigate the role of endogenous PTEN, PTEN siRNA, a small inhibitor RNA (siRNA) construct designed specifically for PTEN, was used, and PTEN SsiRNA, a scrambled control. PTEN siRNA, but not PTEN SsiRNA, reduces PTEN protein levels in hippocampal neurons. PTEN siRNA increases the number of axons at the expense of dendrites. When the level of PTEN protein was examined, the level of PTEN was reduced by more than 50% in PTEN siRNA neurons with multiple axons, whereas PTEN was reduced less than 20% in PTEN siRNA neurons with single axons. Significant reduction of PTEN expression therefore correlates with the formation of multiple axons (Jiang, 2005).
To test for a functional relationship between PTEN and GSK-3ß, two kinds of experiments were performed. The first was to transfect neurons with PTEN and also treat them with SB415286. SB415286 almost completely reverses the effect of PTEN overexpression on axon-dendrite polarity, resulting in multiple axons at the expense of dendrites. The second kind of experiment was to cotransfect PTEN siRNA and GSK-3ß S9A. The effect of PTEN siRNA on multiple axon formation could be inhibited by an S0A mutant of GSK-3ß in which Ser 9 was replaced with alanine. These results indicate that GSK-3ß is downstream of PTEN in axon-dendrite polarity formation because GSK-3ß manipulations dominate over PTEN manipulations (Jiang, 2005).
One role of PTEN does not seem to be downstream of GSK-3ß. When neurons are cotransfected with PTEN siRNA and GSK-3ß S9A, GSK-3ß S9A could not eliminate axons, suggesting that either PTEN siRNA can antagonize the effect of GSK-3ß S9A in axon formation or that GSK-3ß S9A has to act in a PTEN-dependent pathway to inhibit axon formation. In the latter scenario, GSK-3ß activation could be upstream of PTEN in inhibiting axon formation (Jiang, 2005).
Although self-renewal is the central property of stem cells, the underlying mechanism remains inadequately defined. Using a murine hematopoietic stem and progenitor cell (HSPC)-specific conditional induction line, a compound genetic model was generated bearing both Pten deletion and β-catenin activation. These double mutant mice exhibit a novel phenotype, including expansion of phenotypic long-term hematopoietic stem cells (LT-HSCs) without extensive differentiation. Unexpectedly, constitutive activation of β-catenin alone results in apoptosis of HSCs. However, together, the Wnt/β-catenin and PTEN/PI3k/Akt pathways interact to drive phenotypic LT-HSC expansion by inducing proliferation while simultaneously inhibiting apoptosis and blocking differentiation, demonstrating the necessity of complementary cooperation between the two pathways in promoting self-renewal. Mechanistically, β-catenin activation reduces multiple differentiation-inducing transcription factors, blocking differentiation partially through up-regulation of Inhibitor of differentiation 2 (Id2). In double mutants, loss of Pten enhances the HSC anti-apoptotic factor Mcl-1. All of these contribute in a complementary way to HSC self-renewal and expansion. While permanent, genetic alteration of both pathways in double mutant mice leads to expansion of phenotypic HSCs, these HSCs cannot function due to blocked differentiation. A pharmacological approach was developed to expand normal, functional HSCs in culture using factors that reversibly activate both Wnt/β-catenin and PI3K/Akt signaling simultaneously. Activation of either single pathway is insufficient to expand primitive HSCs, but in combination, both pathways drive self-renewal and expansion of HSCs with long-term functional capacity (Perry, 2011).
Two-hybrid searches with the tumor suppressor MMAC1/PTEN have isolated the proteins hDLG and hMAST205. Further two-hybrid analysis and microtiter plate binding assays localized the sites of interaction to PDZ domains from hDLG and hMAST205 and the PDZ binding domain at the COOH terminus of MMAC1/PTEN. A synthetic peptide derived from the MMAC1/PTEN PDZ binding domain (MMAC1/PTEN-PDZBD) was used to coprecipitate proteins from A431 human cell lysate. The recovered proteins were resolved by SDS-PAGE and immobilized on a nitrocellulose membrane. Treatment of this membrane with an anti-hDLG antibody identified a Mr 140,000 band, consistent with the size of hDLG. Treatment of this membrane with the MMAC1/PTEN-PDZBD peptide identified a single prominent band of slightly larger than Mr 200,000 (Mr 200,000 kDa). Threonine phosphorylation of the MMAC1/ PTEN-PDZBD peptide inhibits both microtiter plate binding to the hDLG and hMAST205 PDZ domains and coprecipitation of the Mr 140,000 and 200,000 proteins, but promotes coprecipitation of proteins of approximately Mr 90,000 and Mr 120,000 from A431 cell lysate. This result suggests phosphorylation of the MMAC1/PTEN PDZ binding domain can both inhibit and promote PDZ interactions (Adey, 2000).
Formation of the apical surface and lumen is a fundamental, yet poorly understood, step in epithelial organ development. PTEN localizes to the apical plasma membrane during epithelial morphogenesis to mediate the enrichment of PtdIns(4,5)P2 at this domain during cyst development in three-dimensional culture. Ectopic PtdIns(4,5)P2 at the basolateral surface causes apical proteins to relocalize to the basolateral (BL) surface. Annexin 2 (Anx2) binds PtdIns(4,5)P2 and is recruited to the apical surface. Anx2 binds Cdc42, recruiting it to the apical surface. Cdc42 recruits aPKC to the apical surface. Loss of function of PTEN, Anx2, Cdc42, or aPKC prevents normal development of the apical surface and lumen. It is concluded that the mechanism of PTEN, PtdIns(4,5)P2, Anx2, Cdc42, and aPKC controls apical plasma membrane and lumen formation (Martin-Belmonte, 2007).
Formation of the apical surface and lumen is a central problem in understanding how epithelial tissues arrange themselves into tubes and other hollow structures, such as cysts. This study has uncovered a molecular mechanism of AP surface and lumen formation. PTEN is needed for segregation of PtdIns(4,5)p2 to the apical plasma membrane (PM) and PtdIns(3,4,5)p3 to the BL PM. Apical PtdIns(4,5)p2 recruits Anx2, which in turn recruits Cdc42 to the apical PM, causing the organization of the sub-apical actin cytoskeleton and formation of the apical surface and lumen. Cdc42 binds and localizes the Par6/aPKC complex to the apical PM to promote establishment of polarity (Martin-Belmonte, 2007).
PtdIns(4,5)p2 is thus a key determinant of the apical surface. Similarly, PtdIns(3,4,5)p3 is a key determinant of the BL surface (Gassama-Diagne, 2006). Together, PtdIns(4,5)p2 and PtdIns(3,4,5)p3 play complementary roles in epithelial polarity. More generally, phosphoinositides have emerged as general determinants of membrane identity. An advantage of epithelia is that exogenous phosphoinositides can be inserted into limited ectopic locations. These gain-of-function experiments provide a direct test of the role of the lipid in specifying domain identity (Martin-Belmonte, 2007).
To exert its effects, PtdIns(4,5)p2 interacts with Anx2, which clusters this lipid with high affinity and specificity. Ectopic PtdIns(4,5)p2 in the BL surface recruits Anx2 to the BL PM. Although loss of Anx2 by RNAi prevents lumen formation, RNAi of Anx2 did not produce as strong a phenotype as expression of the DN Anx2CM or RNAi of PTEN or Cdc42. One explanation could be the existence of >20 annexin family members, which might have redundancy with each other. Indeed, Anx2 knockout (KO) mice are viable. Alternatively, even low levels of Anx2 might suffice to exert its function (Martin-Belmonte, 2007).
Cdc42 interacts with Anx2 in a GTP-dependent manner. Cdc42 is activated during cystogenesis. Most activated Cdc42 relocalizes from cell-cell contacts to the apical pole as lumens form. RNAi of Cdc42 caused malformation of the central lumen in cysts but did not affect polarization of MDCK cells in 2D monolayers. This effect of Cdc42 depletion in cysts highlights the importance of using 3D models in analysis of lumen formation. Interestingly, an intracellular accumulation of Anx2 was seen in the cells with reduced Cdc42, suggesting a potential positive feedback loop whereby Cdc42 and Anx2 each promote the localization of the other at the lumen of mature cysts. Anx2 might work by recruiting Cdc42 or a GEF for Cdc42, and this GEF may in turn activate Cdc42 at this location. In contrast, active Cdc42 might promote the exocytosis of Anx2 and other apical proteins (Martin-Belmonte, 2007).
Formation of the apical surface and lumen has been suggested to be mediated by exocytosis of large intracellular vacuoles, termed vacuolar apical compartment (VAC). Formation of endothelial lumens occurs by vacuolar exocytosis. Cdc42 is needed for the exocytosis of secretory vesicles from neuroendocrine cells, apparently via rearrangement of the actin cytoskeleton, and this may be analogous to the fusion of VACs or smaller vesicles with the apical surface. Accumulation of apparent VACs was seen when Cdc42 was depleted. Similarly, DN Cdc42 blocks capillary lumen formation. Perhaps during normal MDCK cyst lumen formation smaller vesicles are rapidly exocytosed to form the lumen. Inhibition of this by Cdc42 depletion may cause the accumulation of larger, more easily detected VACs. Indeed, this may be the defect underlying the phenotypes observed with loss of function of PTEN, Anx2, Cdc42, or aPKC. Cdc42 is also needed for exit of apical and BL proteins from the trans-Golgi network (TGN), so Cdc42 may act at multiple levels in the formation of the apical surface (Martin-Belmonte, 2007).
Localized active Cdc42 may promote formation of the apical surface and lumen by additional mechanisms. Active Cdc42 binds to Par6, a member of the Par3/Par6/aPKC complex that regulates TJ and polarity formation. In Drosophila, Par6/aPKC functions at the apical PM independently of Par3, which is associated with the junctional complex. Indeed, Par6/aPKC localizes at the apical PM of MDCK cysts independently of Par3, and the disruption of aPKC function inhibits normal lumen formation. Mutation of zebrafish aPKCλ causes defects in lumen formation in the intestine. These data suggest the existence of two distinct Par complexes for the establishment of epithelial polarity: a complex of Par6/aPKC localized to the apical PM and involved in the formation of this domain; and a complex that also includes Par3, localized at the TJs and required for their formation (Martin-Belmonte, 2007).
It has been reported that inhibition of Rac1 or β1-integrin in cysts leads to inversion of polarity orientation and abnormal organization of laminin. Rac1, β1-integrin, and laminin may be part of a pathway that determines orientation of the axis of polarity, while PTEN, PtdIns(4,5)p2, Anx2, Cdc42, and Par6/aPKC are part of a pathway that controls formation of the apical surface and lumen. The Rac1/β1-integrin/laminin pathway might be upstream and/or parallel to the PTEN/PtdIns(4,5)p2/Anx2/Cdc42 pathway, and it determines the location of the apical surface. Because activation of Rac1 at the primordial adhesions of epithelial cells controls the association and activation of the Par3/Par6/aPKC complex to induce TJ biogenesis and cell polarity, one potential connection between these pathways might be the targeting of PTEN to the apical domain through its interaction with TJs. PTEN localizes to the adherens junctions in fly epithelium through its interaction with Bazooka/Par3. This study shows that PTEN is needed for apical PM and lumen formation during cyst development and that Par3 localizes specifically to the TJs in MDCK cyst. This observation is consistent with previous studies showing that the expression of DN Par-3 cells disrupted MDCK cyst morphogenesis, causing the lack of a central lumen (Martin-Belmonte, 2007).
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