The tumor suppressor phosphatase PTEN is a key regulator of cell growth and apoptosis that interacts with PDZ domains from regulatory proteins, including MAGI-1/2/3, hDlg, and MAST205. Here we identified novel PTEN-binding PDZ domains within the MAST205-related proteins, syntrophin-associated serine/threonine kinase and MAST3, characterized the regions of PTEN involved in its interaction with distinctive PDZ domains, and analyzed the functional consequences on PTEN of PDZ domain binding. Using a panel of PTEN mutations, as well as PTEN chimeras containing distinct domains of the related protein TPTE, we found that the PTP and C2 domains of PTEN do not affect PDZ domain binding and that the C-terminal tail of PTEN (residues 350 -403) provides selectivity to recognize specific PDZ domains from MAGI-2, hDlg, and MAST205. Binding of PTEN to the PDZ-2 domain from MAGI-2 increased PTEN protein stability. Furthermore, binding of PTEN to the PDZ domains from microtubule-associated serine/ threonine kinases facilitated PTEN phosphorylation at its C terminus by these kinases. Our results suggest an important role for the C-terminal region of PTEN in the selective association with scaffolding and/or regulatory molecules and provide evidence that PDZ domain binding stabilizes PTEN and targets this tumor suppressor for phosphorylation by microtubule-associated serine/ threonine kinases.Alterations in the function of the PTEN phosphatase tumor suppressor protein are of major relevance for the incidence of a wide variety of human cancers, as well as for the occurrence of inherited growth disorders, grouped as PTEN hamartoma tumor syndromes (1). Structurally, PTEN protein is composed of an N-terminal phosphatase catalytic domain and a C-terminal phospholipid-binding C2 domain; the integrity of both domains is required for full PTEN phosphatase activity and binding to membranes (2). The analysis of tumor specimens, tumor cell lines, and model organisms defective in PTEN protein expression has shown that the 3-phosphoinositide phosphatase activity of PTEN toward the phospholipid phosphatidylinositol 3,4,5-trisphosphate is crucial for the control of cell growth, cell cycle, cell motility and migration, and apoptosis (3-6). In addition, some PTEN biological functions have been attributed to its protein phosphatase activity (7-10), and a PTEN phosphatase independent effect on the regulation of p53 stability and transcriptional activity has been reported (11). A major level of regulation of PTEN functions is related with its phosphorylation status, which has been involved in maintaining PTEN protein stability and in the control of PTEN subcellular location and/or its association with regulatory molecules (12-21). In this regard, PTEN possesses a C-terminal tail (last 54 amino acids; residues 350 -403), which harbors at its far C terminus a functional PDZ domain-binding motif (residues Thr 401 -Lys 402 -Val 403 -COOH). PDZ domains are modular protein interaction domains that in most cases recognize C-terminal motifs on their target pr...

The targeting of the tumor suppressor PTEN protein to distinct subcellular compartments is a major regulatory mechanism of PTEN function, by controlling its access to substrates and effector proteins. Here, we investigated the molecular basis and functional consequences of PTEN nuclear/cytoplasmic distribution. PTEN accumulated in the nucleus of cells treated with apoptotic stimuli. Nuclear accumulation of PTEN was enhanced by mutations targeting motifs in distinct PTEN domains, and it was dependent on an N-terminal nuclear localization domain. Coexpression of a dominant negative Ran GTPase protein blocked PTEN accumulation in the nucleus, which was also affected by coexpression of importin ␣ proteins. The lipid-and protein-phosphatase activity of PTEN differentially modulated PTEN nuclear accumulation. Furthermore, catalytically active nuclear PTEN enhanced cell apoptotic responses. Our findings indicate that multiple nuclear exclusion motifs and a nuclear localization domain control PTEN nuclear localization by a Ran-dependent mechanism and suggest a proapoptotic role for PTEN in the cell nucleus. INTRODUCTIONPTEN is a tumor suppressor phosphatase involved in the control of cell growth and cell cycle traverse, apoptosis, cell size, and cell migration Waite and Eng, 2002;Leslie and Downes, 2004;Parsons, 2004;Sansal and Sellers, 2004). The PTEN gene is mutated or lost in a wide variety of human tumors, including malignant glioblastomas, an aggressive tumor of the CNS in humans (Bonneau and Longy, 2000;Eng, 2003). The major tumor suppressor function of PTEN is mediated by the dephosphorylation of phosphatidylinositol-3,4,5-triphosphate (PIP3; Maehama and Dixon, 1998). Through this lipid phosphatase activity, PTEN counteracts the action of the prosurvival proto-oncogenes, the phosphatidylinositol 3-kinases, and protein kinase B/Akt, which control the function of key downstream effectors of this pathway, including cell cycle regulators (such as p27Kip1, cyclin D1, and CHK1) and transcription factors (such as NF-B and FKHR;Li and Sun, 1998;Nakamura et al., 2000;Gustin et al., 2001;Weng et al., 2001;Radu et al., 2003;Puc et al., 2005). To control the PIP3 levels at the plasma membrane, PTEN possesses, in addition to its N-terminal phosphatase catalytic domain (residues 14 -185), a C-terminal phospholipidbinding C2 domain (residues 186 -350), which is critical for optimal binding to membranes and PIP3 dephosphorylation (Lee et al., 1999). In addition, the N-terminus of PTEN contains a phosphatidylinositol-4,5-diphosphate (PIP2) binding motif (residues 6 -15), which is also essential for PTEN membrane binding and activity Funamoto et al., 2002;Iijima and Devreotes, 2002;Campbell et al., 2003;McConnachie et al., 2003;Iijima et al., 2004;Walker et al., 2004;Vazquez et al., 2006). On the other hand, PTEN possesses a C-terminal tail (residues 350 -403) that plays a major role in the stabilization of the molecule and that is the target of posttranslational modifications, including phosphorylation by the protein kinase CK2 ...

The signaling pathways involving class I phosphatidylinositol 3-kinases (PI3K) and the phosphatidylinositol-(3,4,5)-trisphosphate phosphatase PTEN regulate cell proliferation and survival. Thus, mutations in the corresponding genes are associated to a wide variety of human tumors. Heterologous expression of hyperactive forms of mammalian p110A and p110B in Saccharomyces cerevisiae leads to growth arrest, which is counterbalanced by coexpression of mammalian PTEN. Using this in vivo yeast-based system, we have done an extensive functional analysis of germ-line and somatic human PTEN mutations, as well as a directed mutational analysis of discrete PTEN functional domains. A distinctive penetrance of the PTEN rescue phenotype was observed depending on the levels of PTEN expression in yeast and on the combinations of the inactivating PTEN mutations and the activating p110A or p110B mutations analyzed, which may reflect pathologic differences found in tumors with distinct alterations at the p110 and PTEN genes or proteins. We also define the minimum length of the PTEN protein required for stability and function in vivo. In addition, a random mutagenesis screen on PTEN based on this system allowed both the reisolation of known clinically relevant PTEN mutants and the identification of novel PTEN loss-of-function mutations, which were validated in mammalian cells. Our results show that the PI3K/PTEN yeast-based system is a sensitive tool to test in vivo the pathologic properties and the functionality of mutations in the human p110 proto-oncogenes and the PTEN tumor suppressor and provide a framework for comprehensive functional studies of these tumor-related enzymes.

The PTEN tumour suppressor is one of the more commonly inactivated proteins in human cancer and a key regulator of the PI3K/Akt survival pathway. Its direct involvement in human disease, as well as its important role in many cellular processes, including cell development and differentiation, cell growth, apoptosis, cell motility, cell size, stem cell survival, and longevity, has made PTEN the focus of attention of many researchers and clinicians. The major biological function of PTEN resides in its phosphatase activity towards the lipid second messenger phosphatydilinositol-3,4,5-triphosphate (PIP3), antagonizing the activity of the PI3K oncoproteins, and the association of PTEN to lipids at the plasma membrane is required to exert this function. [1][2][3] In addition, the presence of PTEN in the nucleus of many different cell types, and the finding of unexpected PTEN functions in the nucleus has revealed that the regulation of its nuclear/cytoplasmic distribution may also be a key mechanism to drive PTEN functions.4-6 Here, we discuss the current models to explain the regulation of PTEN nuclear accumulation, and the molecular linkage between the targeting of PTEN to the nucleus and to lipid membranes. The distinct pathways that mediate nuclear PTEN functions in the control of cell growth and apoptosis are also discussed. Molecular Mechanisms of PTEN Nuclear AccumulationThe tumour suppressor PTEN protein is present in the nucleus of different cell types, including cell lines and tissue cells.7-10 However, PTEN amino-acid sequence lacks obvious canonical nuclear localization signal sequences (NLS) or nuclear export sequences (NES) that could account for the targeting of the protein in or out of the nucleus, making elusive the molecular basis of PTEN nuclear/cytoplasmic distribution. Recent findings, however, suggest that PTEN entry and accumulation in the nucleus may be controlled by a variety of mechanisms. Chung et al.11 have reported that, in the MCF-7 breast carcinoma cell line, PTEN may be transported into the nucleus using several putative NLS-like sequences located in the PTP and the C2 domains of the protein. Mutating such NLS-like sequences individually did not affect PTEN nuclear/ cytoplasmic distribution. However, combined mutations of the NLS-like sequences caused defective PTEN nuclear accumulation. Based on the differential interaction of the major vault protein (MVP) with wild-type PTEN and with the NLS-like mutants, as well as on the nuclear localization pattern of MVP, the authors propose a model of PTEN nuclear import mediated by MVP. Interestingly, PTEN was found in this study to interact with importin a and b proteins, although such interaction was also observed in the NLS-like mutants that displayed defective nuclear accumulation. A second model of PTEN nuclear entry has been proposed by Liu et al.12 Using PTEN fusion proteins of variable size, the authors of this study noticed that large PTEN fusion proteins (4100 kDa) did not show nuclear localization, whereas PTEN alone (47 kDa) or a GFP-P...

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