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 ...

PTEN phosphatase is one of the most commonly targeted tumor suppressors in human cancers and a key regulator of cell growth and apoptosis. We have found that PTEN is cleaved by caspase-3 at several target sites, located in unstructured regions within the C terminus of the molecule. Cleavage of PTEN was increased upon TNF␣-cell treatment and was negatively regulated by phosphorylation of the C-terminal tail of PTEN by the protein kinase CK2. The proteolytic PTEN fragments displayed reduced protein stability, and their capability to interact with the PTEN interacting scaffolding protein S-SCAM/MAGI-2 was lost. Interestingly, S-SCAM/ MAGI-2 was also cleaved by caspase-3. Our findings suggest the existence of a regulatory mechanism of protein stability and PTEN-protein interactions during apoptosis, executed by caspase-3 in a PTEN phosphorylationregulated manner.The PTEN gene, one of the tumor suppressor genes most frequently mutated in human cancers, encodes a protein of 403 amino acids with phosphatase activity (1-4). PTEN contains tyrosine phosphatase activity toward phosphorylated peptides and proteins in vitro (5, 6). It has been proposed that PTEN controls cell motility and invasiveness by tyrosine dephosphorylation of the focal adhesion kinase, Fak, and the adapter protein Shc (7). On the other hand, PTEN possesses lipid phosphatase activity toward 3-phosphoinositides, counteracting the action of the oncogenic phosphatidylinositol 3-kinases and blocking the activation of the proto-oncogene protein kinase B/Akt (8, 9). Structurally, PTEN is composed of two domains, an N-terminal phosphatase catalytic domain and the C-terminal C2 domain, which binds phospholipids in vitro and is thought to mediate the binding of the molecule to membranes (10). In addition, PTEN contains three unstructured regions: first, the first seven residues form part of a phosphatidylinositol (4,5)-diphosphate binding motif; second, an internal loop within the C2 domain (residues 286 -310) of unknown function; third, a C-terminal tail (residues 354 -403) that contains a region phosphorylated by the protein kinase CK2, as well as a PDZ binding motif located at the far C terminus (1-4). We and others have reported that the phosphorylation of PTEN by CK2 regulates its function and protein stability to proteasome-mediated degradation (11-13). The C-terminal PDZ binding motif of PTEN has been shown to interact with the second PDZ domain of the scaffolding proteins S-SCAM/MAGI-2 and MAGI-3. This interaction has been shown to enhance the inhibitory effect of PTEN on protein kinase B activation (14,15).Caspases (cysteinyl-directed aspartate-specific proteases) are a family of highly specific proteases that play a key role during the apoptotic cell death. Caspases, constitutively expressed as inactive zymogens, are activated in a proteolytic cascade, in which initiator caspases, such as caspase-8, are activated by autoproteolysis in response to apoptotic cell stimulation. Initiator caspases then proteolytically activate downstream executioner caspa...

APC and PTEN are tumor suppressor proteins that bind through their C-termini to the PDZ domain containing-hDlg scaffolding protein. We have found that co-expression of PTEN and hDlg enhanced the negative regulation of the PI3K/Akt pathway by PTEN, indicating the physiologic importance of these interactions. APC and PTEN share other PDZ domain containing-interacting partners, including the MAGI scaffolding proteins and the MAST family of protein kinases. Mutational analysis revealed that the C-terminal PDZ-binding motifs from APC and PTEN were differentially recognized by distinct PDZ domains. APC bound to the three PDZ domains from hDlg, whereas PTEN mainly bound to PDZ-2/hDlg. This indicates the existence of overlapping, but distinct PDZ-domain recognition patterns by APC and PTEN. Furthermore, a ternary complex formed by APC, PTEN, and hDlg was detected, suggesting that hDlg may serve as a platform to bring in proximity APC and PTEN tumor suppressor activities. In line with this, tumor-related mutations targeting the PDZ-2/hDlg domain diminished its interaction with APC and PTEN. Our results expand the PDZ-domain counterparts for the tumor suppressor APC, show that APC and PTEN share PDZ-domain partners but have individual molecular determinants for specific recognition of PDZ domains, and suggest the participation of the tumor suppressors APC, PTEN, and hDlg in PDZ-domain interaction networks which may be relevant in oncogenesis.

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