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...