Error bar indicates standard deviation (n=3), P 0.05 for all three sets of PNUTS shRNA; students em t-test. /em (e) A prostate epithelial BPH1 parental cell line along with stably expressing BPH1-PNUTS wt or PNUTS W401A mutant or Delta TF2S PNUTS cells (1104 cells) were seeded in 100mm dishes. important tumor suppressor, which has major roles in cell survival, proliferation, migration and cell death (1-3). PTEN was initially identified as a gene located in the chromosomal locus 10q23; one of the most frequently mutated or deleted loci in human cancers. Evidence for loss or mutations Edoxaban (tosylate Monohydrate) Edoxaban (tosylate Monohydrate) of the gene in many diverse tumor types (4) and high susceptibility of heterozygous mice to a wide range of tumors (5, 6) strongly support the status of PTEN as an important tumour suppressor for many types of cancers. Functionally, PTEN is a dual specific phosphatase that acts on both lipid and protein substrates (2). The tumor suppressor activity of PTEN is mostly mediated through its lipid substrates (7). One such crucial substrate is phosphatidylinositol 3,4,5-trisphosphate (PIP3), which is converted to phosphatidylinositol 4,5-bisphosphate (PIP2) by PTEN at the cellular membrane (8). PIP3 generated by PI3-Kinase is required for the downstream activation of AKT pathway, which further promotes cell growth and survival. Thus, PTEN keeps a check on tumorigenesis by negatively regulating Edoxaban (tosylate Monohydrate) AKT pathway through down regulating the cellular levels of PIP3. PTEN being a very crucial tumor suppressor, it is important to mechanistically understand its regulation during normal and disease conditions. Although PTEN functions were extensively studied, the regulation of PTEN is less understood. To elucidate potential regulators of PTEN, we recently performed Edoxaban (tosylate Monohydrate) a tandem affinity purification using PTEN stable cell line and identified several PTEN associated proteins (9). We repeatedly found PNUTS as one of the potential PTEN associated proteins. PNUTS (Protein phosphatase-1 nuclear targeting subunit), also called PPP1R10, CAT53 Rabbit Polyclonal to eNOS and p99, was originally isolated as a nuclear protein that forms a stable complex with PP1 and PP1 in mammalian cells (10, 11). PNUTS binds to PP1 and potently decreases the catalytic activity of PP1 towards exogenous substrates such as Retinoblastoma (Rb) protein in vitro, and reduced expression of PNUTS in mammalian cells affects cell viability (12, 13). However, the exact function of PNUTS remains to be elucidated. Materials and Methods Plasmids Full length PNUTS and PTEN were cloned into mammalian expressing S-protein/FLAG/SBP (streptavidin binding protein) – triple tagged destination vector, and MYC-tagged destination vector using Gateway cloning system (Invitrogen). PNUTS domain deletions and PTEN domain deletions were cloned into S-protein/FLAG/SBP (Streptavidin binding protein) – triple tagged destination vector. Bacterially expressing GST-tagged PTEN, GST-tagged PNUTS, MBP-tagged PTEN and MBP-tagged PNUTS vectors were generated by transferring their coding sequences into destination vectors by using gateway cloning system. Antibodies Rabbit anti-PNUTS, anti-Foxo3a, anti-PNUTS (IHC specific) (Bethyl Laboratories), Monoclonal anti-PTEN clone 6H2.1 (cascade biosciences), Monoclonal anti-MBP (New England Bio Labs), anti-GST, anti-Myc clone 9E10, anti-p53, anti-HDAC2 (all from Santacruz Biotechnologies), Rabbit anti-pAkt (ser-473), anti-Akt, anti-PTEN, anti-pFoxo3a (ser-253), (all from Cell Signalling technology), anti-HDAC2 (Biomol, used in figure 2C), anti-GAPDH (Imgenex), anti-Rad51 (Calbiochem), anti-Flag, anti-Actin (Sigma), antibodies were used in this study. Open in a separate window Figure 2 PNUTS sequesters PTEN in the nucleus(a) HeLa cells were transfected with Flag-PTEN alone, in combination with GFP-PNUTS, GFP-Delta TF2S PNUTS, GFP-PNUTS W401A mutant or PTEN D1 mutant alone Edoxaban (tosylate Monohydrate) and in combination with GFP-PNUTS. Flag-PHLPP1 together with GFP-PNUTS was used as a control. The localization of PTEN and PHLPP was detected by immunofluorescence staining using anti-FLAG antibody. (b) HeLa cells were transfected with Myc-tagged PNUTS and 24 hours post transfections the cytoplasmic (CE) and nuclear extracts (NE) were prepared. PTEN localization was detected by immunoblotting. GAPDH.