Akt but not PI3-kinase rescues PTEN/MMAC1-mediated suppression of gene expression from the CMV promoter. (A) Transfections were performed as described in Fig. 3A using CMV-luciferase as the reporter. (B) Transfections were performed as described in Fig. 3B using CMV-enhanced GFP and visualized by fluorescence microscopy.

PTEN/MMAC1 blocks phosphorylation of the translation repressor 4E-BP1, a downstream target of the PI3-kinase/Akt pathway. (A) 293T cells were transfected with 4E-BP1 in the presence or absence of wild-type PTEN/MMAC1 and either Myr-Akt or constitutively active PI3-kinase then starved of serum for 48 hr. Phosphorylation of 4E-BP1 was monitored by altered electrophoretic mobility in immunoblot assays of whole-cell lysates using an anti-HA antibody. Expression of PTEN/MMAC1, PI3-kinase, and Myr-Akt was monitored by immunoblot using anti-PTEN antibody, anti-Myc antibody, and anti-HA antibody respectively. (B) 293T cells were transfected with 4E-BP1 in conjunction with plasmids noted at the top of each lane. Phosphorylation of 4E-BP-1 was monitored as described in B.

PTEN/MMAC1 impairs growth factor-induced activation of Akt. (A) 293T cells were transfected with vector or wild-type PTEN/MMAC1. Endogenous levels of phosphorylated (Top) and total (Bottom) Akt protein levels were measured by immunoblot using an antibody that specifically recognizes Ser-473 (Top) and a pan-Akt antibody (Bottom). (B) Populations of Rat-1 cells stably expressing a Neo control vector, wild-type PTEN/MMAC1 or PTEN/MMAC1 C124S were established by retroviral infection and drug selection in G418. The expression of PTEN/MMAC1 was detected using an antibody against endogenous PTEN/MMAC (Top).The basal levels of activated Erk were analyzed by using an antibody that specifically recognizes phosphorylated, activated Erk (Middle). Total Erk protein was measured by using a pan-Erk antibody (Bottom). (C) Whole-cell lysates were prepared from Rat-1 cells stably expressing neo vector, wild-type PTEN, or PTEN C124S that were starved of serum for 24 hr and after re-exposure to serum for 30 min. Phosphorylated (Top) and total (Bottom) Akt levels were measured by immunoblot as in A.

Enhanced levels of Akt activation in human prostate cancers lacking PTEN expression. Whole-cell lysates were prepared from the human prostate cancer cell lines LNCaP, DU145, PC3, and prostate cancer xenografts LAPC-4 and LAPC-9, which were passaged as subcutaneous tumors in male severe combined immunodeficient mice, and from normal human prostate tissue from three individual patients. The levels of phosphorylated (Top) and total (Bottom) Akt were measured by using antibodies as described in Fig. 1. The status of the PTEN gene in each tumor is designated + (wild type) or − (mutant) as determined (1, 2, 4).

PTEN/MMAC1 suppresses gene expression from the PSA and CMV promoters. (A) LNCaP prostate cancer cells were transfected with PSA enhancer (PSE)-luciferase or CMV-luciferase in conjunction with vector, wild-type PTEN/MMAC1 or PTEN/MMAC1 C124S. Luciferase activity in PSE luciferase-transfected cells was analyzed 48 hr after stimulation with 1 nM concentration of the synthetic androgen R1881 (DuPont) vs. control. CMV-luciferase-transfected cells were not stimulated. (B) LNCaP cells were transfected with CMV-enhanced GFP and vector, wild-type PTEN/MMAC1, or PTEN/MMAC1 C124S and examined by fluorescence microscopy after 48 hr. Flow cytometry analysis confirmed successful transfection of all plates with reduced brightness in the wild-type PTEN/MMAC1-transfected cells (data not shown). (C) LNCaP cells were transfected with CMV-luciferase and analyzed at 6 and 18 hr after transfection. (D) LNCaP cells transfected with CMV-GFP and vector or wild-type PTEN/MMAC1 were analyzed after 48 hr for GFP expression by immunoblot (Top) or Northern blot analysis (Middle). 28S RNA levels were measured by ethidium bromide staining. Immunoblot analysis for GFP protein expression was performed on whole- cell lysates using a commercially available antibody (CLONTECH).