CREB is a novel nuclear target of PTEN phosphatase

Abstract

PTEN phosphatase is a potent tumor suppressor that regulates multiple cellular functions. In the cytoplasm, PTEN dephosphorylates its primary lipid substrate, phosphatidylinositol 3,4,5-trisphosphate, to antagonize the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway. It has also become increasingly evident that PTEN functions in the nucleus and may play an important part in transcription regulation, but its nuclear targets remain elusive. In this report, we demonstrate the transcription factor cyclic AMP response element-binding protein (CREB) is a protein target of PTEN phosphatase and that PTEN deficiency leads to CREB phosphorylation independent of the PI3K/AKT pathway. Using confocal immunofluorescence and reciprocal immunoprecipitation, we further show that PTEN colocalizes with CREB and physically interacts with CREB. Moreover, we use both in vitro and in vivo experiments to show PTEN can dephosphorylate CREB in a phosphatase-dependent manner, suggesting that CREB is a substrate of PTEN nuclear phosphatase. Loss of Pten results in an elevated RNA level of multiple CREB transcriptional targets and increased cell proliferation, which can be reversed by a nonphosphorylatable CREB mutant or knockdown of CREB. These data reveal a mechanism for PTEN modulation of CREB-mediated gene transcription and cell growth. Our study thus characterizes PTEN as a nuclear phophatase of a transcription factor and identifies CREB as a novel protein target of PTEN phosphatase, which contributes to better understanding of PTEN function in the nucleus.

Figure 1

Loss of PTEN leads to upregulation of CREB phosphorylation, independent of PI3K/AKT activation. A, Western analysis of CREB phosphorylation in Pten^+/+ and Pten^−/− MEFs. B, phosphorylation status of CREB in different PTEN-deficient cell lines with counterpart controls as indicated. C, inhibition of the PI3K/AKT pathway does not affect CREB phosphorylation in Pten-null cells. Pten^−/− MEFs treated with different doses of LY294002 were evaluated by Western blot for phosphorylation of CREB and AKT.

Figure 2

PTEN colocalizes and interacts with CREB. A, colocalization of PTEN with CREB in MEFs. Pten^+/+ and Pten^−/− cells were immunostained for CREB (green) and PTEN (red), followed by confocal analysis of their colocalization. The yellow signal in the merged image of Pten^+/+ MEFs represents spatial overlap of CREB and PTEN. Scale bars, 7.5 μm. B, reciprocal coimmunoprecipitation of PTEN and CREB in Pten^+/+ and Pten^−/− MEFs and HeLa cells containing PTEN siRNA. C, in vitro binding between PTEN and CREB. A His-tagged irrelevant protein (His-control) was included as a control.

Figure 3

PTEN dephosphorylates CREB in vitro and in vivo. A, in vitro phosphatase assay. Purified His-CREB was incubated with 3 forms of His-PTEN proteins or a control His-protein prior to evaluation of CREB phosphorylation. B, Western analysis of CREB phosphorylation in Pten^−/− MEFs containing ectopic expression of wild-type or phosphatase-deficient PTEN. Phosphorylation of Akt is also shown to indicate PTEN phosphatase activity.

Figure 4

CREB activation is responsible for upregulated CREB transcriptional targets and increased cell growth in Pten null cells. Pten^−/− MEFs were transfected with CREB siRNA or a nonphosphorylatable CREB mutant, CREB^S133A. A, quantitative real-time PCR analysis of Bcl2 and c-Myc expression. The expression levels of c-Myc and Bcl2 were normalized to GAPDH. B, measurement of cell growth by MTT assay. Data represent the mean ± SEM; **, P < 0.01 as compared with Pten^−/− cells.