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Specific inactivation and nuclear anchoring of extracellular signal-regulated kinase 2 by the inducible dual-specificity protein phosphatase DUSP5.

ABSTRACT: The mechanisms which determine the nuclear accumulation and inactivation of the extracellular signal-regulated kinase 1 (ERK1) or ERK2 mitogen-activated protein (MAP) kinases are poorly understood. Here we demonstrate that DUSP5, an inducible nuclear phosphatase, interacts specifically with ERK2 via a kinase interaction motif (KIM) within its amino-terminal noncatalytic domain. This binding determines the substrate specificity of DUSP5 in vivo, as it inactivates ERK2 but not Jun N-terminal protein kinase or p38 MAP kinase. Using green fluorescent protein fusions, we identify within this same domain of DUSP5 a functional nuclear localization signal (NLS) which functions independently of the KIM. Moreover, we demonstrate that the expression of DUSP5 causes both nuclear translocation and sequestration of inactive ERK2. Nuclear anchoring is ERK2 specific and requires both interactions between the DUSP5 KIM and the common docking site of ERK2 and a functional NLS within DUSP5. Finally, the expression of a catalytically inactive mutant of DUSP5 also tethers ERK2 within the nucleus. Furthermore, this nuclear ERK2 is phosphorylated by MAP kinase kinase in response to growth factors and also activates transcription factor Elk-1. We conclude that DUSP5 is an inducible nuclear ERK-specific MAP kinase phosphatase that functions as both an inactivator of and a nuclear anchor for ERK2 in mammalian cells. In addition, our data indicate that the cytoplasm may not be an exclusive site of MAP kinase activation.

SUBMITTER: Mandl M

PROVIDER: S-EPMC549372 | biostudies-other | 2005 Mar

REPOSITORIES: biostudies-other

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Project description:BackgroundCellular senescence is an aging-related process found in cancer cells that contributes to irreversible growth arrest and tumor aggressiveness. Recently, calmodulin-regulated spectrin-associated protein 3 (CAMSAP3), a minus-end microtubule-stabilizing protein, has received increasing attention in cancer cell biology. However, the biological role of CAMSAP3 on senescence in human lung cancer remains incompletely understood.MethodsThe function of CAMSAP3 on the regulation of cellular senescence-associated phenotypes in human non-small cell lung cancer H460 cells were determined in CAMSAP3 deletion (H460/C3ko) cells. The effects of CAMSAP3 on cell proliferation were investigated using MTT and colony formation assays. The cell cycle activity was evaluated by flow cytometry and the senescence-associated phenotypes were observed by SA-β-Gal staining. Quantitative RT-PCR and westen blot were used to evaluate the expression of cell cycle and senescence markers. Moreover, the interaction of CAMSAP3-ERK1/2 and possible partner protein was quantified using immunoprecipitation/mass spectrometry and immunofluorescence. Lastly, an xenograft model were performed.ResultsCAMSAP3 knockout promotes lung cancer cell senescence-associated phenotypes and induces G1 cell cycle arrest. Mechanistic investigation revealed that phosphorylated ERK (p-ERK) was markedly downregulated in CAMSAP3-deleted cells, suppressing cyclin D1 expression levels, and full-length CAMSAP3 abrogated these phenotypes. Proteomic analysis demonstrated that vimentin, an intermediate filament protein, is required as a scaffold for CAMSAP3-modulating ERK signaling. Furthermore, an in vivo tumor xenograft experiment showed that tumor initiation is potentially delayed in CAMSAP3 knockout tumors with the downregulation of p-ERK and cyclin D1, resulting in a senescence-like phenotype.ConclusionThis study is the first to report an intriguing role of CAMSAP3 in lung carcinoma cell senescence-associated phenotypes via the modulation of p-ERK/cyclin D1 signaling.

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Specific inactivation and nuclear anchoring of extracellular signal-regulated kinase 2 by the inducible dual-specificity protein phosphatase DUSP5.

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