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{beta}-Arrestin-2 Mediates Anti-apoptotic Signaling through Regulation of BAD Phosphorylation.

ABSTRACT: beta-Arrestins, originally discovered as terminators of G protein-coupled receptor signaling, have more recently been appreciated to also function as signal transducers in their own right, although the consequences for cellular physiology have not been well understood. Here we demonstrate that beta-arrestin-2 mediates anti-apoptotic cytoprotective signaling stimulated by a typical 7-transmembrane receptor the angiotensin ATII 1A receptor, expressed endogenously in rat vascular smooth muscle cells or by transfection in HEK-293 cells. Receptor stimulation leads to concerted activation of two pathways, ERK/p90RSK and PI3K/AKT, which converge to phosphorylate and inactivate the pro-apoptotic protein BAD. Anti-apoptotic effects as well as pathway activities can be stimulated by an angiotensin analog (SII), which has been previously shown to activate beta-arrestin but not G protein-dependent signaling, and are abrogated by beta-arrestin-2 small interfering RNA. These findings establish a key role for beta-arrestin-2 in mediating cellular cytoprotective functions by a 7-transmembrane receptor and define the biochemical pathways involved.

SUBMITTER: Ahn S

PROVIDER: S-EPMC2659243 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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{beta}-Arrestin-2 Mediates Anti-apoptotic Signaling through Regulation of BAD Phosphorylation.

Ahn Seungkirl S Kim Jihee J Hara Makoto R MR Ren Xiu-Rong XR Lefkowitz Robert J RJ

The Journal of biological chemistry 20090126 13

beta-Arrestins, originally discovered as terminators of G protein-coupled receptor signaling, have more recently been appreciated to also function as signal transducers in their own right, although the consequences for cellular physiology have not been well understood. Here we demonstrate that beta-arrestin-2 mediates anti-apoptotic cytoprotective signaling stimulated by a typical 7-transmembrane receptor the angiotensin ATII 1A receptor, expressed endogenously in rat vascular smooth muscle cell ...[more]

PMID: 19171933

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Project description:The Kruppel-like transcription factors (KLFs) 4 and 5 (KLF4/5) are coexpressed in mouse embryonic stem cells, where they function redundantly to maintain pluripotency. In mammary carcinoma, KLF4/5 can each impact the malignant phenotype, but potential linkages to drug resistance remain unclear. In primary human breast cancers, we observed a positive correlation between KLF4/5 transcript abundance, particularly in the human epidermal growth factor receptor 2 (HER2)-enriched subtype. Furthermore, KLF4/5 protein was rapidly upregulated in human breast cancer cells following treatment with the HER2/epidermal growth factor receptor inhibitor, lapatinib. In addition, we observed a positive correlation between these factors in the primary tumors of genetically engineered mouse models (GEMMs). In particular, the levels of both factors were enriched in the basal-like tumors of the C3(1) TAg (SV40 large T antigen transgenic mice under control of the C3(1)/prostatein promoter) GEMM. Using tumor cells derived from this model as well as human breast cancer cells, suppression of KLF4 and/or KLF5 sensitized HER2-overexpressing cells to lapatinib. Indicating cooperativity, greater effects were observed when both genes were depleted. KLF4/5-deficient cells had reduced basal mRNA and protein levels of the anti-apoptotic factors myeloid cell leukemia 1 (MCL1) and B-cell lymphoma-extra large (BCL-XL). Moreover, MCL1 was upregulated by lapatinib in a KLF4/5-dependent manner, and enforced expression of MCL1 in KLF4/5-deficient cells restored drug resistance. In addition, combined suppression of KLF4/5 in cultured tumor cells additively inhibited anchorage-independent growth, resistance to anoikis and tumor formation in immunocompromised mice. Consistent with their cooperative role in drug resistance and other malignant properties, KLF4/5 levels selectively stratified human HER2-enriched breast cancer by distant metastasis-free survival. These results identify KLF4 and KLF5 as cooperating protumorigenic factors and critical participants in resistance to lapatinib, furthering the rationale for combining anti-MCL1/BCL-XL inhibitors with conventional HER2-targeted therapies.

Project description:beta-Arrestin-mediated signaling downstream of seven transmembrane receptors (7TMRs) is a relatively new paradigm for signaling by these receptors. We examined changes in protein phosphorylation occurring when HEK293 cells expressing the angiotensin II type 1A receptor (AT1aR) were stimulated with the beta-arrestin-biased ligand Sar(1), Ile(4), Ile(8)-angiotensin (SII), a ligand previously found to signal through beta-arrestin-dependent, G protein-independent mechanisms. Using a phospho-antibody array containing 46 antibodies against signaling molecules, we found that phosphorylation of 35 proteins increased upon SII stimulation. These SII-mediated phosphorylation events were abrogated after depletion of beta-arrestin 2 through siRNA-mediated knockdown. We also performed an MS-based quantitative phosphoproteome analysis after SII stimulation using a strategy of stable isotope labeling of amino acids in cell culture (SILAC). We identified 1,555 phosphoproteins (4,552 unique phosphopeptides), of which 171 proteins (222 phosphopeptides) showed increased phosphorylation, and 53 (66 phosphopeptides) showed decreased phosphorylation upon SII stimulation of the AT1aR. This study identified 38 protein kinases and three phosphatases whose phosphorylation status changed upon SII treatment. Using computational approaches, we performed system-based analyses examining the beta-arrestin-mediated phosphoproteome including construction of a kinase-substrate network for beta-arrestin-mediated AT1aR signaling. Our analysis demonstrates that beta-arrestin-dependent signaling processes are more diverse than previously appreciated. Notably, our analysis identifies an AT1aR-mediated cytoskeletal reorganization network whereby beta-arrestin regulates phosphorylation of several key proteins, including cofilin and slingshot. This study provides a system-based view of beta-arrestin-mediated phosphorylation events downstream of a 7TMR and opens avenues for research in a rapidly evolving area of 7TMR signaling.

Dataset Information

{beta}-Arrestin-2 Mediates Anti-apoptotic Signaling through Regulation of BAD Phosphorylation.

Publications

{beta}-Arrestin-2 Mediates Anti-apoptotic Signaling through Regulation of BAD Phosphorylation.

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