The p53 tumor suppressor targets a novel regulator of G protein signaling - PubMed (original) (raw)

The p53 tumor suppressor targets a novel regulator of G protein signaling

L Buckbinder et al. Proc Natl Acad Sci U S A. 1997.

Abstract

Heterotrimeric G proteins transduce multiple growth-factor-receptor-initiated and intracellular signals that may lead to activation of the mitogen-activated or stress-activated protein kinases. Herein we report on the identification of a novel p53 target gene (A28-RGS14) that is induced in response to genotoxic stress and encodes a novel member of a family of regulators of G protein signaling (RGS) proteins with proposed GTPase-activating protein activity. Overexpression of A28-RGS14p protein inhibits both Gi- and Gq-coupled growth-factor-receptor-mediated activation of the mitogen-activated protein kinase signaling pathway in mammalian cells. Thus, through the induction of A28-RGS14, p53 may regulate cellular sensitivity to growth and/or survival factors acting through G protein-coupled receptor pathways.

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Figures

Figure 1

Figure 1

Regulation of A28-RGS14 gene expression by p53 and genotoxic stress. (A) Regulation by exogenous p53. The metallothionein-promoter driven p53 transgene in EB1 colon carcinoma cells (9) was induced by the addition of CdCl2 (6 μM) to the cell culture medium. Total RNA or protein was prepared from cells at the times (in hours) indicated. Northern blots (A) were prepared and analyzed by hybridization with 32P-labeled cDNA probes corresponding to A28-RGS14, p21WAF1, p53, or GAPDH. (B) Induction of A28-RGS14 expression by endogenous p53. Northern blot analysis of RKO (colon carcinoma cells, wild-type p53) and clonal RKO-E6 cells (express human papillomavirus E6 viral protein and consequently do not express significant levels of p53) treated with increasing concentrations of doxorubicin for 16 h. (C) Induction of A28-RGS14 transcripts in human cells that are wild type (WT) (MCF-7, breast carcinoma cells; MCF-7 subclone 6; U87, astrocytoma cells) but not mutant (T98G; glioblastoma cells, MCF-7Adr.; doxorubicin-resistant MCF7 subclone) or null (HL-60, promyelocytic leukemia cells) for p53. Cell lines were treated with (lanes D) or without (lanes C) doxorubicin (1 μM) for 16 h. RNA was prepared and analyzed by Northern blot as described in A.

Figure 2

Figure 2

A28-RGS14 gene encodes a novel conserved RGS protein. (A) Predicted protein sequences of human and mouse A28-RGS14p protein. (B) Alignment of A28-RGS14p amino acid sequence and that of related mammalian, yeast, and C. elegans RGS protein family members. Shaded areas indicate a 130-amino acid core domain with highest sequence conservation and a predicted α-helical structure. (C) Tissue expression pattern of the A28-RGS14 gene. A human multitissue Northern blot (CLONTECH) was hybridized with a 32P-labeled A28-RGS14 cDNA probe as described in Fig. 1_A_. (D) In vitro interaction of A28-RGS14p with Gαi2, but not Gαs subunits of heterotrimeric G proteins. The _in vitro_-translated 35S-labeled Gα proteins were coimmunoprecipitated with purified His-tagged A28-RGS14p (His-A28) and anti-A28-RGS14p antibody 8D12 (Ab-8D12; Y.C., L.B., and N.K., unpublished results). Lanes: 1, _in vitro_-translated Gα; 2, coimmunoprecipitation of Gα with His-A28-RGS14p using Ab-8D12; 3, coimmunoprecipitation of Gα without His-A28-RGS14p; 4, coimmunoprecipitation of _in vitro_-translated Gα with purified His-tagged p53 (His-p53) and its specific antibody (Ab-1, Oncogene Science); 5, coimmunoprecipitation of _in vitro_-translated hdm2 protein with His-A28-RGS14p and Ab-8D12. m.w.m., Molecular mass markers.

Figure 3

Figure 3

(A) A28-RGS14 coexpression blocks agonist-induced activation of ERK2 in M1- or M2-transfected COS-7 cells. Plasmids encoding M1 or M2 muscarinic receptors were cotransfected with a pcDNA3-A28-RGS14 cDNA vector or empty expression vector, as indicated, and a plasmid encoding HA-tagged MAPK (ERK2). Cells were exposed to Carbachol (100 μM) or phorbol 12-myristate 13-acetate (100 ng/ml) for 5 min and lysed, and ERK activity was assayed on anti-HA immunoprecipitates. After autoradiography, radioactivity incorporated into myelin basic protein (MBP) was quantitated with a Molecular Dynamics PhosphorImager. Data represent the mean ± SEM of six to eight experiments, expressed as fold increase with respect to vector-transfected cells (control). Fifty micrograms of total lysate proteins was subjected to Western blot analysis using anti-HA or anti-A28-RGS14 mouse monoclonal antibodies (Ab-1C5; Y.C., L.B., and N.K., unpublished results). (B) Induction of A28-RGS14 expression in response to mitogenic signals—a potential role as negative feedback regulator. NIH 3T3 cells expressing the muscarinic M1 receptor (10) were grown to confluence, transferred to serum-free medium (DMEM containing 0.1% BSA) for 16 h, and then stimulated with or without Carbachol (50 μM) or fetal bovine serum (10%) for 6 h. RNA prepared from the samples was examined by Northern blot analysis using 32P-labeled probes for mouse A28-RGS14 and GAPDH (as in Fig. 1_A_). Autoradiograms were analyzed by laser densitometric scanning (Applied Biosystems) and the signal was normalized to GAPDH control.

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