The RIIbeta regulatory subunit of protein kinase A binds to cAMP response element: an alternative cAMP signaling pathway

Abstract

cAMP, through the activation of cAMP-dependent protein kinase (PKA), is involved in transcriptional regulation. In eukaryotic cells, cAMP is not considered to alter the binding affinity of CREB/ATF to cAMP-responsive element (CRE) but to induce serine phosphorylation and consequent increase in transcriptional activity. In contrast, in prokaryotic cells, cAMP enhances the DNA binding of the catabolite repressor protein to regulate the transcription of several operons. The structural similarity of the cAMP binding sites in catabolite repressor protein and regulatory subunit of PKA type II (RII) suggested the possibility of a similar role for RII in eukaryotic gene regulation. Herein we report that RIIbeta subunit of PKA is a transcription factor capable of interacting physically and functionally with a CRE. In contrast to CREB/ATF, the binding of RIIbeta to a CRE was enhanced by cAMP, and in addition, RIIbeta exhibited transcriptional activity as a Gal4-RIIbeta fusion protein. These experiments identify RIIbeta as a component of an alternative pathway for regulation of CRE-directed transcription in eukaryotic cells.

Figure 1

Increased complex formation between CRE oligonucleotide and nuclear factor(s) in RIIβ-overexpressing cells. (A) Gel retardation assay with a trioctamer-CRE (11) oligonucleotide. Nuclear extracts from parental DT (lanes 2–6) and DTRIIβ (lanes 7–11) cells untreated (lanes C) or treated with 8-Cl-cAMP (5 μM, 18 hr) (lanes 8-Cl) were incubated with or without anti-RIIβ antiserum (13) or anti-CREB antibody (Santa Cruz Biotechnology). Lane 1 shows probe only. Arrows indicate DNA–protein complexes. (B) Competition experiment. Gel retardation assays were performed as described in A. Unlabeled CRE oligonucleotide (0.01–10 ng) or unlabeled simian virus 40 (SV40) oligonucleotide (10 ng) was used as indicated. PIS, preimmune serum. (C) Gel retardation assay with a single octamer-CRE oligonucleotide. Nuclear extracts from DT cells, untreated control (lanes C) and treated with 8-Cl-cAMP (5 μM, 18 hr; lanes 8-Cl) were incubated in the absence and presence of unlabeled competitor (lanes 1–3). Lanes 4–6 show Sp1 binding assayed in the same nuclear extracts. (D) Photoaffinity labeling and immunoprecipitation of R subunits. Lanes: 1 and 5, photoaffinity labeling only; 2–4 and 6–8, photoaffinity labeling followed by immunoprecipitation with anti-RIα, RIIα, or RIIβ antiserum (13), as indicated. RIα, 48-kDa RI (Sigma); RIIα, 56-kDa RII (Sigma). The data represent one of three to five experiments that gave similar results.

Figure 2

Southwestern blot analysis and UV cross-linking assay demonstrate RIIβ binding to CRE oligonucleotide. (A) Southwestern blot analysis. Lanes: 2 and 4, DT and DTRIIβ cell nuclear extracts; 3 and 5, immunoprecipitated RIIβ from DT and DTRIIβ nuclear extracts; 1, immunoprecipitated CREB from DT nuclear extract; M, ¹⁴C-labeled marker proteins. (B) UV cross-linking assay of DT and DTRIIβ cell nuclear extracts. Lanes: 2 and 4, DT and DTRIIβ cell nuclear extracts; 3 and 5, immunoprecipitated RIIβ from DT and DTRIIβ nuclear extracts; 1, immunoprecipitated CREB from DTRIIβ nuclear-extract; RIα, photoaffinity-labeled 48-kDa RI; RIIα, photoaffinity-labeled 56-kDa RII (Sigma). (C) UV cross-linking assay of DTRIIβ and DTRIIβ-P cell nuclear extracts. Lanes: 1 and 2, DTRIIβ-P and DTRIIβ nuclear extracts; 3 and 4, immunoprecipitated RIIβ from DTRIIβ-P and DTRIIβ nuclear extracts. The data represent one of three to five experiments that gave similar results.

Figure 3

GST-RIIβ fusion protein binding to the CRE. (A) Gel retardation assay of R subunits (N-terminal parts)-GST proteins. The purified RIIβ-, RIIα-, and RIα-GST proteins were used in the assays. Lanes: 1–4, ³²P-labeled CRE probe; 5–8, ³²P-labeled AP-1 probe; 1 and 5, 0.5 μg of CREB (CREB-1, bZIP, Santa Cruz Biotechnology); 2 and 6, 3 μg of GST-RIIβ; 3 and 7, 3 μg of GST-RIIα; 4 and 8, 3 μg of GST-RIα. (B) Gel retardation assay of GST-RIIβ_L (RIIβ whole molecule) protein. Lanes: 1–5, GST-RIIβ_L (500 ng) CRE-monomer-³²P probe in the absence (lane 1) and presence (lanes 2–5) of competitor as indicated; lanes 6–8 contained thrombin-digested GST-RIIβ_L (500 ng), CRE-trioctamer ³²P-probe in the absence (lane 6) and presence (lanes 7 and 8) of competitor as indicated. (C) Gel retardation assay of GST-RIIβ_L in the presence of GST-CREB. ³²P-labeled CRE-monomer was used. Lanes: 2 and 3, GST-CREB (10 ng) in the absence and presence, respectively, of anti-CREB antibody (Santa Cruz Biotechnology); 4 and 5, GST-RIIβ_L (100 ng) in the absence and presence, respectively, of anti-RIIβ antibody (Transduction Laboratories, Lexington, KY); 6–9, GST-CREB (10 ng) plus GST-RIIβ_L (100 ng) in the absence and presence of anti-CREB antibody or anti-RIIβ antibody as indicated. (D) Gel retardation assay of GST-RIIβ_L in the presence of CREB. ³²P-labeled probes are as follows. Lanes: 1–4, Δ−71-CRE; 5–8, α-Gly promoter with one CRE [CRE(1)]; 9–12, α-Gly promoter with CRE dimer [CRE(2)]. All lanes contained 10 ng of CREB (CREB-1, bZIP, Santa Cruz Biotechnology) and the indicated amounts of GST-RIIβ protein; lanes 1, 5, and 9 contained CREB only. Bands I-III, GST-RIIβ- or CREB-CRE complexes; F, free probe. The data represent one of three to five experiments that gave similar results.

Figure 4

RIIβ induction of CRE-directed and Gal4-directed gene transcription. (A) Transient somatostatin-CAT assay. DT, DTRIIβ, and DTRIIβ-P were transfected with Δ−71 SS-CAT plasmid (20 μg), treated or untreated with forskolin (18 hr, 10 μM), and assayed for CAT activity and β-galactosidase activity. (B) Transient somatostatin-CAT assay of cells treated or untreated with 8-Cl-cAMP. DT, DT-RIIβ, and DT-RIIβ-P cells transfected with Δ−71 SS-CAT plasmid, and treated or untreated with 8-Cl-cAMP, and assayed for CAT activity as described in A. (C) Gal4 experiment. COS cells were transfected with GalUAS reporter construct, pG5E4CAT, pJATLACZ, and pGalCREB-1 or pGalRIIβ and assayed for CAT activity as described for A. CAT activities normalized to β-galactosidase activities are shown as mean values of percent conversion derived from three or four experiments.