The DNA Binding Activity of the RIPE3b1 Transcription Factor of Insulin Appears to Be Influenced by Tyrosine Phosphorylation

Abstract

The RIPE3b1 DNA binding factor plays a critical role in pancreatic islet ␤ cell-specific and glucose-regulated transcription of the insulin gene. Recently it was shown that RIPE3b1 binding activity in ␤ cell nuclear extracts is reduced by treatment with either calf intestinal alkaline phosphatase (CIAP) or a brain-enriched phosphatase preparation (BPP) (Zhao, L., Cissell, M. A., Henderson, E., Colbran, R., and Stein, R. (2000) J. Biol. Chem. 275, 10532-10537). Evidence is presented here suggesting that a tyrosine phosphatase(s) influences the ability of RIPE3b1 to bind to the insulin C1 element in ␤ cells. We found that RIPE3b1 binding was inhibited upon incubating ␤ cell nuclear extracts at 30°C. In contrast, PDX-1 and MLTF-1 transcription factor binding activity was unaffected under these conditions. The loss in RIPE3b1 binding activity was prevented by inhibitors of tyrosine phosphatases (sodium orthovanadate and sodium molybdate) but not by inhibitors of serine/threonine phosphatases (sodium fluoride, okadaic acid, and microcystin LR). CIAP-and BPP-catalyzed inhibition of RIPE3b1 binding was also blocked by these tyrosine phosphatase inhibitors. Collectively, the data suggested that removal of a tyrosine(s) within RIPE3b1 prevented activator binding to insulin C1 control element sequences. The presence of a key phosphorylated tyrosine(s) within this transcription factor was further supported by the ability of the 4G10 anti-phosphotyrosine monoclonal antibody to immunoprecipitate RIPE3b1 DNA binding activity. We discuss how tyrosine phosphorylation, a very rare and highly significant regulatory modification, may control RIPE3b1 activator function.Insulin is a polypeptide hormone that plays a critical role in glucose homeostasis by stimulating the uptake of glucose into cells. Expression of insulin in adults is limited to pancreatic islet ␤ cells primarily because of the recognition, by specific positive-acting transcription factors, of its enhancer region, which is located between nucleotides Ϫ340 and Ϫ91 relative to the insulin gene transcription start site (1-3). Selective activation from this region is predominantly mediated by the C2 (Ϫ317 to Ϫ311 base pairs) (2), A3 (Ϫ205 to Ϫ189 base pairs) (4 -6), C1 (Ϫ115 to Ϫ107 base pairs) (7-9), and E elements (Ϫ100 to Ϫ91 base pairs) (4, 7, 10). (These insulin cis-elements are labeled in accordance with the nomenclature proposed by German et al. (11).) The factors that act at A3, C1, and E also control glucose-inducible transcription (5,8,(12)(13)(14)(15), the primary metabolic regulator of insulin gene expression in vivo.The C2 (i.e. PAX6 (2)) and A3 (i.e. PDX-1 (6, 16, 17)) activators are islet ␤ cell-enriched homeodomain proteins, whereas a basic helix-loop-helix-containing protein controls E element stimulation (i.e. BETA2 (18)). Strikingly, these proteins regulate gene expression within islet cell types and during pancreogenesis. Thus, an inactivating mutation in the pdx-1 locus affects a very early development step that prevents both exocrine and endocrine p...

The Islet ॆ Cell-enriched RIPE3b1/Maf Transcription Factor Regulates pdx-1 Expression

“…5A). Complex A mobility was shifted upon incubating the ␤ cell nuclear extract at 30°C with both the B4/5 and InsC1 probes, presumably because of the actions of an endogenous tyrosine phosphatase (40). CIAP treatment reduced binding to each probe, an effect blocked by addition of NaPPi or Na 3 VO 4 (Fig.…”

“…Anti-phosphotyrosine Immunoprecipitation-Immunoprecipitations using anti-Tyr(P) (4G10, Upstate Biotechnology, Lake Placid, NY) were performed as described previously (40). Briefly, SDS was added to a final concentration of 0.5% (w/v) to ␤TC-3 nuclear extract (100 g protein) in a buffer containing 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 10% glycerol, 1 mM Na 3 VO 4 , and 2 mM dithiothreitol (final concentrations) and then heated to 65°C.…”

“…(Fig. 5B, compare 4G10 immunoprecipitate binding to InsC1 (40) and B4/5). Collectively, these results strongly suggest that RIPE3b1 binds to both the pdx-1 B4/5 and InsC1 elements.…”

“…RIPE3b1 binding activity is inhibited by the actions of a tyrosine phosphatase (40). To test whether complex A formation on B4/5 is also regulated in this manner, Min6 nuclear extracts were incubated in the presence or absence of CIAP and a general (sodium pyrophosphate, NaPPi) or phosphotyrosinespecific (sodium orthovanadate, Na 3 VO 4 ) phosphatase inhibitor.…”

NLRR1 Enhances EGF-Mediated MYCN Induction in Neuroblastoma and Accelerates Tumor Growth In Vivo

“…S3A). CIAP treatment is reported to block recruitment of phosphotranscriptional factors on genomic DNA (35). We also used Mithramycin-A (Mit-A), a well-known Sp1 inhibitor (34).…”