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

Taka-Aki Matsuoka,Li Zhao,Roland Stein

doi:10.1074/jbc.m010321200

Taka-Aki Matsuoka, Li Zhao + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m010321200

Copy DOI

Abstract

The RIPE3b1 DNA binding factor plays a critical role in pancreatic islet beta cell-specific and glucose-regulated transcription of the insulin gene. Recently it was shown that RIPE3b1 binding activity in beta 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 beta cells. We found that RIPE3b1 binding was inhibited upon incubating beta cell nuclear extracts at 30 degrees 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.

Highlights

Insulin is a polypeptide hormone that plays a critical role in glucose homeostasis by stimulating the uptake of glucose into cells
RIPE3b1 binding activity is reduced by calf intestinal alkaline phosphatase (CIAP) and brain-enriched phosphatase preparation (BPP) treatment, suggesting that activation of this insulin gene transcription factor is mediated by phosphorylation, of the 46-kDa DNA binding subunit [9]
It is well established that the phosphorylation status of transcription factors, including those involved in insulin gene expression, can play an essential role in activation

Summary

Introduction

Insulin is a polypeptide hormone that plays a critical role in glucose homeostasis by stimulating the uptake of glucose into cells. The ability of islet ␤ cells to produce insulin in sufficient amounts to meet the needs of the body is compromised in type 2 diabetes mellitus patients with an inactivating mutation within one allele of the BETA2 [22] and pdx-1 genes [23, 24] These results established a central role for each of the isolated insulin gene transcription factors in islet cell development and function. RIPE3b1 DNA binding is mediated by a ␤ cellenriched protein(s) of ϳ46 kDa, whose activity is reduced by either calf intestinal alkaline phosphatase (CIAP) or a brainenriched phosphatase preparation (BPP) treatment [9] These results suggested that RIPE3b1 binding is regulated by phosphorylation, a posttranslational modification mechanism utilized in controlling PDX-1 activation [25, 26]. Our findings strongly suggest the RIPE3b1 activation is influenced by phosphorylation of a tyrosine residue(s) within the 46-kDa DNA-binding protein

Methods

Results

Conclusion