Han Song-Iee, Tsunekage Yukino, Kataoka Kohsuke
Laboratory of Molecular Medical Bioscience, Graduate School of Medical Life Science, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama, 230-0045, Japan.
Department of Internal Medicine (Endocrinology and Metabolism), Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, 305-8575, Japan.
Acta Diabetol. 2016 Aug;53(4):651-60. doi: 10.1007/s00592-016-0853-1. Epub 2016 Mar 26.
MafA is a critical regulator of insulin expression and mature β-cell function. MafA binds to the insulin promoter through its carboxyl-terminal basic domain-leucine zipper (bZip) region and activates transcription synergistically with the β-cell-enriched transactivators Beta2 (NeuroD1) and Pdx1. MafA protein is highly phosphorylated in β-cells, and phosphorylation at multiple sites within its amino-terminal region is critical for its DNA-binding and transactivating abilities, as well as for regulation of its degradation. Here, we investigated whether phosphorylation of MafA affects its interaction with Beta2.
By mutational analysis, we identified interaction domains of MafA and Beta2. Using in situ proximity ligation assay (PLA), we explored mechanism of phosphorylation-dependent binding of MafA with Beta2. We also searched for a pathophysiological condition that would induce lower levels of MafA phosphorylation.
Mutational analysis revealed that the phosphorylation sites within the amino-terminal region of MafA were not necessary for interaction with Beta2. In situ PLA suggested that phosphorylation induces conformational or configurational changes in MafA, thereby regulating the interaction with Beta2. We also found that long-term culture of the MIN6 insulinoma cell line under high-glucose conditions resulted in a decrease in β-cell-specific transcripts including insulin, along with a decrease in MafA phosphorylation and DNA binding.
Phosphorylation of MafA plays a critical role in β-cell function by regulating multiple functionalities, including binding to DNA, interaction with Beta2, and transactivation.
MafA是胰岛素表达和成熟β细胞功能的关键调节因子。MafA通过其羧基末端碱性结构域-亮氨酸拉链（bZip）区域与胰岛素启动子结合，并与富含β细胞的反式激活因子Beta2（NeuroD1）和Pdx1协同激活转录。MafA蛋白在β细胞中高度磷酸化，其氨基末端区域内多个位点的磷酸化对其DNA结合和反式激活能力以及对其降解的调节至关重要。在此，我们研究了MafA的磷酸化是否影响其与Beta2的相互作用。
通过突变分析，我们确定了MafA和Beta2的相互作用结构域。使用原位邻近连接分析（PLA），我们探索了MafA与Beta2磷酸化依赖性结合的机制。我们还寻找了一种能诱导MafA磷酸化水平降低的病理生理状况。
突变分析表明，MafA氨基末端区域内的磷酸化位点对于与Beta2的相互作用并非必需。原位PLA表明，磷酸化诱导MafA发生构象或构型变化，从而调节与Beta2的相互作用。我们还发现，在高糖条件下长期培养MIN6胰岛素瘤细胞系会导致包括胰岛素在内的β细胞特异性转录本减少，同时MafA磷酸化和DNA结合也减少。
MafA的磷酸化通过调节多种功能，包括与DNA结合、与Beta2相互作用和反式激活，在β细胞功能中起关键作用。
