Phosphorylation of MafA Is Essential for Its Transcriptional and Biological Properties

We previously described the identification of quail MafA, a novel transcription factor of the Maf bZIP (basic region leucine zipper) family, expressed in the differentiating neuroretina (NR). In the present study, we provide the first evidence that MafA is phosphorylated and that its biological properties strongly rely upon phosphorylation of serines 14 and 65, two residues located in the transcriptional activating domain within a consensus for phosphorylation by mitogen-activated protein kinases and which are conserved among Maf proteins. These residues are phosphorylated by ERK2 but not by p38, JNK, and ERK5 in vitro. However, the contribution of the MEK/ERK pathway to MafA phosphorylation in vivo appears to be moderate, implicating another kinase. The integrity of serine 14 and serine 65 residues is required for transcriptional activity, since their mutation into alanine severely impairs MafA capacity to activate transcription. Furthermore, we show that the MafA S14A/S65A mutant displays reduced capacity to induce expression of QR1, an NR-specific target of Maf proteins. Likewise, the integrity of serines 14 and 65 is essential for the MafA ability to stimulate expression of crystallin genes in NR cells and to induce NR-to-lens transdifferentiation. Thus, the MafA capacity to induce differentiation programs is dependent on its phosphorylation.

我们此前曾报道鹌鹑MafA的鉴定工作，该蛋白是Maf碱性亮氨酸拉链（basic region leucine zipper, bZIP）家族的新型转录因子，在分化中的神经视网膜（neuroretina, NR）中表达。本研究首次证实MafA可被磷酸化，且其生物学活性高度依赖丝氨酸14与65位的磷酸化修饰；这两个残基位于转录激活结构域内，符合丝裂原活化蛋白激酶（mitogen-activated protein kinases, MAPK）的磷酸化共有基序，且在所有Maf家族蛋白中均保守。体外实验显示，细胞外调节蛋白激酶2（ERK2）可对这两个位点进行磷酸化，而p38、JNK及ERK5则无此活性。但体内实验表明，丝裂原活化蛋白激酶激酶/细胞外调节蛋白激酶（MEK/ERK）通路对MafA磷酸化的贡献较为有限，提示存在其他激酶参与该过程。丝氨酸14与65位的完整性是MafA具备转录活性的必要条件：将这两个位点突变为丙氨酸会严重削弱MafA激活转录的能力。此外，我们发现MafA S14A/S65A突变体诱导QR1，Maf蛋白的神经视网膜特异性靶基因，表达的能力显著降低。同样，丝氨酸14与65位的完整性对于MafA刺激神经视网膜细胞中晶状体蛋白基因的表达，以及诱导神经视网膜向晶状体转分化均至关重要。综上，MafA诱导细胞分化程序的能力依赖于其磷酸化修饰状态。