akt依赖性磷酸化通过抑制DNA结合活性负向调节dHAND的转录活性。

Masao Murakami, Keiichiro Kataoka, Shigetomo Fukuhara, Osamu Nakagawa, Hiroki Kurihara
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引用次数: 18

摘要

HAND2/dHAND是一种基本的螺旋-环-螺旋转录因子,在胚胎发生过程中在心脏和神经嵴衍生物中表达。虽然dHAND对鳃弓、心血管和肢体发育至关重要,但其靶基因尚未确定。dHAND功能的调控机制也相对未知。Akt/PKB是一种丝氨酸/苏氨酸蛋白激酶,参与细胞存活、生长和分化,磷酸化dHAND并抑制dHAND介导的转录。在缺乏激酶抑制剂的情况下,293T细胞中表达的AU5-dHAND被磷酸化,可能是在其Akt磷酸化基元上,而磷脂酰肌醇3-激酶抑制剂wortmannin和Akt抑制剂NL-71-101,而p70 S6激酶抑制剂rapamycin则没有显著降低dHAND的磷酸化。HA-Akt的共表达增强了dHAND主要位于bHLH结构域的多个丝氨酸和苏氨酸残基的磷酸化,从而降低了dHAND的转录活性。同样,模拟非磷酸化状态的丙氨酸突变消除了Akt对dHAND的抑制作用,而模拟磷酸化状态的天冬氨酸突变导致dHAND转录活性丧失。这些dHAND转录活性的变化与DNA结合活性的变化而不是与二聚化活性的变化平行。这些结果表明,在胚胎发生过程中,akt介导的信号可能通过调节dHAND的DNA结合活性来调节dHAND的转录活性。
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Akt-dependent phosphorylation negatively regulates the transcriptional activity of dHAND by inhibiting the DNA binding activity.

HAND2/dHAND is a basic helix-loop-helix transcription factor expressed in the heart and neural crest derivatives during embryogenesis. Although dHAND is essential for branchial arch, cardiovascular and limb development, its target genes have not been identified. The regulatory mechanisms of dHAND function also remain relatively unknown. Here we report that Akt/PKB, a serine/threonine protein kinase involved in cell survival, growth and differentiation, phosphorylates dHAND and inhibits dHAND-mediated transcription. AU5-dHAND expressed in 293T cells became phosphorylated, possibly at its Akt phosphorylation motif, in the absence of kinase inhibitors, whereas the phosphatidylinositol 3-kinase inhibitor wortmannin and the Akt inhibitor NL-71-101, but not the p70 S6 kinase inhibitor rapamycin, significantly reduced dHAND phosphorylation. Coexpression of HA-Akt augmented dHAND phosphorylation at multiple serine and threonine residues mainly located in the bHLH domain and, as a result, decreased the transcriptional activity of dHAND. Consistently, alanine mutation mimicking the nonphosphorylation state abolished the inhibitory effect of Akt on dHAND, whereas aspartate mutation mimicking the phosphorylation state resulted in a loss of dHAND transcriptional activity. These changes in dHAND transcriptional activity were in parallel with changes in the DNA binding activity rather than in dimerization activity. These results suggest that Akt-mediated signaling may regulate dHAND transcriptional activity through the modulation of its DNA binding activity during embryogenesis.

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