Phosphorylation and SCF-mediated degradation regulate CREB-H transcription of metabolic targets.
Phosphorylation and SCF-mediated degradation regulate CREB-H transcription of metabolic targets.
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DOI：
10.1091/mbc.e15-04-0247
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发表时间：
2015-08-15
影响因子：
3.3
通讯作者：
O'Hare P
中科院分区：
文献类型：
--
作者：
Barbosa S;Carreira S;Bailey D;Abaitua F;O'Hare P
CREB 3转录因子是一组特殊的调节蛋白，其通常锚定在内质网中并将功能扰动中继回细胞核。磷酸化和降解调节一种这样的因子（CREB-H）的反应，其参与分泌和代谢稳态。CREB-H是一种内质网锚定的转录因子，在调节分泌以及代谢和炎症途径中起着关键作用，但其活性如何调节仍不清楚。我们研究了核活性形式的加工，并确定了S87-S90周围的基序与DSG型磷酸降解决定子同源。我们发现，这一地区是受多重磷酸化，调节CREB-H的稳定性，通过将其定位到SCFFbw 1a E3泛素连接酶。磷酸酶处理，使用磷酸特异性抗体，和丝氨酸残基的取代的数据表明在该地区的候选丝氨酸的磷酸化，与核心S87/S90基序代表一个关键的决定因素，促进蛋白酶体介导的降解。候选激酶CKII和GSK-3b在体外磷酸化CREB-H，对不同丝氨酸具有特异性。先前在一个或多个相邻丝氨酸处用GSK-3磷酸化显著增加了CKII的S87/S90依赖性磷酸化。显性负性Cul 1的体内表达增强了CREB-H的稳态水平，Fbw 1a增强了这种作用。CREB-H以磷酸化依赖的方式与Fbw 1a直接相互作用。最后，当掺入全长蛋白质中时，磷酸降解决定子内的突变导致组成性切割的核蛋白水平增加以及关键内源性靶基因载脂蛋白A IV的转录和分泌增加。
The CREB3 transcription factors are a specialized set of regulatory proteins that are normally anchored in the endoplasmic reticulum and relay perturbation of function back to the nucleus. Phosphorylation and degradation modulate the response of one such factor (CREB-H), which is involved in secretion and metabolic homeostasis. CREB‑H, an endoplasmic reticulum–anchored transcription factor, plays a key role in regulating secretion and in metabolic and inflammatory pathways, but how its activity is modulated remains unclear. We examined processing of the nuclear active form and identified a motif around S87–S90 with homology to DSG-type phosphodegrons. We show that this region is subject to multiple phosphorylations, which regulate CREB-H stability by targeting it to the SCFFbw1a E3 ubiquitin ligase. Data from phosphatase treatment, use of phosophospecific antibody, and substitution of serine residues demonstrate phosphorylation of candidate serines in the region, with the core S87/S90 motif representing a critical determinant promoting proteasome-mediated degradation. Candidate kinases CKII and GSK-3b phosphorylate CREB-H in vitro with specificities for different serines. Prior phosphorylation with GSK-3 at one or more of the adjacent serines substantially increases S87/S90-dependent phosphorylation by CKII. In vivo expression of a dominant-negative Cul1 enhances steady-state levels of CREB‑H, an effect augmented by Fbw1a. CREB-H directly interacts with Fbw1a in a phosphorylation-dependent manner. Finally, mutations within the phosphodegron, when incorporated into the full-length protein, result in increased levels of constitutively cleaved nuclear protein and increased transcription and secretion of a key endogenous target gene, apolipoprotein A IV.