Phosphorylation-dependent Regulation of Connecdenn/DENND1 Guanine Nucleotide Exchange Factors*
Phosphorylation-dependent Regulation of Connecdenn/DENND1 Guanine Nucleotide Exchange Factors*
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Connecdenn/DENND1 鸟嘌呤核苷酸交换因子的磷酸化依赖性调节*
DOI：
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发表时间：
2015
影响因子：
4.8
通讯作者：
P. McPherson
中科院分区：
文献类型：
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作者：
Gopinath Kulasekaran;Nadya Nossova;Andrea L. Marat;I. Lund;C. Cremer;Maria S. Ioannou;P. McPherson
背景：连接是Rab35的gef， Rab35是一种控制内吞循环的GTPase。connecdenn功能的调控尚不清楚。结果：连接蛋白1和连接蛋白2的GEF活性受到胰岛素刺激下游Akt的激活调控。结论：Akt介导的连接蛋白磷酸化可能是控制货物循环的一种机制。意义：信号通路影响膜转运通路。Connecdenn 1/2是DENN（在正常细胞和肿瘤细胞中差异表达）结构域承载蛋白，作为小GTPase Rab35的GEFs（鸟嘌呤核苷酸交换因子）。connecdenn/Rab35功能的破坏会导致从细胞功能改变的核内体中回收多种货物蛋白的缺陷，但connecdenn GEF活性的调节尚未被探索。我们现在证明connecdenn 1/2是自抑制的，因此纯化的全长蛋白的Rab35结合和GEF活性明显低于分离的DENN结构域。这两种蛋白都被磷酸化，在连接蛋白1的残基500和600之间有显著的磷酸化位点。大规模的蛋白质组学筛选显示，connecdenn 1在Ser-536和Ser-538残基上以akt依赖的方式磷酸化，以响应胰岛素对脂肪细胞的刺激。有趣的是，我们发现Akt抑制剂在胰岛素治疗脂肪细胞后减少了连接蛋白1与Rab35的相互作用。值得注意的是，Ser-536/Ser-538旁边的肽结合了连接蛋白1的DENN结构域，而类似磷的肽则没有。此外，connecdenn 1与14-3-3蛋白相互作用，这种相互作用也被Akt抑制和Ser-536/Ser-538突变破坏。我们认为Akt磷酸化胰岛素激活下游的connecdenn 1通过分子内相互作用调节connecdenn 1功能。
Background: Connecdenns are GEFs for Rab35, a GTPase controlling endocytic recycling. Regulation of connecdenn function is unknown. Results: The GEF activity of connecdenn 1 and 2 are autoinhibited, and this is regulated by Akt activation downstream of insulin stimulation. Conclusion: Phosphorylation of connecdenn by Akt may provide a mechanism to control cargo recycling. Significance: Signaling pathways impinge on membrane-trafficking pathways. Connecdenn 1/2 are DENN (differentially expressed in normal and neoplastic cells) domain-bearing proteins that function as GEFs (guanine nucleotide exchange factors) for the small GTPase Rab35. Disruption of connecdenn/Rab35 function leads to defects in the recycling of multiple cargo proteins from endosomes with altered cell function, yet the regulation of connecdenn GEF activity is unexplored. We now demonstrate that connecdenn 1/2 are autoinhibited such that the purified, full-length proteins have significantly less Rab35 binding and GEF activity than the isolated DENN domain. Both proteins are phosphorylated with prominent phosphorylation sites between residues 500 and 600 of connecdenn 1. A large scale proteomics screen revealed that connecdenn 1 is phosphorylated at residues Ser-536 and Ser-538 in an Akt-dependent manner in response to insulin stimulation of adipocytes. Interestingly, we find that an Akt inhibitor reduces connecdenn 1 interaction with Rab35 after insulin treatment of adipocytes. Remarkably, a peptide flanking Ser-536/Ser-538 binds the DENN domain of connecdenn 1, whereas a phosphomimetic peptide does not. Moreover, connecdenn 1 interacts with 14-3-3 proteins, and this interaction is also disrupted by Akt inhibition and by mutation of Ser-536/Ser-538. We propose that Akt phosphorylation of connecdenn 1 downstream of insulin activation regulates connecdenn 1 function through an intramolecular interaction.
影响因子：
16
作者：
Allaire PD;Marat AL;Dall'Armi C;Di Paolo G;McPherson PS;Ritter B
通讯作者：
Ritter B