14-3-3 cooperates with LKB1 to regulate the activity and localization of QSK and SIK
14-3-3 cooperates with LKB1 to regulate the activity and localization of QSK and SIK
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DOI：
10.1242/jcs.02670
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发表时间：
2005-12-01
影响因子：
4
通讯作者：
Alessi, DR
中科院分区：
文献类型：
--
作者：
Al-Hakim, AK;Göransson, O;Alessi, DR
LKB1 肿瘤抑制激酶磷酸化并激活许多属于 AMP 激活蛋白激酶 (AMPK) 亚家族的蛋白激酶。我们使用改进的串联亲和纯化策略来鉴定与 AMPK α 相互作用的蛋白质，以及由 LKB1 激活的 12 种 AMPK 相关激酶。 AMPK β 和 AMPK γ 调节亚基与 AMPK α 相关，但与任何 AMPK 相关激酶无关，这解释了为什么 AMP 不影响这些酶的活性。此外，我们还鉴定了与一种或多种 AMPK 亚家族酶相互作用的新型结合伴侣，包括脂肪面/泛素特异性蛋白酶 9 (USP9)、AAA-ATPase-p97、腺嘌呤核苷酸转位酶、蛋白磷酸酶 2A 全酶和磷酸蛋白结合接头 14-3-3 的亚型。有趣的是，在 QSK 和 SIK 被 LKB1 磷酸化后，14-3-3 亚型直接与 T 环 Thr 残基结合。与此一致的是，在 LKB1 敲除肌肉或不表达 LKB1 的 HeLa 细胞中，14-3-3 亚型未能与非磷酸化 QSK 和 SIK 相互作用。此外，LKB1 磷酸化的 T 环 Thr 突变可阻止 QSK 和 SIK 在体外与 14-3-3 相互作用。 14-3-3 与 QSK 和 SIK 的结合增强了对 TORC2 蛋白和 AMARA 肽的催化活性，并且是 SIK 的细胞质定位和 QSK 定位到细胞质内的点状结构所必需的。据我们所知，这项研究提供了第一个 14-3-3 直接与蛋白激酶 T 环结合并影响其催化活性和细胞定位的例子。
The LKB1 tumour suppressor kinase phosphorylates and activates a number of protein kinases belonging to the AMP-activated protein kinase (AMPK) subfamily. We have used a modified tandem affinity purification strategy to identify proteins that interact with AMPK alpha, as well as the twelve AMPK-related kinases that are activated by LKB1. The AMPK beta and AMPK gamma regulatory subunits were associated with AMPK alpha, but not with any of the AMPK-related kinases, explaining why AMP does not influence the activity of these enzymes. In addition, we identified novel binding partners that interacted with one or more of the AMPK subfamily enzymes, including fat facets/ubiquitin specific protease-9 (USP9), AAA-ATPase-p97, adenine nucleotide translocase, protein pbosphatase 2A holoenzyme and isoforms of the phospho-protein binding adaptor 14-3-3. Interestingly, the 14-3-3 isoforms bound directly to the T-loop Thr residue of QSK and SIK, after these were phosphorylated by LKB1. Consistent with this, the 14-3-3 isoforms failed to interact with non-phosphorylated QSK and SIK, in LKB1 knockout muscle or in HeLa cells in which LKB1 is not expressed. Moreover, mutation of the T-loop Thr phosphorylated by LKB1, prevented QSK and SIK from interacting with 14-3-3 in vitro. Binding of 14-3-3 to QSK and SIK, enhanced catalytic activity towards the TORC2 protein and the AMARA peptide, and was required for the cytoplasmic localization of SIK and for localization of QSK to punctate structures within the cytoplasm. To our knowledge, this study provides the first example of 14-3-3 binding directly to the T-loop of a protein kinase and influencing its catalytic activity and cellular localization.