LATS1 and LATS2 phosphorylate CDC26 to modulate assembly of the tetratricopeptide repeat subcomplex of APC/C.
LATS1 and LATS2 phosphorylate CDC26 to modulate assembly of the tetratricopeptide repeat subcomplex of APC/C.
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DOI：
10.1371/journal.pone.0118662
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Kuninaka S
中科院分区：
文献类型：
--
作者：
Masuda K;Chiyoda T;Sugiyama N;Segura-Cabrera A;Kabe Y;Ueki A;Banno K;Suematsu M;Aoki D;Ishihama Y;Saya H;Kuninaka S
在芽殖酵母中，有丝分裂退出网络（MEN）通过Dbf 2-Cdc 14信号级联调节后期促进复合物/细胞核小体（APC/C）。Dbf 2激酶磷酸化并激活Cdc 14磷酸酶，从而消除APC/C辅因子Cdh 1的抑制性磷酸化。虽然MEN的每个组分在进化过程中都高度保守，但目前没有证据支持大肿瘤抑制激酶1（LATS 1）直接磷酸化CDC 14，LATS 1是Dbf 2的人类对应物;因此，目前还不清楚LATS 1如何调节APC/C。在这里，我们证明LATS 1直接磷酸化APC/C组分CDC 26的Thr 7（T7）残基。Nocodazole诱导的T7磷酸化通过在HeLa细胞中敲低LATS 1和LATS 2而减少，表明这两种激酶都有助于体内CDC 26的磷酸化。CDC 26的T7残基对于其与APC/C的含有三肽重复的亚基APC 6的相互作用是关键的，并且该残基突变为Asp（T7 D）降低了CDC 26与APC 6的相互作用。用外源性磷酸化模拟T7 D突变的CDC 26替换HeLa细胞中的内源性CDC 26增加了凝胶过滤测定中APC/C亚基的洗脱大小，这意味着在CDC 26磷酸化后APC/C组装的变化。此外，T7 D突变的CDC 26促进了polo样激酶1的泛素化，polo样激酶1是APC/C的一种众所周知的底物。总之，这些结果表明，LATS 1/2是参与APC/C磷酸化的新型激酶，并表明LATS 1/2和APC/C之间的直接调节联系。
In budding yeast, the Mitotic Exit Network (MEN) regulates anaphase promoting complex/cyclosome (APC/C) via the Dbf2-Cdc14 signaling cascade. Dbf2 kinase phosphorylates and activates Cdc14 phosphatase, which removes the inhibitory phosphorylation of the APC/C cofactor Cdh1. Although each component of the MEN was highly conserved during evolution, there is presently no evidence supporting direct phosphorylation of CDC14 by large tumor suppressor kinase 1 (LATS1), the human counterpart of Dbf2; hence, it is unclear how LATS1 regulates APC/C. Here, we demonstrate that LATS1 phosphorylates the Thr7 (T7) residue of the APC/C component CDC26 directly. Nocodazole-induced phosphorylation of T7 was reduced by knockdown of LATS1 and LATS2 in HeLa cells, indicating that both of these kinases contribute to the phosphorylation of CDC26 in vivo. The T7 residue of CDC26 is critical for its interaction with APC6, a tetratricopeptide repeat-containing subunit of APC/C, and mutation of this residue to Asp (T7D) reduced the interaction of CDC26 with APC6. Replacement of endogenous CDC26 in HeLa cells with exogenous phosphor-mimic T7D-mutated CDC26 increased the elution size of APC/C subunits in a gel filtration assay, implying a change in the APC/C assembly upon phosphorylation of CDC26. Furthermore, T7D-mutated CDC26 promoted the ubiquitination of polo-like kinase 1, a well-known substrate of APC/C. Overall, these results suggest that LATS1/2 are novel kinases involved in APC/C phosphorylation and indicate a direct regulatory link between LATS1/2 and APC/C.