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| Protein degradation: Merlin performs a double Akt
The protein kinase Akt promotes both the phosphorylation and ubiquitin-mediated degradation of the tumor-suppressor protein merlin. The tumor-suppressor protein merlin inhibits cell growth by modulating the activity of its interaction partners, including the small GTPase PIKE-L, the cell surface glycoprotein CD44 and the membrane–cytoskeleton linker protein ezrin. The regulation of merlin is achieved through phosphorylation, which attenuates these protein–protein interactions, and ubiquitination, which targets merlin for proteasomal degradation. However, a link between these two regulatory mechanisms has not yet been found. In Nature Cell Biology, Xiaoling Tang et al. now report that Akt phosphorylates merlin, and that Akt-mediated phosphorylation promotes the ubiquitination and degradation of merlin.
Previous studies showed that the p21-activated kinases (PAKs) could phosphorylate merlin at Ser 518. In this study, in vitro kinase assays uncovered a role for Akt in phosphorylating merlin at Thr 230 and Ser 315. Phosphorylation of Ser 315 was detected in vivo as well. Expression of constitutively active Akt promoted merlin phosphorylation, and small-interfering RNA-based depletion of Akt completely eliminated merlin phosphorylation, showing that Akt is both necessary and sufficient for phosphorylation of merlin. The intramolecular and protein–protein interactions of merlin are mediated by its amino-terminal (NTD) and carboxy-terminal (CTD) domains. PAK-mediated phosphorylation of merlin promotes a conformational change that precludes further intramolecular and intermolecular associations. Akt-mediated phosphorylation of merlin likewise disrupted the NTD/CTD interaction and also inhibited merlin binding to PIKE-L, CD44 and ezrin. Furthermore, phosphorylation altered the biological activity of merlin, as expression of a phosphomimetic merlin mutant increased cell motility and proliferation in a rat schwannoma cell line. In contrast, expression of a mutant that was unable to undergo phosphorylation (T230A/S315A) inhibited cell growth and motility. Thus, Akt-mediated phosphorylation of merlin disrupts the formation of intramolecular and intermolecular interactions and represses merlin-mediated growth inhibition. The NTD of merlin contains an F1 ubiquitin-like motif that stimulates both ubiquitination and proteasomal degradation. Surprisingly, inhibition of the proteasome did not affect total merlin protein levels, but led to a marked increase of phospho-Ser 315 merlin. The authors found that Akt-phosphorylated merlin was rapidly ubiquitinated, presumably by spectrin, which binds to merlin and possesses ubiquitin-conjugating and ubiquitin-ligase function. Accordingly, tumors that possessed high levels of phospho-Akt exhibited low levels of merlin. Together, these data present a novel role for Akt in promoting phosphorylation and subsequent degradation of merlin. Loss of merlin has been linked to schwannomas and other nervous system tumors, and these results highlight a potential use for inhibitors of the phosphatidylinositol-3-OH kinase (PI(3)K)–Akt pathway in restoring merlin function in tumors. Emily J. Chenette
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