釐清SLK是否不依賴其激酶功能調控Ezrin與actin

Abstract

本研究起始於利用shRNA和訊息傳遞路徑藥物的合作篩選策略，初步揭示Ste20-like kinase(SLK)與ROCK會共同合作，幫助細胞遷移。隨後進一步研究釐清SLK與ROCK沒有直接的訊號傳遞交互作用，推測是透過SLK控制actomyosin結構，ROCK調控actomyosin contractility的協同模式合作。我們因而進一步研究SLK如何在細胞遷移時，影響actomyosin的結構。文獻回顧指出SLK為serine/threonine kinase，可能透過磷酸化ERM蛋白（Ezrin、Radixin、Moesin)以調節與細胞遷移相關的細胞骨架。然而，我們的初步實驗發現：單獨敲除SLK與Ezrin表現均增加細胞骨架，而抑制其磷酸化則減少，提示SLK-Ezrin可能以非磷酸化方式調控細胞骨架。 為驗證此假說，我們分別使用Ezrin T567位點磷酸化突變株及SLK激酶活性突變株，結果皆導致細胞骨架組裝減少，顯示磷酸化並非必要機制。延續先前發現，SLK敲除會誘導Ezrin異常進入細胞核，而抑制Ezrin磷酸化則無此現象，說明SLK可能透過非磷酸化方式影響Ezrin的膜定位。我們進一步比較不同Ezrin T567位點磷酸化突變株，其結果差異性並不明顯，支持非磷酸化調控。 此外，過度表達無法結合細胞膜的Ezrin突變株顯示與野生型Ezrin對細胞骨架的組裝有相反的表現，顯示Ezrin分布對細胞骨架重塑具功能性影響。最後，我們初步實驗指出SLK–Ezrin可能透過空間競爭，影響具有結合細胞膜能力的細胞骨架調控因子Cofilin，參與細胞骨架動態組裝，未來將進一步釐清此路徑在細胞遷移中的功能角色。 綜合以上結果，我們提出一個新的調控路徑：SLK並非經由磷酸化，而是以調控Ezrin的分布為核心機制，進而改變細胞骨架的組裝動態，對理解細胞骨架調控機制提供了新的視角。

This study began with a combinatorial screen using shRNA and pathway inhibitors, which preliminarily identified Ste20-like kinase (SLK) and the ROCK pathway as potential regulators of cell migration. Subsequent analyses revealed that SLK and ROCK function through distinct signaling pathways. Despite the lack of crosstalk between the two, they appear to cooperate through a modular collaboration, wherein SLK regulates the structural organization of the actomyosin network, while ROCK modulates its contractile activity. Based on this observation, we further investigated how SLK contributes to actomyosin remodeling during migration. Literature suggests that SLK is a serine/threonine kinase that may phosphorylate ERM proteins (Ezrin, Radixin, Moesin) to regulate cytoskeletal dynamics. However, our initial experiments showed that knockdown of either SLK or Ezrin increased contractile actin, whereas inhibition of Ezrin phosphorylation reduced them— suggesting a phosphorylation-independent mechanism. To test this hypothesis, we employed Ezrin T567 phosphorylation mutants and SLK kinase-dead constructs. Both manipulations led to reduced actin filaments, supporting that phosphorylation is not essential for SLK–Ezrin–mediated cytoskeletal regulation. Moreover, SLK knockdown—but not Ezrin phosphorylation inhibition—induced nuclear accumulation of Ezrin, implying that SLK influences Ezrin membrane localization via a non-phosphorylation mechanism. Comparative analysis of Ezrin T567 mutants revealed minimal differences in distribution, further supporting this model. Overexpression of a membrane-binding–deficient Ezrin mutant resulted in actin accumulation patterns opposite to those seen with wild-type Ezrin, highlighting the functional importance of Ezrin’s subcellular localization in actin remodeling. Finally, our preliminary data suggest that SLK–Ezrin may modulate actin dynamics by spatially competing with membrane-associated cytoskeletal regulator Cofilin. Together, these findings support a novel model in which SLK regulates the actin cytoskeleton not through phosphorylation, but by controlling Ezrin localization. This work provides new insights into non-canonical mechanisms of cytoskeletal regulation.