PDF(Pubmed)
Abstract：
LIM domain kinases (LIMK) are important regulators of actin cytoskeletal remodeling. These protein kinases phosphorylate the actin depolymerizing factor cofilin to suppress filament severing, and are key nodes between Rho GTPase cascades and actin. The two mammalian LIMKs, LIMK1 and LIMK2, contain consecutive LIM domains and a PDZ domain upstream of the C-terminal kinase domain. The roles of the N-terminal regions are not fully understood, and the function of the PDZ domain remains elusive. Here, we determine the 2.0 Å crystal structure of the PDZ domain of LIMK2 and reveal features not previously observed in PDZ domains including a core-facing arginine residue located at the second position of the \'x-Φ-G-Φ\' motif, and that the expected peptide binding cleft is shallow and poorly conserved. We find a distal extended surface to be highly conserved, and when LIMK1 was ectopically expressed in yeast we find targeted mutagenesis of this surface decreases growth, implying increased LIMK activity. PDZ domain LIMK1 mutants expressed in yeast are hyperphosphorylated and show elevated activity in vitro. This surface in both LIMK1 and LIMK2 is critical for autoregulation independent of activation loop phosphorylation. Overall, our study demonstrates the functional importance of the PDZ domain to autoregulation of LIMKs.
摘要：
LIM结构域激酶（LIMK）是肌动蛋白细胞骨架重塑的重要调节因子。这些蛋白激酶磷酸化肌动蛋白解聚因子cofilin以抑制丝切断，是RhoGTPase级联和肌动蛋白之间的关键节点。这两种哺乳动物的LIMK,LIMK1和LIMK2含有连续的LIM结构域和C末端激酶结构域上游的PDZ结构域。N端区域的作用尚未完全理解，并且PDZ结构域的功能仍然难以捉摸。这里,我们确定了LIMK2的PDZ结构域的2.0µ晶体结构，并揭示了以前在PDZ结构域中未观察到的特征，包括位于\'x-Φ-G-Φ'基序第二位置的面向核心的精氨酸残基，并且预期的肽结合裂缝是浅的且保守性差。我们发现远端延伸表面高度保守，当LIMK1在酵母中异位表达时，我们发现该表面的靶向诱变降低了生长，暗示LIMK活性增加。在酵母中表达的PDZ结构域LIMK1突变体被过度磷酸化并在体外显示出升高的活性。LIMK1和LIMK2中的该表面对于独立于活化环磷酸化的自动调节是关键的。总的来说,我们的研究证明了PDZ结构域对LIMK的自动调节的功能重要性。
