Phosphorylation of α-Actinin 4 upon Epidermal Growth Factor Exposure Regulates Its Interaction with Actin
Phosphorylation of α-Actinin 4 upon Epidermal Growth Factor Exposure Regulates Its Interaction with Actin
复制标题
DOI：
10.1074/jbc.m109.035790
复制
发表时间：
2010-01-22
影响因子：
4.8
通讯作者：
Wells, Alan
中科院分区：
文献类型：
--
作者：
Shao, Hanshuang;Wu, Chuanyue;Wells, Alan
广泛表达的α-辅肌动蛋白家族桥接肌动蛋白丝以稳定粘附，这是在生长因子诱导的细胞迁移期间被破坏的过程。在肌动蛋白细胞骨架的溶解过程中，肌动蛋白在酪氨酸上被磷酸化，尽管其后果尚不清楚。我们在表达人表皮生长因子受体（EGFR）的鼠成纤维细胞中表达了人α-辅肌动蛋白的两种亚型，发现在用EGF刺激后，α-辅肌动蛋白1（ACTN 1）和α-辅肌动蛋白4（ACTN 4）的酪氨酸残基都被磷酸化，尽管ACTN 4被磷酸化的程度更大。这需要Src蛋白酪氨酸激酶和p38-MAPK（和部分磷酸肌醇三磷酸激酶）的激活，而不是MEK/ERK或Rac 1，如抑制剂所确定的。EGF诱导的ACTN 4磷酸化位点定位于酪氨酸4（主要位点）和酪氨酸31（次要位点）。截短诱变显示，ACTN 4的C-末端结构域（氨基酸300 - 911），其交联肌动蛋白结合头部结构域，作为肌动蛋白结合和EGF介导的磷酸化的抑制结构域。这两个属性是相互排斥的，去除C末端增强肌动蛋白结合的ACTN 4突变体，同时限制EGF诱导的磷酸化，并反过来EGF刺激的ACTN 4磷酸化降低其亲和力肌动蛋白。有趣的是，酪氨酸265的磷酸化模拟物（可在癌细胞中发现，位于引起局灶节段性肾小球硬化的K255 E突变附近）表现出肌动蛋白结合活性增加和ACTN 4对钙蛋白酶介导的裂解的敏感性;这种变体也延缓了细胞扩散。值得注意的是，无论是用低浓度的latrunculin A处理细胞，它已被证明去磷酸化F-肌动蛋白，或删除肌动蛋白结合结构域（100 - 252个氨基酸）的ACTN 4 Y265 E恢复EGF诱导的磷酸化。F-actin体外结合试验表明，与野生型（WT）相比，二磷酸化的ACTN 4的模拟物Y 4 E/Y31 E与F-actin的结合轻微。重要的是，EGF介导的ACTN 4在酪氨酸4和31的磷酸化显著抑制过表达ACTN 4的增殖NR 6 WT成纤维细胞的多核化。这些结果表明，EGF调节肌动蛋白结合活性的ACTN 4诱导酪氨酰定向磷酸化。
The ubiquitously expressed family of alpha-actinins bridges actin filaments to stabilize adhesions, a process disrupted during growth factor-induced migration of cells. During the dissolution of the actin cytoskeleton, actinins are phosphorylated on tyrosines, although the consequences of this are unknown. We expressed the two isoforms of human alpha-actinin in murine fibroblasts that express human epidermal growth factor receptor (EGFR) and found that both alpha-actinin 1 (ACTN1) and alpha-actinin 4 (ACTN4) were phosphorylated on tyrosine residues after stimulation with EGF, although ACTN4 was phosphorylated to the greater extent. This required the activation of Src protein-tyrosine kinase and p38-MAPK (and phosphoinositide trisphosphate kinase in part) but not MEK/ERK or Rac1, as determined by inhibitors. The EGF-induced phosphorylation sites of ACTN4 were mapped to tyrosine 4, the major site, and tyrosine 31, the minor one. Truncation mutagenesis showed that the C-terminal domains of ACTN4 (amino acids 300 - 911), which cross-link the actin binding head domains, act as an inhibitory domain for both actin binding and EGF-mediated phosphorylation. These two properties were mutually exclusive; removal of the C terminus enhanced actin binding of ACTN4 mutants while limiting EGF-induced phosphorylation, and conversely EGF-stimulated phosphorylation of ACTN4 decreased its affinity to actin. Interestingly, a phosphomimetic of tyrosine 265 (which can be found in carcinoma cells and lies near the K255E mutation that causes focal segmental glomerulosclerosis) demonstrated increased actin binding activity and susceptibility of ACTN4 to calpain-mediated cleavage; this variant also retarded cell spreading. Remarkably, either treatment of cells with low concentrations of latrunculin A, which has been shown to depolymerize F-actin, or the deletion of the actin binding domain (100 - 252 amino acids) of ACTN4Y265E restored EGF-induced phosphorylation. An F-actin binding assay in vitro showed that Y4E/Y31E, a mimetic of diphosphorylated ACTN4, bound F-actin slightly compared with wild type (WT). Importantly, the EGF-mediated phosphorylation of ACTN4 at tyrosine 4 and 31 significantly inhibited multinucleation of proliferating NR6WT fibroblasts that overexpress ACTN4. These results suggest that EGF regulates the actin binding activity of ACTN4 by inducing tyrosyl-directed phosphorylation.