Transducible heat shock protein 20 (HSP20) phosphopeptide alters cytoskeletal dynamics
Transducible heat shock protein 20 (HSP20) phosphopeptide alters cytoskeletal dynamics
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DOI：
10.1096/fj.04-2911fje
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发表时间：
2004-12-01
期刊：
影响因子：
4.8
通讯作者：
Panitch, A
中科院分区：
文献类型：
--
作者：
Dreiza, CM;Brophy, CM;Panitch, A
环核苷酸依赖性信号通路的激活导致平滑肌松弛、培养细胞的细胞骨架改变和HSP 20磷酸化的增加。为了确定磷酸化HSP 20对肌动蛋白细胞骨架的影响，合成了HSP 20的磷酸肽类似物。这些肽含有1）围绕HSP 20磷酸化位点的氨基酸序列，2）磷酸丝氨酸，和3）蛋白转导结构域。瑞士3 T3细胞与磷酸化肽类似物的HSP 20的治疗导致肌动蛋白应力纤维和粘着斑复合物的损失所证明的免疫细胞化学，干涉反射显微镜，和球状肌动蛋白的生化定量。用HSP 20的磷酸肽类似物处理也导致肌动蛋白解聚蛋白cofilin的去磷酸化。下拉分析表明，14-3-3蛋白与HSP 20的磷酸肽类似物（但不是丝氨酸未磷酸化的肽类似物）相关。14-3-3蛋白与HSP 20的磷酸肽类似物的结合阻止了cofilin与14-3-3的缔合。这些数据表明，热休克蛋白20可以调节肌动蛋白细胞骨架动力学与肌动蛋白解聚蛋白cofilin的结合支架蛋白14-3-3的竞争。有趣的是，这种效应并不需要整个蛋白质，这表明这种关联是由HSP 20的磷酸肽基序调节的。这些数据还表明，环核苷酸依赖性平滑肌松弛的可能性，可以介导的细丝（肌动蛋白）的监管过程。最后，这些数据表明，蛋白质转导可以作为一种工具来阐明蛋白质的肽基序的特定功能。
Activation of cyclic nucleotide dependent signaling pathways leads to relaxation of smooth muscle, alterations in the cytoskeleton of cultured cells, and increases in the phosphorylation of HSP20. To determine the effects of phosphorylated HSP20 on the actin cytoskeleton, phosphopeptide analogs of HSP20 were synthesized. These peptides contained 1) the amino acid sequence surrounding the phosphorylation site of HSP20, 2) a phosphoserine, and 3) a protein transduction domain. Treatment of Swiss 3T3 cells with phosphopeptide analogs of HSP20 led to loss of actin stress fibers and focal adhesion complexes as demonstrated by immunocytochemistry, interference reflection microscopy, and biochemical quantitation of globular-actin. Treatment with phosphopeptide analogs of HSP20 also led to dephosphorylation of the actin depolymerizing protein cofilin. Pull-down assays demonstrated that 14-3-3 proteins associated with phosphopeptide analogs of HSP20 ( but not peptide analogs in which the serine was not phosphorylated). The binding of 14-3-3 protein to phosphopeptide analogs of HSP20 prevented the association of cofilin with 14-3-3. These data suggest that HSP20 may modulate actin cytoskeletal dynamics by competing with the actin depolymerizing protein cofilin for binding to the scaffolding protein 14-3-3. Interestingly, the entire protein was not needed for this effect, suggesting that the association is modulated by phosphopeptide motifs of HSP20. These data also suggest the possibility that cyclic nucleotide dependent relaxation of smooth muscle may be mediated by a thin filament ( actin) regulatory process. Finally, these data suggest that protein transduction can be used as a tool to elucidate the specific function of peptide motifs of proteins.