REGULATION OF ORGANIZATION/FUNCTION OF CYTOSKELETAL PROTEINS BY PHOSPHORYLATION

通过磷酸化调节细胞骨架蛋白的组织/功能

• 批准号: 6163122
• 负责人: R K SIHAG
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6163122
• 关键词: calcineurin; electron microscopy; enzyme mechanism; gel electrophoresis; neurofilament; neurofilament proteins; phosphorylation; protein kinase A; protein structure function; synthetic peptide

Previous studies using retinal ganglion cell neurons as a model system have demonstrated that the phosphate groups on the N-terminal head domain of the 145 kDa middle subunit of neurofilaments (NF-M) in vivo are added by second messenger-dependent protein kinases. In the present study, ser-23 was identified as a specific protein kinase A phosphorylation site on native NF-M subunit and on two synthetic peptides S1 (14RRVPTETRSSF24) and S2 (21RSSFSRVSGSPSSGFRSQSWS41) localized within the N-terminal head domain region. Ser-23 was the major site of phosphorylation for protein kinase A on the 32P-labeled peptides from native NF-M subunit and on both the S1 and S2 peptides. The synthetic peptide S2 also showed low level phosphorylation of ser-28 and ser-32 by protein kinase A. Ser-23, ser-25, ser-28, ser-32 and a threonine residue were phosphorylated by protein kinase C but neither of the site was phosphorylated stoichiometrically. The analyses of the phosphorylated synthetic peptides by LC/MS/MS also showed that protein kinase A phosphorylated only 1 site on peptide S1and ions containing up to 3 phosphates were detected on peptide S2. By comparison, 2 and 4 sites were phosphorylatable by protein kinase C on peptide S1 and S2, respectively. These data showed that ser-23 is a major protein kinase A phosphorylation site on the amino-terminal head domain of NF-M. Since phosphorylation of neurofilaments by protein kinase A in vitro is known to regulate neurofilament assembly/disassembly, phosphorylation of ser- 23 on the amino-terminal head domain may play a critical role in neurofilament dynamics during axonal transport. This hypothesis was supported by a functional assay developed to study how individual protein kinases or protein phosphatases may regulate the assembly/disassembly of various cytoskeletal elements into a supramolecular structure. In this assay, the Triton-insoluble cytoskeleton preparations are treated with 1-3 M urea to release high molecular weight complexes (5 - 20 million daltons) from the cytoskeletal preparation which are then fractionated on a Sephacryl S- 500 column and analyzed by gel electrophoresis and by electron microscopy of rotary shadowed or negatively stained oligomers. For example, the treatment of cytoskeletal preparation with 1.25 M urea resulted in the elution of neurofilament proteins in peaks containing heteropolymers of neurofilament subunits. NF-H eluted in a peak at 8-12 million daltons and NF-M and NF-L peaks eluted at 6-10 million daltons. Similarly, spectrin containing oligomers eluted into two peaks at > 12 million and 4-8 million daltons. Dephosphorylation of the cytoskeletal preparations by calcineurin resulted in more than 100% decrease in the amount of neurofilament proteins dissociated into high molecular weight oligomers. The dephosphorylation-mediated decreased dissociation of neurofilament proteins was partially restored by phosphorylation by protein kinase A but not by protein kinase C both of which phosphorylate only the N-terminal head domain regions of neurofilament proteins. These data support the hypothesis that the supramolecular organization of the axonal cytoskeleton is regulated by site-specific phosphorylation/ dephosphorylation of the constituent cytoskeletal proteins.

以前的研究使用视网膜神经节细胞神经元作为模型系统 已经证明N-末端头部的磷酸基团 体内145 kDa神经丝中亚基（NF-M）的结构域 由第二信使依赖性蛋白激酶添加。本 Ser-23是一种特异性蛋白激酶A 天然NF-M亚基和两个合成的NF-M亚基上的磷酸化位点 肽S1（14 RRVPTETRSSF 24）和S2（21 RSSFSRVSGSPSSGFRSQSWS 41） 位于N-末端头部结构域区域内。23号爵士是少校 32 P标记肽上蛋白激酶A的磷酸化位点 来自天然NF-M亚基和在S1和S2肽上。的 合成肽S2也显示低水平的Ser-28磷酸化， ser-32蛋白激酶A。Ser-23、Ser-25、Ser-28、Ser-32和a 苏氨酸残基被蛋白激酶C磷酸化，但 的位点被化学计量地磷酸化。的分析 通过LC/MS/MS的磷酸化合成肽也显示蛋白质 激酶A仅磷酸化肽S1上的1个位点， 在肽S2上检测到3个磷酸。相比之下，2和4 肽S1和S2上的位点可被蛋白激酶C磷酸化， 分别这些数据表明ser-23是一种主要的蛋白激酶 NF-M的氨基末端头部结构域上的磷酸化位点。以来 已知在体外通过蛋白激酶A磷酸化神经丝 调节神经丝的组装/拆卸，丝氨酸蛋白酶的磷酸化， 23在氨基末端头部结构域上可能起关键作用， 轴突运输过程中的神经丝动力学。 这一假设得到了一项功能测定的支持，该测定旨在研究 单个蛋白激酶或蛋白磷酸酶如何调节 将各种细胞骨架元件组装/拆卸成 超分子结构在该测定中，Triton不溶性的 用1-3 M尿素处理细胞骨架制备物， 分子量复合物（5 - 20百万道尔顿）来自 细胞骨架制备物，然后在Sephacryl S- 500柱，并通过凝胶电泳和电子显微镜分析。 旋转阴影或负染色寡聚体的显微镜检查。为 例如，用1.25 M尿素处理细胞骨架制剂 导致神经丝蛋白在含有 神经丝亚单位的杂聚体。NF-H在8-12 μ m处洗脱， NF-M和NF-L峰在6-10百万道尔顿洗脱。 类似地，含有血影蛋白的寡聚体在> 12 400万和400 - 800万道尔顿。细胞骨架的去磷酸化 钙调神经磷酸酶制剂导致超过100%的减少， 解离成高分子量的神经丝蛋白的量 低聚物去磷酸化介导的解离减少， 神经丝蛋白通过磷酸化部分恢复， 蛋白激酶A而不是蛋白激酶C，两者都磷酸化 只有神经丝蛋白的N-末端头部区域。这些 数据支持的假设，超分子组织的 轴突细胞骨架受位点特异性磷酸化调节 构成细胞骨架蛋白的去磷酸化。