HSP27 Association with Thin Filaments in Colonic Muscle

HSP27 与结肠肌细丝的关联

• 批准号: 7077637
• 负责人: KHALIL N BITAR
• 金额: $ 32.05万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-06-15 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7077637
• 关键词: SDS polyacrylamide gel electrophoresis; actins; calcium flux; caldesmon; calponin; colon; gastrointestinal motility /pressure; genetically modified animals; heat shock proteins; immunoprecipitation; laboratory rabbit; muscle contraction; myosin light chain kinase; myosins; phosphorylation; polymerase chain reaction; protein kinase C; protein protein interaction; smooth muscle; tissue /cell culture; transfection; tropomyosin

DESCRIPTION (provided by the applicant): Emerging data suggest that impaired motility of the colon could be associated with the absence of phosphorylation of the small heat shock protein HSP27: (1) Transgenic mice over-expressing non-phospho-HSP27 showed inhibition of acetylcholine-induced contraction (decrease in cell length), (2) Smooth muscle cells from the colons of aged rats, exhibited 50% decreased contraction with concomitant decreased phosphorylation of HSP27 and decreased association of actin with myosin. HSP27 phosphorylation seems to be at the core of the PKC-mediated sliding of filaments in smooth muscle cells of the colon, by modulating the interaction of the thin filament proteins (tropomyosin, caldesmon and calponin): (1) association of tropomyosin with HSP27 is modulated by phosphorylation of HSP27; (2) non-phosphomimic HSP27 transfected colonic smooth muscle show: (a) reduced association of tropomyosin with HSP27, (b) reduced association of actin with myosin, (c) decreased phosphorylation of caldesmon, and (d) inhibition of the decreased association of tropomyosin with phospho-caldesmon. Our preliminary data also indicate that we were able to reinstate the association of HSP27 with tropomyosin in non-phosphomimic HSP27 transfected colonic smooth muscle cells by introducing phosphomimic HSP27. Specifically we propose to study the role of PKC-mediated phosphorylated HSP27, in modulating the activities of thin filament regulatory proteins (leading to contraction of circular colon smooth muscle). The coordinated phosphorylation and interactions of thin filament regulatory proteins with each other, with actin as well as with HSP27 may modulate thin filament based regulation of contraction in circular smooth muscle cells of colon. We therefore propose to: (1) Examine PKC-mediated phosphorylation of tropomyosin, caldesmon, and calponin. (2) Examine the effect of PKC-mediated phosphorylated HSP27 on the phosphorylation of caldesmon, calponin, and tropomyosin. Investigate the role of phosphorylated HSP27 in coordinated phosphorylation and interactions of thin filament regulatory proteins with each other and with actin. Investigate the effect of phosphorylated HSP27 on their individual and coordinated role in actomyosin interaction. And (3) Use the phospho/non-phospho-HSP27 mutant transfected circular colonic smooth muscle cells and smooth muscle cells from the colons of phospho/non-phospho-HSP27 mutant transgenic mice, to examine the effect of phosphorylated HSP27 on: (a) the PKC-mediated phosphorylation of tropomyosin, caldesmon and calponin, (b) their interaction, (c) modulation of myosin light chain phosphorylation.

描述（由申请人提供）：新出现的数据表明，结肠运动受损可能与小热休克蛋白 HSP27 磷酸化的缺失有关：（1）过度表达非磷酸化 HSP27 的转基因小鼠表现出乙酰胆碱抑制作用-诱导收缩（细胞长度减少），（2）来自老年大鼠结肠的平滑肌细胞表现出收缩减少 50%，同时 HSP27 和磷酸化水平降低肌动蛋白与肌球蛋白的结合减少。 HSP27 磷酸化似乎是结肠平滑肌细胞中 PKC 介导的细丝滑动的核心，通过调节细丝蛋白（原肌球蛋白、钙结合蛋白和钙调蛋白）的相互作用：(1) 原肌球蛋白与 HSP27 的关联是由 HSP27 磷酸化调节； (2)非拟磷酸化HSP27转染的结肠平滑肌显示：(a)原肌球蛋白与HSP27的关联减少，(b)肌动蛋白与肌球蛋白的关联减少，(c)钙结合蛋白的磷酸化减少，以及(d)对减少的关联的抑制原肌球蛋白与磷酸钙结合蛋白。我们的初步数据还表明，通过引入拟磷HSP27，我们能够在非拟磷HSP27转染的结肠平滑肌细胞中恢复HSP27与原肌球蛋白的关联。具体来说，我们建议研究 PKC 介导的磷酸化 HSP27 在调节细丝调节蛋白的活性（导致环形结肠平滑肌收缩）中的作用。细丝调节蛋白彼此之间、与肌动蛋白以及与HSP27的协调磷酸化和相互作用可以调节结肠环形平滑肌细胞中基于细丝的收缩调节。因此，我们建议：（1）检查 PKC 介导的原肌球蛋白、caldesmon 和 calponin 的磷酸化。 (2)检查PKC介导的磷酸化HSP27对caldesmon、calponin和原肌球蛋白磷酸化的影响。研究磷酸化 HSP27 在协调磷酸化以及细丝调节蛋白彼此之间以及与肌动蛋白的相互作用中的作用。研究磷酸化 HSP27 对肌动球蛋白相互作用中个体和协调作用的影响。 (3)使用磷酸化/非磷酸化HSP27突变体转染的环状结肠平滑肌细胞和来自磷酸化/非磷酸化HSP27突变体转基因小鼠结肠的平滑肌细胞，来检查磷酸化HSP27对：（a ) PKC 介导的原肌球蛋白、caldesmon 和 calponin 的磷酸化，(b) 它们的相互作用，(c) 肌球蛋白轻链磷酸化的调节。