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.

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与伴随的Hsp27磷酸化和肌动蛋白与肌球蛋白的关联降低的收缩。 HSP27磷酸化似乎是通过调节薄丝蛋白（tropomyosin，caldesmon and calponin and calponin）的相互作用，是在结肠平滑肌细胞中PKC介导的细丝滑动的核心：（1）通过HSP27与HSP27的关联，由HSP27进行了调节。 （2）非磷酸HSP27转染的结肠平滑肌显示：（a）肌动蛋白与肌球蛋白与肌球蛋白的缔合降低，（c）降低Caldesmon磷酸化的磷酸化降低，（d）抑制抑制phospomyosin与磷酸层的降低。我们的初步数据还表明，我们能够通过引入磷酸化的HSP27来恢复非磷酸HSP27转染的结肠平滑肌细胞中HSP27与肌球蛋白的关联。具体而言，我们建议研究PKC介导的磷酸化HSP27的作用，在调节细丝调节蛋白（导致圆形结肠平滑肌的收缩）的活性中。与肌动蛋白以及HSP27相互调节的薄丝调节蛋白的协调磷酸化和相互作用可能调节结肠圆形平滑肌细胞中基于薄丝的收缩调节。因此，我们建议：（1）检查pkc介导的tropomyosin，caldesmon和calponin的磷酸化。 （2）检查PKC介导的磷酸化HSP27对Caldesmon，Calponin和Tropomyosin的磷酸化的影响。研究磷酸化的Hsp27在协调的磷酸化以及薄丝调节蛋白与彼此和肌动蛋白之间的相互作用中的作用。研究磷酸化的Hsp27对其个体和肌动菌素相互作用的协调作用的影响。 （3）使用磷酸/非磷酸-HSP27突变体从磷酸/非磷酸/非磷酸-HSP27突变体转基因小鼠的结肠中转染的圆肠平滑肌细胞和平滑肌细胞，检查磷酸化的Hsp27 On的效果： （c）调节肌球蛋白轻链磷酸化。