HSP27 Association with Thin Filaments in Colonic Muscle

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HSP 27磷酸化的缺乏有关：（1）过表达非磷酸化HSP 27的转基因小鼠对乙酰胆碱引起的收缩有抑制作用（细胞长度减少），（2）来自老年大鼠结肠的平滑肌细胞，表现出50%的收缩减少，伴随着HSP 27磷酸化的减少和肌动蛋白与肌球蛋白的结合减少。HSP 27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，通过调节细丝蛋白的相互作用（原肌球蛋白、钙调蛋白和钙调蛋白）：（1）原肌球蛋白与HSP 27的结合受HSP 27的磷酸化调节;（2）非磷酸模拟HSP 27转染的结肠平滑肌显示：（a）减少原肌球蛋白与HSP 27的结合，（B）减少肌动蛋白与肌球蛋白的结合，（c）减少钙调蛋白的磷酸化，和（d）抑制原肌球蛋白与磷酸钙调蛋白的结合。我们的初步数据还表明，我们能够恢复协会的HSP 27与原肌球蛋白在非磷酸模拟HSP 27转染结肠平滑肌细胞通过引入磷酸模拟HSP 27。具体而言，我们建议研究PKC介导的磷酸化HSP 27的作用，在调节细丝调节蛋白的活动（导致环形结肠平滑肌收缩）。细丝调节蛋白相互之间、与肌动蛋白以及与HSP 27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，钙调蛋白和钙调蛋白的磷酸化。(2)检测PKC介导的磷酸化HSP 27对钙调蛋白、钙调蛋白和原肌球蛋白磷酸化的影响。研究磷酸化的HSP 27在细纤维调节蛋白相互之间以及与肌动蛋白的协调磷酸化和相互作用中的作用。研究磷酸化HSP 27对肌动球蛋白相互作用中的个体和协调作用的影响。（3）用磷酸化/非磷酸化HSP 27突变体转染的结肠平滑肌细胞和磷酸化/非磷酸化HSP 27突变体转基因小鼠结肠平滑肌细胞，研究磷酸化HSP 27对PKC介导的原肌球蛋白、钙调蛋白和钙调蛋白磷酸化的影响，（B）它们之间的相互作用，（c）对肌球蛋白轻链磷酸化的调节。

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.