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HSP27磷酸化缺失有关。(1)过表达非磷酸化HSP27的转基因小鼠对乙酰胆碱诱导的收缩有抑制作用（细胞长度减少）；(2)老龄大鼠结肠平滑肌细胞收缩减少50%，同时HSP27磷酸化降低，肌动蛋白与肌球蛋白的关联减少。HSP27的磷酸化似乎是pkc介导的结肠平滑肌细胞纤维滑动的核心，通过调节细丝蛋白（原肌球蛋白，caldesmon和calponin）的相互作用：(1)原肌球蛋白与HSP27的关联通过HSP27的磷酸化调节；(2)非磷系HSP27转染的结肠平滑肌显示：(a)原肌球蛋白与HSP27的关联降低，(b)肌动蛋白与肌球蛋白的关联降低，(c) caldesmon磷酸化降低，(d)原肌球蛋白与磷酸化caldesmon的关联降低受到抑制。我们的初步数据还表明，通过引入磷酸化HSP27，我们能够在非磷酸化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活性（导致环状结肠平滑肌收缩）中的作用。细丝调节蛋白与肌动蛋白和HSP27的协同磷酸化和相互作用可能调节细丝对结肠圆形平滑肌细胞收缩的调节。因此，我们建议：(1)检测pkc介导的原肌球蛋白、钙调蛋白和钙钙蛋白的磷酸化。(2)检测pkc介导的磷酸化HSP27对钙调素、钙钙蛋白和原肌球蛋白磷酸化的影响。研究磷酸化的HSP27在细丝调节蛋白相互及与肌动蛋白的协调磷酸化和相互作用中的作用。研究磷酸化的HSP27对它们在肌动球蛋白相互作用中的个体和协调作用的影响。(3)利用phospho/non-phospho-HSP27突变体转染的圆形结肠平滑肌细胞和来自phospho/non-phospho-HSP27突变体转基因小鼠结肠的平滑肌细胞，研究磷酸化的HSP27对pkc介导的原肌球蛋白、caldesmon和calponin磷酸化的影响，(b)它们之间的相互作用，(c)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.