HSP27 Association with Thin Filaments in Colonic Muscle

描述(申请人提供)：新数据表明，结肠的运动功能受损可能与小型热休克蛋白HSP27缺乏磷酸化有关：(1)过度表达非磷酸HSP27的转基因小鼠显示出抑制了乙酰胆碱诱导的收缩(细胞长度减少)，(2)老年大鼠结肠平滑肌细胞的收缩能力下降了50%，同时HSP27的磷酸化水平降低，肌动蛋白与肌球蛋白的结合减少。HSP27的磷酸化似乎是PKC介导的结肠平滑肌细胞细丝滑动的核心，通过调节细丝蛋白(原肌球蛋白、钙结合蛋白和钙粘蛋白)的相互作用：(1)原肌球蛋白与HSP27的结合受到HSP27磷酸化的调节；(2)非磷酸化的HSP27基因转染的结肠平滑肌显示：(A)原肌球蛋白与HSP27的结合减少，(B)肌动蛋白与肌球蛋白的结合减少，(C)钙结合蛋白的磷酸化减少，以及(D)原肌球蛋白与磷酸钙结合蛋白的减少。我们的初步数据还表明，通过引入磷酸化的HSP27，我们能够在非磷酸化的HSP27转基因的结肠平滑肌细胞中恢复HSP27与原肌球蛋白的联系。具体地说，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以及与HSP27的协同磷酸化和相互作用可能调节结肠环状平滑肌细胞基于细丝的收缩调节。因此，我们建议：(1)研究PKC介导的原肌球蛋白、钙调蛋白和钙粘蛋白的磷酸化。(2)检测PKC介导的磷酸化HSP27对钙调蛋白、钙蛋白和原肌球蛋白磷酸化的影响。研究磷酸化的HSP27在细丝调节蛋白之间的协调磷酸化和相互作用以及与肌动蛋白的相互作用中的作用。研究磷酸化的HSP27在肌动球蛋白相互作用中对其个体和协调作用的影响。(3)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突变小鼠结肠的平滑肌细胞，检测磷酸化的HSP27对：(A)PKC介导的原肌球蛋白、钙键蛋白和钙蛋白的磷酸化，(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.