Tyr-phosphorylation of PKC-delta and myocyte function

描述(由申请人提供)：本提案的总体目标是确定酪氨酸残基磷酸化在调节心肌组织中蛋白激酶C增量(PKCd)中的作用。需要检验的假设是，激动剂特异性激活PKCd涉及酪氨酸磷酸化，它改变了PKC肌丝底物的特异性和激酶活性。实验将表征在不同刺激下PKCd上的酪氨酸磷酸化状态以及对心肌细胞机械功能的影响。此外，还将确定在铰链、假底物和激活环区用不可磷酸化或假磷酸化的氨基酸替换酪氨酸残基对PKCd定位和底物特异性的影响。最后，实验将确定PKCd激活是否促进参与调节病理生理过程(肥大或凋亡)的其他信号转导通路的活性。令人信服的初步证据表明，PKCd的特定酪氨酸残基的磷酸化改变了底物的特异性，特别是针对肌钙蛋白(TN)复合体成分。此外，磷酸化酪氨酸(Py)可能会改变激活脂类非依赖活性的激酶的辅因子要求。结果将提供有关刺激特异性PKCd的激活和活性以及对心肌肌丝磷酸化和心肌细胞功能的影响的新信息。

DESCRIPTION (provided by applicant): The general objective of this proposal is to define the role of tyrosine residue phosphorylation in the regulation of protein kinase C delta (PKCd) in myocardial tissue. The hypothesis to be tested is that agonist-specific activation of PKCd involves tyrosine phosphorylation which modifies PKC myofilament substrate specificity and kinase activity. Experiments will characterize tyrosine phosphorylation status on PKCd with various stimuli and the impact on myocyte mechanical function. In addition, the effect of tyrosine residue replacement with unphosphorylatable or pseudo-phosphorylated amino acids in the hinge, pseudosubstrate, and activation loop domains on PKCd localization and substrate specificity will be determined. Finally, experiments will resolve whether PKCd activation promotes the activity of other signal transduction pathways involved in regulation pathophysiological processes (hypertrophy or apoptosis. Compelling preliminary evidence indicates phosphorylation of specific tyrosine residues of PKCd alters substrate specificity particularly in respect to troponin (Tn) complex components. In addition, phosphorylated tyrosine (pY) may change the cofactor requirements of the kinase enabling lipid independent activity. Results will provide novel information regarding stimuli specific PKCd activation and activity as well as the effect on cardiac myofilament phosphorylation and myocyte functional capabilities.