BIOCHEMISTRY OF CONTRACTILE PROTEINS

平滑肌是大多数中空器官的收缩成分，如脉管系统和胃肠道，对生理功能至关重要。控制平滑肌收缩活性的一个重要机制是肌球蛋白的磷酸化。肌球蛋白磷酸化水平反映了肌球蛋白轻链激酶和肌球蛋白磷酸酶（MP） 2种酶的活性。一个关键的发现是MP可以调节，提出抑制和激活。MP的调节在正常和异常（如高血压）中起着至关重要的作用，顺利地了解涉及MP亚基的相互作用对于在分子水平上理解MP功能至关重要。在sp4特异性Aim 1中，以肌球蛋白磷酸酶靶（MYPT1）亚基为中心的相互作用将被检查，包括：与底物的相互作用；MYPT1与c端之间的相互作用；以及与RhoA.GTP的相互作用。这些目标应该阐明基本MP机制的一个重要方面，并将解决抑制MP活性的分子基础。一些参与MYPT1抑制位点磷酸化的激酶被确定，但没有关于逆转磷酸酶反应的数据。它不太可能涉及内源性MP磷酸酶（PP1c），因为它被MYPT1的相互作用阻断。特异性目标2的目的是确定阻止PP1c作用的相互作用，并确定体内参与的磷酸酶。解决这一重要问题的实验方案将利用体外分析和对斯皮纳平滑肌纤维的研究。众所周知，RhoA和cAMP/cGMP通路具有相反的作用。这种拮抗作用涉及cAMP/ cgmp依赖性激酶磷酸化MYPT1的可能性将在Specific Aim 3中进行评估，并与涉及RhoA磷酸化的调节机制进行比较。几种激酶可以磷酸化MOYTI并抑制MP活性，从而提高了可能涉及不同信号转导途径的可能性。特异性Aim 4将研究两种内源性激酶，即80 kD未知激酶和ziplike激酶。以上研究将使我们更详细地了解MAP在平滑肌和非肌细胞中的功能和调控，并为了解异常功能奠定基础。

Smooth muscle is the contractile element in most hollow organs, e.g. the vasculature and gastrointestinal tract and is essential for physiological function. An important mechanism controlling contractile activity in smooth muscle is phosphorylation of myosin. The level of phosphorylated myosin reflects the activities of 2 enzymes: myosin light chain kinase and myosin phosphatase (MP). A key discovery was that MP could be regulated, both inhibition and activation are proposed. The regulation of MP plays a crucial role in normal and abnormal (e.g. hypertension) smooth appreciation of the interactions involving the MP subunits is essential for an understanding of MP function at a molecular level. In Sp4ecific Aim 1 interactions centered on the myosin phosphatase target (MYPT1) subunit will be examined, including: interaction with substrate; interaction between the and C-terminal parts of MYPT1; and interaction with RhoA.GTP. These aims should clarify an important aspect of basic MP mechanism and will address the molecular basis for inhibition of MP activity. Some of the kinases involved in phosphorylation of the inhibitory site on MYPT1 are identified but there are no data on the reverse phosphatase reaction. It is unlikely to involve the endogenous MP phosphatase (PP1c) since this is blocked by interactions of MYPT1. The objectives of Specific Aim 2 are to define those interactions preventing PP1c action and to identify the phosphatase involved in vivo. The experimental protocol to address this important issue will utilize both in-vitro assays and studies with skinner smooth muscle fibers. It is known that the RhoA and cAMP/cGMP pathways have opposing effects. The possibility that this antagonism involves phosphorylation of MYPT1 by cAMP/cGMP-dependent kinases will be evaluated in Specific Aim 3 and compared to a regulatory mechanism involving phosphorylation of RhoA. Several kinases can phosphorylate MOYTI and inhibit MP activity thus raising the possibility that different signal transduction pathways could be implicated. Two endogenous kinases will be investigated in Specific Aim 4, i.e. an 80 kD unidentified kinase and ZIP-like kinase. The above studies will lead to a more detailed understanding of MAP FUNCTION and regulation in smooth muscle and non-muscle cells and will form a basis for appreciation of abnormal function.