BIOCHEMISTRY OF CONTRACTILE PROTEINS

平滑肌是大多数中空器官，如血管和胃肠道的收缩元件，是生理功能所必需的。肌球蛋白的磷酸化是控制平滑肌收缩活动的一个重要机制。肌球蛋白磷酸化水平反映了两种酶的活性：肌球蛋白轻链激酶和肌球蛋白磷酸酶(MP)。一项关键发现是MP是可以调节的，提出了抑制和激活两种方法。MP的调节在正常和异常(如高血压)中起着至关重要的作用，正确认识MP亚单位之间的相互作用对于从分子水平上理解MP的功能至关重要。在Sp4特异性目标1中，将研究以肌球蛋白磷酸酶靶标(MYPT1)亚单位为中心的相互作用，包括：与底物的相互作用；MYPT1与C末端部分的相互作用；以及与RhoA.GTP的相互作用。这些目的将阐明MP基本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上抑制位点磷酸化的激酶已被鉴定，但没有关于反向磷酸酶反应的数据。它不太可能涉及内源性MP磷酸酶(PP1c)，因为它被MYPT1的相互作用所阻断。具体目标2的目的是定义阻止PP1c作用的那些相互作用，并确定体内涉及的磷酸酶。解决这一重要问题的实验方案将利用体外分析和对斯金纳平滑肌纤维的研究。已知RhoA和cAMP/cGMP途径具有相反的作用。这种拮抗作用涉及cAMP/cGMP依赖的激酶对MYPT1的磷酸化的可能性将在特定的目标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的活性，从而增加了不同信号转导途径相互作用的可能性。在特定的目标4中，将研究两种内源性激酶，即一个80kD的未知激酶和ZIP样激酶。上述研究将使我们对MAP在平滑肌和非肌肉细胞中的功能和调控有更详细的了解，并为评价异常功能奠定基础。

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.