BIOCHEMISTRY OF CONTRACTILE PROTEINS

平滑肌是大多数中空器官（例如脉管系统和胃肠道）中的收缩元件，并且对于生理功能是必不可少的。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。磷酸化肌球蛋白的水平反映了2种酶的活性：肌球蛋白轻链激酶和肌球蛋白磷酸酶（MP）。一个关键的发现是MP可以被调节，提出了抑制和激活。MP的调节在正常和异常（例如高血压）中起着至关重要的作用。对MP亚基相互作用的顺利理解对于在分子水平上理解MP功能至关重要。在特异性Aim 1中，将检查以肌球蛋白磷酸酶靶（MYPT 1）亚基为中心的相互作用，包括：与底物的相互作用; 和MYPT 1的C-末端部分;以及与RhoA. GTP的相互作用。这些目标应该澄清一个重要方面的基本MP机制，并将解决抑制MP活性的分子基础。一些参与MYPT 1抑制位点磷酸化的激酶被鉴定出来，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（PP 1c），因为它被MYPT 1的相互作用阻断。具体目标2的目的是定义那些阻止PP 1c作用的相互作用，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测定和对皮肤平滑肌纤维的研究。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依赖性激酶对MYPT 1磷酸化的可能性将在特异性目的3中进行评价，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。将在特异性目的4中研究两种内源性激酶，即80 kD未鉴别激酶和ZIP样激酶。上述研究将导致更详细地了解MAP功能和平滑肌和非肌肉细胞的调节，并将形成评价异常功能的基础。

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.