Structure-directed investigations into the regulation of Ste20 kinases

描述(由申请人提供)： 蛋白激动组的不育-20(Ste20)家族包括p21激活的激酶(PAK)和生发中心激酶III(GCKIII)家族。这些蛋白质对多种细胞功能至关重要，包括细胞极化和迁移、细胞周期、细胞凋亡和轴突生长和再生。Ste20激酶的异常在癌症中经常被观察到，并且可能是脑海绵状畸形的一个重要的致病因素。因此，了解Ste20激酶调控的分子基础具有重要的生物学和临床意义。在这项建议中，我们将研究Ste20激酶的两个重要的亚家族，即II型PAKs和GCKIII激酶的正常和异常调节的结构基础，并将研究这两个亚家族之间特异性差异的分子水平决定因素。在目标1中，我们将研究II型PAK的调节。II型PAK包括蛋白PAK4、PAK5和PAK6。虽然I型PAK(PAK1、PAK2和PAK3)如何通过分子间自身抑制来调节的分子基础已经被很好地建立，但II型PAK不是由相同的机制调节的。II型PAK不包括保守的AID(自动抑制域)，尽管一些研究表明II型PAK与GTP酶的相互作用对定位很重要，但也有有趣的数据表明II型PAK的N端区域可以在自动调节中发挥作用。因此，在目标1中，我们将研究II型PAK的分子基础 监管。在目标2中，我们研究了GCKIII类蛋白激酶：MST4、STK24和STK25的调节。这一蛋白激酶家族的调节在分子水平上还不是很清楚。然而，这些蛋白中的每一个都使用一个C末端区域直接与大脑海绵状畸形相关蛋白CCM3相互作用，这可能是一种调节相互作用。因此，在目标2中，我们将确定GCKI与CCM3结合的结构基础，以及CCM3介导的GCKIII激酶活性的调节。在目标3中，我们建议通过X射线结晶学和结构导向突变相结合的方法来探索Ste20激酶底物选择性的结构决定因素。Ste20家族包括具有截然不同的磷酸化基序的密切相关的激酶，这为通过引入交换突变体来探索底物识别的结构要求提供了一个独特的、前所未有的机会。总体而言，这里提出的研究将显著提高我们对Ste20激酶的调节和特异性的理解，并将更好地理解在癌症和脑海绵状畸形中被破坏的原子水平的机制。

DESCRIPTION (provided by applicant): The Sterile-20 (Ste20) family of the protein kinome includes both the p21-activated kinase (PAK) and germinal center kinase III (GCKIII) families. These proteins are critical for a variety o cellular functions including cell polarization and migration, cell cycle, apoptosis and axon outgrowth and regeneration. Dysregulation of Ste20 kinases is frequently observed in cancer and may be an important causative factor in cerebral cavernous malformations. Consequently, understanding the molecular basis for regulation of the Ste20 kinases is biologically and clinically important. In this proposal we will investigate the structural basis for normal and alteed regulation of two important sub-families of the Ste20 kinases, the type II PAKs and the GCKIII kinases, and will investigate the molecular level determinants of differences in specificity between the two sub-families. In Aim 1 we will investigate the regulation of type II PAKs. Type II PAKs encompass the proteins PAK4, PAK5 and PAK6. Whereas the molecular basis for how type I PAKs (PAK1, PAK2 and PAK3) are regulated by inter- molecular autoinhibition is well established, the type II PAKs are not regulated by the same mechanism. Type II PAKs do not include a conserved AID (autoinhibitory domain), and although some studies suggest that interaction of type II PAKs with GTPases is important for localization but not kinase activity, there is also intriguing data suggesting that the N-terminal region of the type II PAKs can play a role in autoregulation. Therefore, in Aim 1 we will investigate the molecular basis for type II PAK regulation. In Aim 2 we investigate the regulation of the GCKIII class of protein kinases: MST4, STK24 and STK25. The regulation of this family of protein kinases is not understood at the molecular level. However, each of these proteins uses a C-terminal region to directly interact with the cerebral cavernous malformation-associated protein CCM3 which may be a regulatory interaction. Therefore in Aim 2 we will determine the structural basis for GCKI association with CCM3, and CCM3-mediated modulation of GCKIII kinase activity. In Aim 3, we propose to probe the structural determinants of substrate selectivity by Ste20 kinases through a combination of X-ray crystallography and structure-guided mutagenesis. The Ste20 family includes closely related kinases having drastically different phosphorylation motifs, providing a unique and unprecedented opportunity to probe the structural requirements for substrate recognition through the introduction of exchange mutants. Overall, the studies proposed here will significantly improve our understanding of Ste20 kinase regulation and specificity, and will provide a better understanding of the atomic-level mechanisms that are disrupted in cancer and cerebral cavernous malformations.