Structure-directed investigations into the regulation of Ste20 kinases

描述（由申请人提供）： 蛋白激酶组的Sterile-20（Ste 20）家族包括p21激活激酶（PAK）和生发中心激酶III（GCKIII）家族。这些蛋白质对于多种细胞功能包括细胞极化和迁移、细胞周期、凋亡和轴突生长和再生是关键的。Ste 20激酶的失调经常在癌症中观察到，并且可能是脑海绵状畸形的重要致病因素。因此，了解调节Ste 20激酶的分子基础在生物学和临床上是重要的。在这个提议中，我们将研究Ste 20激酶的两个重要亚家族，II型PAK和GCKIII激酶的正常和改变调节的结构基础，并将研究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包括蛋白质PAK 4、PAK 5和PAK 6。尽管I型PAK（PAK 1、PAK 2和PAK 3）如何通过分子间自抑制调节的分子基础已被充分确立，但II型PAK不通过相同的机制调节。II型PAK不包括保守的AID（自抑制结构域），尽管一些研究表明II型PAK与GTP酶的相互作用对于定位而不是激酶活性是重要的，但也有有趣的数据表明II型PAK的N-末端区域可以在自调节中发挥作用。因此，在目标1中，我们将研究II型PAK的分子基础 调控在目标2中，我们研究了GCK III类蛋白激酶：MST 4，STK 24和STK 25的调节。该蛋白激酶家族的调节在分子水平上还不清楚。然而，这些蛋白质中的每一个都使用C-末端区域与脑海绵状畸形相关蛋白CCM 3直接相互作用，这可能是一种调节性相互作用。因此，在目标2中，我们将确定GCKI与CCM 3相关的结构基础，以及CCM 3介导的GCKIII激酶活性调节。在目标3中，我们建议通过结合X射线晶体学和结构导向诱变来探测Ste 20激酶的底物选择性的结构决定因素。Ste 20家族包括具有截然不同的磷酸化基序的密切相关的激酶，通过引入交换突变体来探测底物识别的结构要求，提供了独特且前所未有的机会。总体而言，本文提出的研究将显著提高我们对Ste 20激酶调节和特异性的理解，并将更好地理解癌症和脑海绵状畸形中被破坏的原子水平机制。

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.