ALPK3 in cardiac function and disease

项目总结 α蛋白激酶3(ALPK3，又称MAK，Midori)是一种新型的高度非典型蛋白激酶 在心肌中表达。ALPK3双等位基因截断突变可导致严重的儿科心肌病。 在基因陷阱突变小鼠中，ALPK3的缺失被报道会导致心肌病。然而，几乎没有什么是 已知ALPK3在心肌细胞中的特定作用，或ALPK3丢失的分子机制 会导致心肌病。此外，ALPK3是否是一种真正的蛋白激酶或 起到了假性激酶的作用。为了解决ALPK3的心脏作用，我们产生了一个带花丝的ALPK3 小鼠品系，并用其产生ALPK3全局敲除(GKO)，以及构造性(CKO)和诱导性 (IcKO)心脏特异性基因敲除小鼠模型。我们的初步数据显示，与已公布的 ALPK3基因陷阱突变、ALPK3 GKO和CKO小鼠发生早发性心肌病。然而，不同于 已报道的ALPK3基因陷阱小鼠、我们的ALPK3 GKO和CKO小鼠表现出更严重的扩张 心肌病(DCM)导致过早死亡。我们还观察到成年ALPK3icKO小鼠发育 扩张型心肌病和心力衰竭。这些观察结果有力地表明，ALPK3在这两种发育过程中都发挥着关键作用 和成年心肌细胞。为了测试ALPK3的活性，我们在体外进行了使用 重组ALPK3激动域。令人惊讶的是，我们没有检测到激酶活性。此外，我们生成了一个 在新的ALPK3敲入小鼠模型中，磷酸化必需的催化赖氨酸(不变赖氨酸1420) 转移活性突变为精氨酸(ALPK3KR/KR)，从而破坏了可能的ALPK3激酶活性。 ALPK3KR/KR小鼠未表现出任何心脏异常。总而言之，这些观察表明， ALPK3假定的磷酸转移活性不是心脏功能所必需的。研究推定者的角色 我们建立了一个突变的小鼠模型，在该模型中，两个与锌离子配位的半胱氨酸残基 α-激酶结构域结构突变为丙氨酸残基(ALPK3CA/CA)。ALPK3CA/CA小鼠 与ALPK3 CKO小鼠观察到的DCM相比，表现为DCM，尽管没有那么严重，而且起病延迟。 ALPK3CA/CA突变体的心脏表型表明，在 ALPK3对心脏功能起关键作用。综上所述，上述观察结果使我们得出这样的假设 ALPK3在调节心脏功能中起着至关重要的作用，虽然缺乏催化活性，但 推测的激酶结构域和/或ALPK3的其他结构域介导对心脏至关重要的蛋白质相互作用 功能。我们的具体目标是：1。阐明ALPK3在发育和成人心肌中的作用。 破译不含激酶的假定ALPK3激酶结构域所需的机制 心脏功能中的活动性。

PROJECT SUMMARY Alpha protein kinase 3 (ALPK3, also known as MAK, MIDORI) is a novel atypical protein kinase highly expressed in cardiac muscle. Biallelic truncating mutations in ALPK3 cause severe pediatric cardiomyopathy. Deficiency of ALPK3 in gene-trap mutant mice has been reported to cause cardiomyopathy. However, little is known as to the specific role of ALPK3 in cardiomyocytes, or molecular mechanisms by which loss of ALPK3 results in cardiomyopathy. Furthermore, it is yet to be determined whether ALPK3 is a true protein kinase or functions as a pseudokinase. To address the cardiac role of ALPK3, we have generated a floxed ALPK3 mouse line and used it to generate ALPK3 global knockout (gKO), as well as constitutive (cKO) and inducible (icKO) cardiac-specific knockout mouse models. Our preliminary data revealed that, similar to the published ALPK3 gene-trap mutant, ALPK3 gKO and cKO mice develop early onset cardiomyopathy. However, unlike the reported ALPK3 gene-trap mice, our ALPK3 gKO and cKO mice exhibited a more severe dilated cardiomyopathy (DCM) leading to premature lethality. We also observed that adult ALPK3 icKO mice develop DCM and heart failure. These observations strongly suggest that ALPK3 plays a critical role in both developing and adult cardiomyocytes. To test the kinase activity of ALPK3, we performed in vitro kinase assays using recombinant ALPK3 kinase domain. Surprisingly, we did not detect kinase activity. Moreover, we generated a novel ALPK3 knock-in mouse model in which the catalytic lysine (invariant lysine 1420) essential for phospho- transfer activity was mutated to arginine (ALPK3KR/KR), thereby disrupting putative ALPK3 kinase activity. ALPK3KR/KR mice did not display any cardiac abnormalities. Together, these observations indicate that the putative phospho-transfer activity of ALPK3 is not required for cardiac function. To study the role of the putative kinase domain, we generated a mutant mouse model in which the two Zn2+-coordinating cysteine residues critical for α-kinase domain structure were mutated to alanine residues (ALPK3CA/CA). ALPK3CA/CA mice displayed DCM, albeit less severe and with delayed onset relative to the DCM observed in ALPK3 cKO mice. The cardiac phenotype of ALPK3CA/CA mutants indicates that a structurally intact putative kinase domain in ALPK3 is critical for cardiac function. Taken together, the foregoing observations lead us to the hypothesis that ALPK3 plays an essential role in regulating cardiac function, and that although devoid of catalytic activity, the putative kinase domain, and/or other domains of ALPK3, mediate protein interactions critical for cardiac function. Our Specific Aims are: 1. Elucidate the role of ALPK3 in developing and adult myocardium and 2. Decipher mechanisms underlying the requirement for the putative ALPK3 kinase domain, devoid of kinase activity, in cardiac function.