ALPK3 in cardiac function and disease

项目摘要 α蛋白激酶3（ALPK 3，also known as MAK，MIDORI）是一种新型的非典型蛋白激酶， 在心肌中表达。ALPK 3的双等位基因截短突变导致严重的小儿心肌病。 据报道，基因陷阱突变小鼠中ALPK 3的缺乏会导致心肌病。然而， 已知ALPK 3在心肌细胞中的特定作用，或ALPK 3丢失的分子机制， 导致心肌病此外，还有待确定ALPK 3是否是真正的蛋白激酶， 作为假激酶发挥作用。为了说明ALPK 3的心脏作用，我们已经产生了一个floxed ALPK 3 小鼠系，并用其产生ALPK 3整体敲除（gKO），以及组成型（cKO）和诱导型（cKO） （icKO）心脏特异性敲除小鼠模型。我们的初步数据显示，与公布的 ALPK 3基因陷阱突变体、ALPK 3 gKO和cKO小鼠发生早发性心肌病。但不同于 报道的ALPK 3基因诱捕小鼠，我们的ALPK 3 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ALPK 3 icKO小鼠 DCM和心力衰竭。这些观察结果有力地表明，ALPK 3在两种发展中起着关键作用， 和成体心肌细胞。为了测试ALPK 3的激酶活性，我们使用 重组ALPK 3激酶结构域。令人惊讶的是，我们没有检测到激酶活性。此外，我们还生成了一个 新的ALPK 3基因敲入小鼠模型，其中磷酸化所必需的催化赖氨酸（不变赖氨酸1420） 转移活性突变为精氨酸（ALPK 3 KR/KR），从而破坏推定的ALPK 3激酶活性。 ALPK 3 KR/KR小鼠未显示任何心脏异常。总之，这些观察结果表明， 推测的ALPK 3磷酸转移活性不是心脏功能所必需的。为了研究假定的 激酶结构域，我们产生了突变小鼠模型，其中两个Zn 2 +-配位半胱氨酸残基 对α-激酶结构域结构至关重要的突变为丙氨酸残基（ALPK 3CA/CA）。ALPK 3CA/CA小鼠 显示DCM，尽管相对于在ALPK 3 cKO小鼠中观察到的DCM不太严重并且具有延迟的发作。 ALPK 3CA/CA突变体的心脏表型表明， ALPK 3对心脏功能至关重要。综上所述，上述观察使我们得出这样的假设， ALPK 3在调节心脏功能中起着重要作用，尽管缺乏催化活性， 假定的激酶结构域和/或ALPK 3的其他结构域介导心脏关键的蛋白质相互作用 功能我们的具体目标是：1。阐明ALPK 3在发育和成年心肌中的作用; 对假定的ALPK 3激酶结构域的需求的潜在机制的解读，缺乏激酶 心脏功能的活动。

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.