Kochi University, Kochi, Japan.
Gene. 2012 Apr 15;497(2):214-27. doi: 10.1016/j.gene.2012.01.036. Epub 2012 Jan 28.
Phosphagen (guanidino) kinases (PK) constitute a family of homologous phosphotransferases catalyzing the reversible transfer of the high-energy phosphoryl group of ATP to naturally occurring guanidine compounds. Prior work has shown that PKs can be phylogenetically separated into two distinct groups- an arginine kinase (AK) subfamily and a creatine kinase (CK) subfamily. The latter includes three CK isoforms- cytoplasmic CK (CyCK), mitochondrial CK (MiCK) and three-domain flagellar CK (fCK). In the present study we identified six unique PK genes from the draft genome sequence of the starlet sea anemone Nematostella vectensis, a well-known model organism for understanding metazoan developmental evolution. Using reverse transcription polymerase chain reaction (RTPCR) methods, full length cDNAs were amplified for all of these PKs. These cDNAs were cloned and expressed in Escherichia coli as 6x His-tagged fusion proteins. The six PKs were identified as the three typical CK isoforms (CyCK, MiCK and fCK), two unusual AKs (a two-domain AK (2DAK) and a three-domain AK (3DAK)) and a PK which phosphorylated arginine. The latter enzyme had a very low AK activity (its apparent V(max) value being less than 0.2% that of 3DAK), lacks several key residues necessary for AK enzyme activity, and was tentatively designated as AK1. As far as we know, this constitutes the first report of an AK with the three fused AK domains. The Bayesian tree suggested that the third domain of 3DAK likely evolved from the gene for domain 2 of typical two-domain AK found widely in cnidarians. Construction of phylogenetic trees and comparison of exon-intron organizations of their respective genes indicated that the N. vectensis three-domain fCK and 3DAK evolved independently, and both enzymes are likely to be targeted to cell membranes since they have a myristoylation signal at their respective N-termini. These results complement prior work on other basal invertebrates showing that multiple CK and AK isoform genes were present at the dawn of the radiation of metazoans. The presence of isoform diversity in an organism lacking in structural complexity reflects an early imperative for targeting of PKs to particular cellular contexts such as muscle fibers, neurons, ciliated/flagellated epithelia and spermatozoa.
磷酸原（胍基）激酶（PK）构成同源磷酸转移酶家族，催化 ATP 的高能磷酸基团可逆地转移到天然胍基化合物上。先前的工作表明，PK 可以从系统发生上分为两个不同的组 - 精氨酸激酶（AK）亚家族和肌酸激酶（CK）亚家族。后者包括三种 CK 同工酶 - 细胞质 CK（CyCK）、线粒体 CK（MiCK）和三结构域鞭毛 CK（fCK）。在本研究中，我们从星状海葵 Nematostella vectensis 的基因组草图中鉴定了六个独特的 PK 基因，Nematostella vectensis 是一种用于理解后生动物发育进化的知名模式生物。使用反转录聚合酶链反应（RT-PCR）方法，我们扩增了所有这些 PK 的全长 cDNA。这些 cDNA 被克隆并在大肠杆菌中作为 6x His 标记的融合蛋白表达。这六个 PK 被鉴定为三种典型的 CK 同工酶（CyCK、MiCK 和 fCK）、两种不寻常的 AK（双结构域 AK（2DAK）和三结构域 AK（3DAK））和一种磷酸化精氨酸的 PK。后一种酶的 AK 活性非常低（其表观 Vmax 值低于 3DAK 的 0.2%），缺乏 AK 酶活性所必需的几个关键残基，并且被暂定命名为 AK1。据我们所知，这是首次报道具有三个融合 AK 结构域的 AK。贝叶斯树表明，3DAK 的第三个结构域可能是从广泛存在于刺胞动物中的典型双结构域 AK 的 2 结构域基因进化而来的。系统发育树的构建和各自基因的外显子-内含子组织比较表明，N. vectensis 的三结构域 fCK 和 3DAK 是独立进化的，并且这两种酶都可能靶向细胞膜，因为它们在各自的 N 末端具有豆蔻酰化信号。这些结果补充了先前对其他基础无脊椎动物的研究工作，表明在后生动物辐射的黎明时期就存在多种 CK 和 AK 同工酶基因。在缺乏结构复杂性的生物体中存在同工酶多样性反映了 PK 靶向特定细胞环境（如肌肉纤维、神经元、纤毛/鞭毛上皮和精子）的早期必要性。
