Hannah Research Institute, Ayr KA6 5HL, Scotland, UK.
Arch Biochem Biophys. 2012 Mar 1;519(1):38-45. doi: 10.1016/j.abb.2012.01.008. Epub 2012 Jan 20.
Multiple isoforms of the cyclic AMP-dependent protein kinase (PK-A) catalytic (C) subunit, arise as a consequence of the use of alternative splicing strategies during transcription of the kin-1 gene in the nematode, Caenorhabditis elegans. N-myristoylation is a common co-translational modification of mammalian PK-A C-subunits; however, the major isoform (N'3), originally characterised in C. elegans, is not N-myristoylated. Here, we show that N'1 isoforms are targets for N-myristoylation in C. elegans. We have demonstrated the in vivo incorporation of radioactivity into N'1 C-subunit isoforms, following incubation of nematodes with [(3)H]-myristic acid. HPLC and MALDI-TOF MS analysis of proteolytic digests of immunoprecipitates confirmed the presence of myristoyl-glycine in the C-subunit. In order to better understand the impact of the N'1 N-terminal sequence, and its myristoylation, on C-subunit activity, a chimerical C-subunit, consisting of the N'1 N-terminus from C. elegans and a murine core and C-terminal sequence was expressed. Myristoylation had no appreciable effect on the catalytic properties of the chimeric protein. However, the myristoylated chimeric protein did exhibit enhanced apolar targeting compared to the myristoylated wild-type murine polypeptide. This behaviour may reflect the inability of the N'1-encoded N-terminus sequence to correctly dock with a hydrophobic domain on the surface of the C-subunit.
在秀丽隐杆线虫（Caenorhabditis elegans）中，由于在转录激酶基因 kin-1 时使用了不同的剪接策略，环腺苷酸依赖的蛋白激酶（PK-A）催化（C）亚基的多个同工型得以产生。N-豆蔻酰化是哺乳动物 PK-A C 亚基的常见共翻译修饰；然而，最初在秀丽隐杆线虫中表征的主要同工型（N'3）不是 N-豆蔻酰化的。在这里，我们表明 N'1 同工型是秀丽隐杆线虫中 N-豆蔻酰化的靶标。我们已经证明，在用 [(3)H]-豆蔻酸孵育线虫后，N'1 C 亚基同工型可以掺入放射性物质。免疫沉淀的蛋白水解消化物的 HPLC 和 MALDI-TOF MS 分析证实了豆蔻酰-甘氨酸在 C 亚基中的存在。为了更好地理解 N'1 N 端序列及其豆蔻酰化对 C 亚基活性的影响，我们表达了一种嵌合 C 亚基，由秀丽隐杆线虫的 N'1 N 端和鼠的核心和 C 端序列组成。豆蔻酰化对嵌合蛋白的催化特性没有明显影响。然而，与豆蔻酰化的野生型鼠多肽相比，豆蔻酰化的嵌合蛋白确实表现出增强的非极性靶向性。这种行为可能反映了 N'1 编码的 N 端序列无法正确与 C 亚基表面的疏水区结合。
