Cátedra de Microbiología, Facultad de Agronomía, Universidad de Buenos Aires, INBA-CONICET, Avda. San Martin 4453, C1417DSE Buenos Aires, Argentina.
Arch Biochem Biophys. 2011 May 1;509(1):66-75. doi: 10.1016/j.abb.2011.03.001. Epub 2011 Mar 5.
cAMP-dependent protein kinase (PKA) catalytic (C) and regulatory (R) subunits from Yarrowia lipolytica are encoded by single genes, TPK1 and RKA1, respectively. Here we performed the heterologous expression, purification and characterization of the R subunit from Y. lipolytica yeast cells, and explored the main biochemical features of the PKA. The purified recombinant R, active and capable to interact with C subunit was used to prepare highly specific polyclonal antiserum. Sucrose-gradient centrifugation and gel filtration analysis of both recombinant and native R revealed the monomeric nature of this subunit. Hydrodynamic parameters of the holoenzyme indicated that Y. lipolytica PKA is a dimer of 90 kDa composed of an R subunit of 42 kDa and a C subunit of 39 kDa. The identification of the N-terminal sequence was carried out by mass spectrometry analysis of the purified native R subunit. The differences between N-terminal sequences of R subunits from Y. lipolytica and other organisms, particularly a short linker that spans the inhibitory site, were discussed as the possible cause of the lack of dimerization. R was identified as a type II subunit since our results indicated that it was phosphorylated in vivo by C at S124 identified by anti-phospho-PKA substrate (RRXS/T) antibody.
酵母脂肪酶 cAMP 依赖性蛋白激酶（PKA）的催化亚基（C）和调节亚基（R）分别由 TPK1 和 RKA1 基因编码。本研究对来自酵母脂肪酶的 R 亚基进行了异源表达、纯化和表征，并探索了 PKA 的主要生化特性。纯化的重组 R 亚基具有活性且能够与 C 亚基相互作用，用于制备高度特异性的多克隆抗血清。蔗糖梯度离心和凝胶过滤分析表明，该亚基为单体。全酶的流体力学参数表明，酵母脂肪酶 PKA 是由一个 42 kDa 的 R 亚基和一个 39 kDa 的 C 亚基组成的 90 kDa 二聚体。通过对纯化的天然 R 亚基的质谱分析进行了 N 端序列鉴定。讨论了 R 亚基的 N 端序列与其他生物体之间的差异，特别是跨越抑制部位的短接头，这可能是缺乏二聚化的原因。由于我们的结果表明它可以在体内被 C 亚基磷酸化，因此 R 被鉴定为 II 型亚基，该磷酸化位点是由抗磷酸化 PKA 底物（RRXS/T）抗体识别的 S124 位点。
