Abdullah K, Cromlish W A, Payette P, Laliberté F, Huang Z, Street I, Kennedy B P
Department of Biochemistry and Molecular Biology, Merck Frosst Center for Therapeutic Research, Merck Frosst Canada Inc., Pointe Claire-Dorval, Quebec.
Biochim Biophys Acta. 1995 May 11;1244(1):157-64. doi: 10.1016/0304-4165(94)00218-m.
Cytosolic PLA2 (cPLA2) has been implicated in the release of the arachidonic acid utilized in the inflammatory cascade. Phosphorylation of cPLA2 on Ser-505 by MAP kinase in response to agonist treatment, is thought to be one of the mechanisms required for activation of the enzyme in the cell. In order to obtain enough material for enzymological studies as well as to investigate the role of phosphorylation in the activation of cPLA2, the human enzyme was overexpressed in insect cells using a recombinant baculovirus. We report here on the characterization of the phosphorylation state of cPLA2 overexpressed in Sf9 cells. The level of overexpressed cPLA2 was shown to peak between 48 and 60 h post-infection, by this time the phosphorylated enzyme could easily be detected because of its reduced mobility on polyacrylamide gels. The reduced mobility or gel-shift has been shown to be due to phosphorylation of Ser-505. To determine whether this was also the case for insect cell overexpressed cPLA2, Ser-505 was replaced by Ala, and this mutant (cPLA2S505A) was expressed in Sf9 cells. Analysis of the overexpressed cPLA2S505A showed that it migrated only as the lower unshifted cPLA2 band confirming that the baculovirus overexpressed cPLA2 is extensively phosphorylated on Ser-505. Furthermore, treatment of infected Sf9 cells expressing the wild-type cPLA2 with phorbol 12-tetradecanoate 13-acetate (TPA) shifted all of the overexpressed cPLA2 to the phosphorylated Ser-505 form. When infected Sf9 cells were labelled with [32P], in addition to labelling of Ser-505 other sites were also labelled. Both cPLA2 and cPLA2S505A were purified from infected Sf9 cells and the specific activity for each of the enzymes was measured in a phosphatidylcholine vesicle fluorescence assay using 1-(10-pyrenedecanyl)arachidonyl-sn-glycero-3-phosphocholine as substrate. Under these conditions the specific activity of cPLA2 was, 2 mumol/min per mg, whereas cPLA2S505A was 7-fold less active. These findings suggest that Sf9 cells have a mechanism for phosphorylating cPLA2 similar to that found in mammalian cells which probably proceeds through a MAP kinase. Thus, insect cell overexpressed cPLA2 is a very good source for the Ser-505 phosphorylated enzyme.
胞质型磷脂酶A2（cPLA2）与炎症级联反应中花生四烯酸的释放有关。在激动剂处理后，丝裂原活化蛋白激酶（MAP激酶）使cPLA2的丝氨酸505位点发生磷酸化，这被认为是该酶在细胞内激活所需的机制之一。为了获得足够的材料用于酶学研究，并研究磷酸化在cPLA2激活中的作用，使用重组杆状病毒在昆虫细胞中过表达人源该酶。我们在此报告在Sf9细胞中过表达的cPLA2的磷酸化状态特征。结果显示，过表达的cPLA2水平在感染后48至60小时达到峰值，此时由于其在聚丙烯酰胺凝胶上迁移率降低，磷酸化的酶很容易被检测到。迁移率降低或凝胶迁移已被证明是由于丝氨酸505位点的磷酸化。为了确定昆虫细胞中过表达的cPLA2是否也是这种情况，将丝氨酸505替换为丙氨酸，该突变体（cPLA2S505A）在Sf9细胞中表达。对过表达的cPLA2S505A的分析表明，它仅以未迁移的较低cPLA2条带形式迁移，这证实杆状病毒过表达的cPLA2在丝氨酸505位点被广泛磷酸化。此外，用佛波醇12 - 十四烷酸酯13 - 乙酸酯（TPA）处理表达野生型cPLA2的感染Sf9细胞，使所有过表达的cPLA2转变为磷酸化的丝氨酸505形式。当用[32P]标记感染的Sf9细胞时，除了丝氨酸505位点被标记外，其他位点也被标记。从感染的Sf9细胞中纯化cPLA2和cPLA2S505A，并使用1-（10 - 芘癸基）花生四烯酰 - sn - 甘油 - 3 - 磷酸胆碱作为底物，通过磷脂酰胆碱囊泡荧光测定法测量每种酶的比活性。在这些条件下，cPLA2的比活性为每毫克2 μmol/分钟，而cPLA2S505A的活性低7倍。这些发现表明，Sf9细胞具有一种使cPLA2磷酸化的机制，类似于在哺乳动物细胞中发现的机制，可能是通过MAP激酶进行的。因此，昆虫细胞中过表达的cPLA2是丝氨酸505磷酸化酶的一个非常好的来源。
