Guergnon Julien, Dessauge Frederic, Traincard François, Cayla Xavier, Rebollo Angelita, Bost Pierre Etienne, Langsley Gordon, Garcia Alphonse
Unité de Chimie Organique-Equipe Phosphatase et Laboratoire de signalisation Immunoparasitaire, URA CNRS 2581, 75015 Paris, France.
Apoptosis. 2006 Aug;11(8):1263-73. doi: 10.1007/s10495-006-7702-6.
T. annulata, an intracellular pathogenic parasite of the Aplicomplexa protozoan family infects bovine B-lymphocytes and macrophages. Parasitized cells that become transformed survive and proliferate independently of exogenous growth factors. In the present study, we used the isogenic non parasitized BL3 and parasitized TBL3 B cell lines, as a model to evaluate the contribution of two-major PI3-K- and PKA-dependent anti-apoptotic pathways in the survival of T. annulata parasitized B lymphocytes. We found that T. annulata increases PKA activity, induces over-expression of the catalytic subunit and down-regulates the pro-survival phosphorylation state of Akt/PKB. Consistent with a role of PKA activation in survival, two pharmacological inhibitors H89 and KT5720 ablate PKA-dependent survival of parasitized cells. To specifically inhibit PKA pro-survival pathways we linked the DPTsh1 peptide shuttle sequence to PKI(5-24) and we generated DPT-PKI, a cell permeable PKI. DPT-PKI specifically inhibited PKA activity in bovine cell extracts and, as expected, also inhibited the PKA-dependent survival of T. annulata parasitized TBL3 cells. Thus, parasite-dependent constitutive activation of PKA in TBL3 cells generates an anti-apoptotic pathway that can protect T. annulata-infected B cells from apoptosis. These results also indicate that DPT-PKI could be a powerful tool to inhibit PKA pathways in other cell types.
环形泰勒虫是顶复门原生动物家族的一种细胞内致病性寄生虫，可感染牛B淋巴细胞和巨噬细胞。被寄生并发生转化的细胞能够独立于外源性生长因子而存活和增殖。在本研究中，我们使用同基因的未被寄生的BL3和被寄生的TBL3 B细胞系作为模型，来评估两条主要的PI3-K和PKA依赖性抗凋亡途径对环形泰勒虫寄生的B淋巴细胞存活的贡献。我们发现，环形泰勒虫可增加PKA活性，诱导催化亚基的过表达，并下调Akt/PKB的促生存磷酸化状态。与PKA激活在存活中的作用一致，两种药理学抑制剂H89和KT5720消除了被寄生细胞的PKA依赖性存活。为了特异性抑制PKA促生存途径，我们将DPTsh1肽穿梭序列与PKI(5-24)连接，并生成了一种细胞可渗透的PKI，即DPT-PKI。DPT-PKI特异性抑制牛细胞提取物中的PKA活性，并且正如预期的那样，也抑制了环形泰勒虫寄生的TBL3细胞的PKA依赖性存活。因此，TBL3细胞中寄生虫依赖性的PKA组成性激活产生了一条抗凋亡途径，可保护环形泰勒虫感染的B细胞免于凋亡。这些结果还表明，DPT-PKI可能是抑制其他细胞类型中PKA途径的有力工具。
