Departamento de Biología Celular y Desarrollo, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Apartado Postal 70-248, México DF 04510, México.
Departamento de Biología Celular y Desarrollo, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Apartado Postal 70-248, México DF 04510, México.
Eur J Pharmacol. 2014 Oct 5;740:88-96. doi: 10.1016/j.ejphar.2014.06.057. Epub 2014 Jul 10.
Phosphoinositide-dependent protein kinase 1 (PDK1) is a key enzyme, master regulator of cellular proliferation and metabolism; it is considered a key target for pharmacological intervention. Using membranes obtained from DDT1 MF-2 cells, phospho-PDK1 was identified by Western blotting, as two major protein bands of Mr 58-68 kDa. Cell incubation with the PDK1 inhibitor, UCN-01, induced a time- and concentration-dependent decrease in the amount of phospho-PDK1 with a concomitant appearance of a ≈42 kDa phosphorylated fragment. Knocking down PDK1 diminished the amount of phospho-PDK1 detected in membranes, accompanied by similarly decreased fragment generation. UCN-01-induced fragment generation was also observed in membranes from cells stably expressing a myc-tagged PDK1 construct. Other PDK1 inhibitors were also tested: OSU-03012 induced a clear decrease in phospho-PDK1 and increased the presence of the phosphorylated fragment in membrane preparations; in contrast, GSK2334470 and staurosporine induced only marginal increases in the amount of PDK1 fragment. Galardin and batimastat, two metalloproteinase inhibitors, markedly attenuated inhibitor-induced PDK1 fragment generation. Metalloproteinases 2, 3, and 9 co-immunoprecipitated with myc-PDK1 under baseline conditions and this interaction was stimulated by UCN-01; batimastat also markedly diminished this effect of the PDK1 inhibitor. Our results indicate that a series of protein kinase inhibitors, namely UCN-01 and OSU-03012 and to a lesser extent GSK2334470 and staurosporine induce PDK1 fragmentation and suggest that metalloproteinases could participate in this effect.
磷酸肌醇依赖性蛋白激酶 1（PDK1）是一种关键酶，是细胞增殖和代谢的主要调节剂；它被认为是药物干预的关键靶点。使用 DDT1 MF-2 细胞获得的膜，通过 Western blot 鉴定磷酸 PDK1，其为 Mr 58-68 kDa 的两个主要蛋白带。用 PDK1 抑制剂 UCN-01 孵育细胞，可诱导磷酸 PDK1 的量呈时间和浓度依赖性下降，同时出现约 42 kDa 的磷酸化片段。敲低 PDK1 会减少膜中检测到的磷酸 PDK1 的量，同时片段生成也会相应减少。在稳定表达 myc 标记的 PDK1 构建体的细胞的膜中也观察到 UCN-01 诱导的片段生成。还测试了其他 PDK1 抑制剂：OSU-03012 可明显降低磷酸 PDK1 的含量，并增加膜制剂中磷酸化片段的存在；相比之下，GSK2334470 和 staurosporine 仅引起 PDK1 片段量的轻微增加。Galardin 和 batimastat 两种金属蛋白酶抑制剂显著减弱了抑制剂诱导的 PDK1 片段生成。在基线条件下，基质金属蛋白酶 2、3 和 9 与 myc-PDK1 共免疫沉淀，而 UCN-01 刺激这种相互作用；batimastat 也显著减弱了 PDK1 抑制剂的这种作用。我们的结果表明，一系列蛋白激酶抑制剂，即 UCN-01 和 OSU-03012，以及在较小程度上 GSK2334470 和 staurosporine，诱导 PDK1 片段化，并表明金属蛋白酶可能参与这种作用。
