Activation of Cell Cycle Arrest and Apoptosis by the Proto-Oncogene Pim-2

Potent survival effects have been ascribed to the serine/threonine kinase proto-oncogene PIM-2. Elevated levels of PIM-2 are associated with various malignancies. In human cells, a single Pim-2 transcript gives rise mainly to two protein isoforms (34, 41 kDa) that share an identical catalytic site but differ at their N-terminus, due to in-frame alternative translation initiation sites. In this study we observed that the 34 kDa PIM-2 isoform has differential nuclear and cytoplasmic forms in all tested cell lines, suggesting a possible role for the balance between these forms for PIM-2's function. To further study the cellular role of the 34 kDa isoform of PIM-2, an N-terminally HA-tagged form of this isoform was transiently expressed in HeLa cells. Surprisingly, this resulted in increased level of G1 arrested cells, as well as of apoptotic cells. These effects could not be obtained by a Flag-tagged form of the 41 kDa isoform. The G1 arrest and apoptotic effects were associated with an increase in T14/Y15 phosphorylation of CDK2 and proteasom-dependent down-regulation of CDC25A, as well as with up-regulation of p57, E2F-1, and p73. No such effects were obtained upon over-expression of a kinase-dead form of the HA-tagged 34 kDa PIM-2. By either using a dominant negative form of p73, or by over-expressing the 34 kDa PIM-2 in p73-silenced cells, we demonstrated that these effects were p73-dependent. These results demonstrate that while PIM-2 can function as a potent survival factor, it can, under certain circumstances, exhibit pro-apoptotic effects as well.

PIM-2（丝氨酸/苏氨酸激酶原癌基因）已被认为具有显著的生存效应。PIM-2水平的升高与多种恶性肿瘤相关。在人类细胞中，单个Pim-2转录本主要产生两种蛋白质异构体（34, 41 kDa），这两种异构体共享相同的催化位点，但在其N端存在差异，这是由于存在内含子中的选择性翻译起始位点所致。在本研究中，我们观察到34 kDa的PIM-2异构体在所有测试的细胞系中存在差异性的核和细胞质形式，这表明这些形式之间的平衡可能在PIM-2的功能中发挥重要作用。为了进一步研究34 kDa PIM-2异构体的细胞作用，我们在HeLa细胞中瞬时表达了一种N端带有HA标签的该异构体形式。令人惊讶的是，这导致G1期停滞细胞以及凋亡细胞的水平增加。这些效应无法通过41 kDa异构体的Flag标签形式获得。G1期停滞和凋亡效应与CDK2的T14/Y15磷酸化增加、CDC25A的蛋白酶体依赖性下调以及p57、E2F-1和p73的上调相关。在过表达带有HA标签的34 kDa PIM-2的激酶失活形式时，并未观察到这些效应。通过使用p73的显性负形式，或者在高表达p73沉默细胞中的34 kDa PIM-2，我们证明了这些效应是依赖于p73的。这些结果证明了PIM-2不仅可以作为有效的生存因子发挥作用，在特定条件下，还可以表现出促凋亡效应。