Regulatory mechanism of PML on STAT-3 transcriptional activity and analyses of PML protein interaction by yeast two hybrid screening

Abstract

A single promyelocytic leukemia (PML) gene, through alternative splicing of exons 4-9 in its C-terminal region, generates several PML isoforms that can interact with specific partners andperform distinct functions, mediated by their specific C-terminal sequence. The PML protein is a tumor suppressor that plays important roles in the induction of apoptosis, control of cytokine signaling, and regulation of cellular senescence by interacting with various proteins. In this study, common characteristic and specific functions of PML isoforms were investigated. The effect of the PML isoforms on OSM-induced STAT-3 transcriptional activity, and the interacting protein that bind to the C-terminal of PML isoforms were investigated using reporter assay and yeast two hybrid screening. To examine the effects of the PML isoforms on STAT-3 transcriptional activity, expression vectors for three different PML isoforms, PML-I, PML-III, and PML-IV, together with STAT-3-luciferase, were transfected into cell lines, including non-cancerous immortalized cell lines (NIH3T3 and HEK293T cells) and glioma cell lines (U87-MG and U251-MG cells). After 24 h of transfection, the cells were stimulated with OSM (10 ng/ml) for 24 h, harvested, and the STAT-3 luciferase activity was determined. The efficiency of PML expression and OSM-induced STAT-3 activation were assessed by immunoblotting. PML influenced OSM-induced STAT-3 activity in a cell type-specific manner, which was dependent on p53 status of the cells. Interestingly, overexpression of PML exerted opposite effects on OSM-induced STAT-3 activity in p53 wild-type and mutant cells. Specifically, overexpression of PML in the cell lines bearing wild-type p53 (NIH3T3 and U87-MG cells) decreased OSM-induced STAT-3 transcriptional activity, whereas overexpression of PML increased OSM-induced STAT-3 transcriptional activity in mutant p53-bearing cell lines (HEK293T and U251-MG cells). When wild-type p53 cells were co-transfected with PML-IV and R273H-p53 mutant, OSM-mediated STAT-3 transcriptional activity was significantly enhanced, compared to that of cells which were transfected with PML-IV expression vector alone; however, when cells bearing mutant p53 were co-transfected with PML-IV and wild-type p53, OSM-induced STAT-3 transcriptional activity was significantly decreased, compared to that of transfected cells with PML-IV alone. These results strongly suggest that PML acts together with wild-type or mutant p53 and influences OSM-mediated STAT-3 activity in a negative or positive manner, resulting in decreased or increased STAT-3 transcriptional activity, respectively. Therefore, the aberrant activation of STAT-3 in cancer cells bearing mutant p53, probably occurs through the interaction of mutant p53 with PML. Further, the difference between the various PML isoforms were investigated by analyzing their amino acid sequences using PONDR, and yeast two hybrid screening was carried out to identify PML isoform-specific binding proteins. PML-III specific C-terminal regions showed relatively conserved disordered regions compared to the C-terminal region of PML-IV. Using yeast two hybrid screening, interacting proteins that specifically bound to PML-III were identified in the human brain library. These results provide evidence that PML isoforms PML-I, PML-III, and PML-IV, exert identical effects on OSM-induced STAT-3 transcriptional activity, which are dependent on the p53 status of the cells. Additionally, the identification of PML-III binding proteins suggest that PML can perform a number of intracellular functions by interacting with various proteins.;하나의 promyelocytic leukemia (PML) 유전자는 C-말단의 exons 4~9의 alternative splicing을 통해 여러 개의 동형 단백질을 생성하며, 각각의 동형 단백질은 특이적인 C-말단 서열을 가지기 때문에 이 부분을 통해 동형 단백질 특이적인 기능을 가질 수 있다. PML 단백질은 종양 억제단백질로서, 다양한 단백질들과 상호작용하여 세포 자살 유도, cytokine의 신호전달 조절, 세포의 노화 조절 등 세포에서 중요한 역할을 한다. 이 연구에서는 PML 동형 단백질의 공통된 기능과 특이적인 기능에 대해 연구하고자 하였다. OSM에 의해 유도되는 STAT-3 전사 활성에 대한 PML 동형 단백질의 효과를 reporter assay로 수행하였으며, yeast two hybrid screening을 통해 PML 동형 단백질과 특이적으로 결합하는 단백질들을 찾고자 하였다. 먼저, PML 동형 단백질의 과발현은 OSM에 의해 유도되는 STAT-3 전사 활성이 세포주에서 다르게 조절되는 것을 확인했다. Wild-type p53을 가지고 있는 세포인 NIH3T3와 U87-MG에서의 PML 과발현은 OSM에 의해 유도되는 STAT-3 전사 활성을 감소시켰고, mutant p53을 가지고 있는 세포인 HEK293T와 U251-MG에서의 PML 과발현은 OSM에 의해 유도되는 STAT-3 전사 활성을 증가시켰다. 이러한 결과는 PML 동형 단백질은 mutant p53과 상호작용하여 OSM에 의해 유도되는 STAT-3 전사 활성을 조절하는데, PML과 p53의 상호작용은 암에서 비정상적인 STAT-3 활성화에 기여할 수 있음을 의미한다. 또한, PML-III 동형 단백질의 특이적인 C-말단 영역은 PML-IV 동형 단백질의 특이적인 C-말단 영역에 비하여 상대적으로 disordered region을 가지고 있음을 알 수 있었다. Yeast two hybrid screening을 이용하여 human brain library에서 PML-III 특이적으로 결합하는 단백질들을 확인하였다. 본 연구를 통해, PML 동형 단백질이 적어도 OSM으로 유도된 STAT-3 전사 활성에 있어서는 동일한 기능을 나타내고, 이러한 기능이 세포의 p53 상태에 의존적임을 밝혔으며, PML-III와 결합하는 단백질들을 밝힘으로써 다양한 단백질과의 상호작용을 통해 세포 내에서 다양한 기능을 수행할 수 있음을 확인하였다.