Purification and characterization of nuclear protein complex containing human POZ-domain transcription factor, Pokemon 1

Abstract

[한글]우리는 BTB/POZ (broad-complex, tramtrack and bric-a-brac/poxvirus and zinc finger) 도메인 계열 단백질 중 nuclear localization signal과 4개의 C-terminal krüppel-like zinc finger motif를 가지고 있는 Pokemon 1의 유전자 전사조절 및 다른 단백질과의 상호작용을 통한 세포기능 조절에 관한 연구를 수행하고 있다. Pokemon 1은 ADH5/FDH promoter와 HIV-1 LTR promoter에 결합하여 전사를 조절할 뿐만 아니라 지방세포로의 분화와 골세포 형성과정, 세포의 발달과 분화과정에 작용한다고 알려져 있다. 최근 발표된 논문에 의하면 과 발현된 Pokemon 1은 종양 억제 유전자인 ARF의 전사를 억제시켜 p53의 발현을 낮추고, 종양형성을 유발한다고 한다.따라서 우리는 Pokemon 1이 전사인자로서 다른 여러 단백질들과의 상호작용을 통하여 종양 형성에 중요한 역할을 할 것 이라고 생각하여, 핵 내에서 Pokemon 1과 상호작용을 하고 있는 단백질체를 분리하여 MALDI-TOF (matrix-assisted laser desorption/ionization time-offlight) mass spectrometry를 수행하였다. 이를 통해MBD3, RFC2, RFC5, KIAA0111, Vimentin, ATP5A1, MSH2, K-Alpha-1, SET, NAP1L1, TRIP1, eEF1A1 등 12개의 단백질들을 동정하였다. 뿐만 아니라, 이들 단백질들이 암화 과정이나 세포사멸, 염색체 형성 및 복제, 세포 증식과 전사과정 등 여러 가지 다양한 세포조절 기능을 가지고 있다는 것을 알게 되었다. 그러므로 Pokemon 1이 이들 단백질들과의 직•간접적인 상호작용을 통하여 세포 내에서 여러 기능을 수행할 것이라 예측할 수 있었다.이와 함께, microarray를 통해서 Pokemon 1, 그리고 Pokemon 1과 구조적으로 매우 유사한 Pokemon 2에 의해 전사가 조절되는 유전자들을 찾아보았다. Microarray분석을 통해 Pokemon 1, 2가 과발현됨으로써 많은 유전자들의 전사가 조절되는 것을 알 수 있었다.

[영문]POK (POZ and Krüppel) family proteins contain N-terminal POZ domain and C-terminal DNA-binding domain made of Krüppel-like zinc fingers. Pokemon 1 (also called as FBI-1; Factor that Binds to the Inducer of short transcripts of human immunodeficiency virus-1) is one of the POK family proteins that plays critical and pleiotropic function in differentiation. Pokemon 1 regulates transcription of various genes and plays important roles in differentiation, oncogenesis and adipocyte differentiation. Pokemon 1 is a proto-oncogenic transcription factor that is expressed in almost all tissues and over expressed in human cancer tissues Recently, Pokemon 1 was shown to have a proto-oncogenic activity and its expression is elevated in many cancer tissues. It physically interacts with other POK family members such as Pokemon 1, PLZF, etc. Pokemon 1 exerts its function by interaction with other regulatory proteins via POZ-domain. We identify other POZ domain proteins and corepressors bind to the POZ-domain of Pokemon 1.To investigate the biological functions of Pokemon 1, we tried to isolate the protein complex containing Pokemon 1 and characterize the complex by proteomic tools. Identification of Pokemon 1-binding proteins could reveal potential signaling targets and previously undescribed functional roles for Pokemon 1. Mammalian expression vector containing Flag-tagged Pokemon 1 were constructed into pcDNA5/FRT/TO© and stably transfected into human kidney 293T cells. The expression of Flag-Pokemon 1 is under the control of tetracycline. After induction of Flag-Pokemon 1 expression with tetracycline analogue, doxycycline, cells were harvested and the complex containing Pokemon 1 was purified by immunoprecipitation with anti-Flag M2 agarose beads. 2-DE and silver staining showed that purified Pokemon 1 complex. Proteins associated with Pokemon 1 are being characterized by MALDI-TOF (matrix-assisted laser desorption/ ionization time-offlight) mass spectrometry. Thus, 12 unique proteins were identified to interaction with Pokemon 1. These proteins are MBD3, RFC2, RFC5, KIAA0111, Vimentin, ATP5A1, MSH2, K-Alpha-1, SET, NAP1L1, TRIP1 and eEF1A1. These proteins related with various human cancers and involved various cellular processes such as apoptosis, chromatin modulation, replication, cell proliferation and transcription. Therefore Pokemon1 is interaction with these proteins participated in various cellular process.And we investigated of Pokemon 1 and Pokemon 2 regulated gene expression using oligonucleotide microarray. Microarray analysis showed that ectopic Pokemon 1, 2 altered transcription of large number of genes, suggesting that Pokemon 1, 2 likely to have variety of biological functions. Two genes wide overall structure and functional similarities can play different functional roles in the cells.