Identification of the binding partners of p63, a p53 homolog

Abstract

〔PARTⅠ〕 The role for p53 in maintaining the integrity of the genome has been well established, but the functions of two p53 homologues, p63 and p73, are unclear. Although p63 and p73 have structural similarity to p53 and have p53-like activities (binding canonical p53 sites and transactivating p53 target genes), the two homologues have been implicated in p53-independent pathways, such as cell proliferation, death and tumorigenesis. p63 is involved in limb, epithelial, and craniofacial development and stem cell biology. p63 has a role in the commitment to stratification and the maintenance of the proliferative potential of epithelial cells. p63 germline mutations in humans lead to inborn abnormalities, which are characterized by abnormal limb development or ectodermal dysplasia. Previous studies identified ΔNp63α as the primary p63 variant expressed in squamous epithelial tissues and ΔNp63α acts antagonistically toward p53 and TAp63 proteins. In this study, to identify proteins interacting with p63, I screened a human prostate yeast two-hybrid cDNA library using the core DNA binding domain of p63 as bait. 38 clones were selected as potential binding partners of p63 from 3.3×10^(6) clones and those include transcription factors, intracellular proteins, and signaling proteins. Since p63 is predominantly localized in the nucleus, the proteins interacting with p63 are expected to be localized in the nucleus. Based on this criteria, as well as the functions of the positives, the following positives are being tested for binding affinity to p63. The most interesting proteins among the positives were: NFYB, a transcription factor that binds with high specificity to CCAAT motifs in promoter regions and NF-Y transcription factors regulate the transcription of Hsp70 by interacting with ΔNp63α; EAF2, a transcription factor which shows the activity in growth inhibition and tumor suppression; RNF135 (ring finger protein 135), an intracellular protein containing a RING finger domain involved in ubiquitination; SNX6, containing a phox (PX) domain involved in intracellular trafficking; BCA3, which is overexpressed in tumor tissues of prostate and breast. By using an in vivo immunoprecipitation assay, I confirmed the interaction between BCA3 and ΔNp63α. This result shows that it is worth to examine whether BCA3 participates in the functions of ΔNp63α through contributing to the stability of ΔNp63α and enhancing the activity of ΔNp63α as a transcription factor.;〔PART Ⅱ〕 Lck has important roles in immunoreceptor signaling. Lck has tyrosine kinase activity but also functions as an adapter protein, facilitating the assembly of macromolecular complexes via interactions through its src homologous (SH)-2 and SH3 domains. Two adaptor proteins, namely LIME and LAD, have been identified as the binding proteins of the Lck SH2 domain. LIME was identified as a transmembrane adaptor protein (TRAP), associating with Lck in T cells. LIME serves as a positive regulator of T-cell activation and is involved in AICD. LAD is a cytosolic adaptor protein associating with Lck. It is phosphorylated upon TCR stimulation and is involved in TCR-mediated IL-2 production. Although, LIME and LAD were originally identified in T cells, recent reports suggest that these proteins are also expressed in other types of cells. Therefore, defining the tissue specific expression of these proteins is important to understanding the additional functions of these proteins. To identify the tissue specific expression of LIME and LAD, I generated transgenic vectors containing a LacZ reporter inserted into the BAC clones of LAD and LIME genes, so that the reporter expression was controlled by the LIME or LAD promoters. For this I used bacterial ET recombination for the targeted modification of LIME/LAD BAC promoter clones in an E. coli BAC host strain DH10B and IRES-tau-LacZ-km/Neo^(r) reporter was knocked into the BAC clones of Lad and LIME genes by homologous recombination. Finally, the expression of these reporter constructs was confirmed by performing X-gal staining after transient transfection in Jurkat T cells.;유전체 보전의 유지 과정에 있어서 p53의 역할은 많이 연구되어왔으나, p53 동족체들인 p63과 p73의 정확한 기능은 잘 알려진 바가 없다. 비록 p63과 p73이 p53과 구조적으로 유사하고 p53과 같은 활성을 갖고있으나 (canonical p53 위치들에 결합하고 p53 target 유전자들의 전사 활성화시킴), 이 두 homologues는 세포 증식, 사멸 그리고 tumorigenesis와 같은, p53과는 독립적인 경로들에도 관여하고있다. 특히, p63은 limb, 상피조직, 그리고 craniofacial의 발달과정과 줄기세포의 조절에 있어서 epithelial cells의 증식 potential의 유지에도 중요한 역할들을 한다. 또한 p63의 germline 돌연변이들을 보유하는 사람들은 이상한 limb 발달 이상 또는 ectodermal dysplasia로 특징 지워진다. 이전의 연구들에서 squamous epithelial 조직에서 primary p63 variant로서 ΔNp63α를 확인하였고 ΔNp63α이 p53과 TAp63 단백질들에 대해 대립적으로 작용함을 밝힌바 있으나 구체적인 분자수준에서의 조절 기작은 불분명하다. 나는 이 연구에서 p63과 결합하는 새로운 단백질들을 찾기 위해 사람의 전립선 cDNA library를 yeast two-hybrid 방법을 수행하였다. p63의 DNA binding domain을 bait로 사용하여, 3.3×10^(6) 개의 clone들에서 결합 단백질들을 38개 찾았고 이것들은 전사인자들과 세포내부의 단백질들과 신호전달 단백질들을 포함한다. p63은 절대적으로 핵 내부에 있으므로 p63과 결합하는 단백질들은 핵 내부에 위치할 것으로 예상되어진다. 이러한 위치적 기준에 기초하여, positive들의 기능들도 함께 고려하여, 다음의 positive들의 p63에 대한 결합 친화력에 대한 시험을 해 볼 것이다. positive들 중에서 가장 의미 있는 단백질들: NFYB, 전사인자로서 promoter 부위에 있는 CCAAT motif들에 가장 특이적으로 결합하고 NF-Y 전사인자들은 ΔNp63α와 결합하여 Hsp70의 전사를 조절함; EAF2, 전사인자로서 증식 억제와 암 억제의 활성을 보여줌; RNF135 (ring finger protein 135), ubiquitination에 관여하는 RING finger domain을 갖고 있는 세포 내부 단백질; SNX6, intracellular trafficking에 관여하는 phox (PX) domain을 갖는 단백질; BCA3, 유방과 전립선 암 조직에서 과발현 되어있다. 특히 BCA3는 유방암과 전립선암과 같은 상피세포암에서 발현이 유도되어있는 단백질이므로 ΔNp63α 기능과의 관련성을 연구하였다. 즉, 나는 세포내에서 면역침강법을 사용하여, BCA3이 ΔNp63α과 결합한다는 것을 확인하였다. BCA3와 ΔNp63α이 전립선 암과 유방암에서의 과발현 된다는 관점에서, BCA3가 ΔNp63α의 안전성을 돕고 전사인자로서의 활성을 증진시킴으로써, ΔNp63α의 oncogenic한 기능에 관여하는지를 앞으로 시험해보는 일은 중요한 의미를 지닐 것이다.