Exploring molecular mechanisms that underlie regulation of transcription factor NFIB by the peptidyl prolyl isomerase pin1

Abstract

Embryonic development entails complex cellular events regulated by a variety of signaling pathways and coordinated actions of numerous transcription factors. One of these transcription factors is Nuclear Factor One B (NFIB) of the NFI family, which includes three other members (NFIA, NFIC, and NFIX) in vertebrates. Each NFI has a well-conserved N-terminal DNA binding and dimerization domain along with a less conserved transactivation and repression domain. Knockout studies have shed light on NFI function during development. The phenotypes of knockout mice demonstrated that the absence of NFIA, NFIB, or NFIX causes severe developmental defects in CNS characterized by enlarged lateral ventricles and abnormal corpus callosum development arising from failure in neural progenitor cell differentiation. Consequently, neural and neuronal differentiation programs are also delayed. In addition, the absence of NFIB leads to respiratory defects in mice due to abnormal lung development, while NFIX deletion results in skeletal muscle system anomalies. NFIA, on the other hand, plays a role in the development of urinary tract and kidney. In contrast to the other members, the absence of NFIC does not apparently affect CNS development. Instead, it regulates osteoblast differentiation and is essential for tooth root development. Overall, these phenotypes indicate that the NFI family is an important regulator of CNS development, as well as other organ systems. However, the precise regulatory mechanisms involving interaction partners are poorly understood. Previously, a yeast two-hybrid (Y2H) screen was performed to find novel interaction partners that regulate NFI function; this screen identified PIN1 as a potential NFIB-binding protein. PIN1, a cis-trans peptidyl-prolyl isomerase, plays an important role in various molecular pathways such as gene expression, DNA repair and it regulates numerous proteins with diverse functions. These include transcription factors, enzymes, tumor suppressors, oncogenic proteins, and other regulatory proteins. Consequently, it has been implicated in control of cell proliferation, differentiation, metabolism, cancer, drug resistance, genomic instability, metastasis, and apoptosis as well as survival. PIN1 recognizes phosphorylated Ser/Thr-Pro residues and modulates protein function, DNA binding affinity, stability, and subcellular localization. Since kinases or phosphatases also bind these recognition sequences, PIN1 serves as an important mediator in the communication of various signaling pathways. Additionally, PIN1 often enhances stability of oncogenic proteins while decreasing the half-life of tumor suppressors. Moreover, elevated PIN1 expression and/or activity is frequently observed during tumorigenesis, distinguishing PIN1 as an oncogene. NFI family members carry PIN1 recognition motifs within their N- and C-terminal domains, both NFIB and PIN1 are expressed in neural progenitor cells during embryonic development, thereby involved in regulation of neurogenesis. Misregulation of both proteins is implicated in development of cancer including glioblastoma multiform. As both proteins are likely to act in the same cellular and developmental pathways, we focused on PIN1 as a potential NFIB upstream regulator. To investigate PIN1-NFIB interaction, I overexpressed both proteins in HEK293T cells and conducted GST pull-down and immunoprecipitation assays. These experiments revealed that when overexpressed, NFIB, along with other NFI members, interacts with PIN1. NFIB-PIN1 interaction requires the conserved NFI N-terminal DBD domain and the WW domain of PIN1. More specifically, the region spanning residues 167-208 on NFIB serves as the main target of PIN1. However, point mutations converting conserved Ser/Thr-Pro motifs to Ala-Pro, including those in the 167-208 region, failed to abolish NFIB-PIN1 interactions. Furthermore, I determined that NFIB-PIN1 binding occurs in a phosphorylation-dependent manner, as CIP treatment significantly decreases the interaction. In summary, I posit that while the N-terminal DBD domain is required for PIN1 binding, non-canonical phosphorylation sequences may also contribute to the interaction. Subsequently, I investigated whether NFIB function is regulated by the presence of PIN1. To this end, I measured luciferase activity driven by GFAP promoter, since NFIB, as well as other NFIs, enhance GFAP expression. I found that GFAP activation decreased in the presence of both NFIB and PIN1, whereas the PIN1 mutant (PIN1-W34A), unable to bind NFIB, did not exhibit such an inhibitory effect. One of the known functions of PIN1 is regulation of protein stability. Therefore, I measured the half-life of NFIB in the presence or absence of PIN1 and found that PIN1 increases that of NFIB's. Collectively, these results suggest that PIN1 induces a conformational change in NFIB, resulting in decreased transcriptional activity while simultaneously increasing its stability. However, the potential involvement of the ubiquitin-proteasome degradation system in regulating NFIB's stability, or the contribution of the PIN1-NFIB-GFAP axis to tumorigenesis remains to be further characterized. The induction of GFAP is one of the hallmarks of glial differentiation. Moreover, its high expression is generally associated with less aggressive tumor characteristics, whereas low GFAP levels are often observed in more aggressive tumors and are associated with poor prognosis in glioma patients. Since both NFIB and PIN1 are required for correct development of CNS and their misregulation of both proteins is also implicated in tumorigenesis, the physiological relevance of the PIN1-NFIB-GFAP axis and its involvement in the etiology of various disease need to be further investigated.