Identification and characterization of an interaction between pp32 and the retinoblastoma protein

• Main Author: Adegbola, Onikepe
• Format: Dissertation
• Language: English
• Subjects: chemistry, biochemistry; health sciences, pathology
• ISBN: 0542100401; 9780542100406

pp32 (ANP32A) is a member of the ANP32 family of acidic, leucine-rich nuclear phosphoproteins found in cells capable of self renewal. pp32 is a nucleocytoplasmic shuttling protein implicated in proliferation, differentiation, apoptosis, tumorigenesis, inhibition of histone acetylation and regulation of mRNA processing by binding to the RNA binding domain of HuR. Like pp32, retinoblastoma protein (Rb), a nuclear phosphoprotein, regulates proliferation, differentiation, and apoptosis. Mutations in the Rb gene or disruption of the Rb pathway are seen in a majority of cancers.
Here I report the characterization of an interaction between pp32 and Rb. I find that a specific form of hyperphosphorylated Rb phosphorylated on T826 interacts with the leucine rich repeat of pp32 but not with closely related family members pp32r1 and pp32r2. Using reporter, apoptosic and colony formation assays, I show that the pp32-Rb interaction results in increased E2F1-mediated transcription, proliferation and survival, suggesting a pro-oncogenic function for Rb.
By affinity purification and mass spectrometry, I identify non-POU domain-containing, octamer binding protein/nuclear RNA-binding protein, 54 kDa (nonO/p54nrb) and polypyrimidine tract-binding protein (PTB)-associated splicing factor (PSF) as components of the pp32-Rb complex. Like pp32 and Rb, nonO/p54nrb and PSF have roles in cancer and differentiation and function as transcriptional coregulators of nuclear receptors. I show that pp32 and Rb together modulate androgen receptor-mediated transcription. Like pp32, nonO/p54nrb and PSF have been implicated in mRNA processing. NonO/p54nrb and PSF contain two RNA binding domains, bind both RNA and DNA, and coordinately regulate nuclear hormone receptor-mediated transcription and splicing. I demonstrate via coimmunoprecipitation that endogenous pp32, Rb, nonO/p54nrb and PSF interact during TPA-induced K562 differentiation. Together, these results suggest that the complex containing pp32, Rb, nonO/p54nrb and PSF may simultaneously regulate transcription and mRNA processing.
In summary, in this thesis, I have identified and characterized an interaction between pp32 and Rb. I have shown the functional significance of the pp32-Rb interaction in proliferation, apoptosis and transcription. I have identified nonO/p54nrb and PSF as components of the pp32-Rb complex. These studies suggest that pp32, Rb, nonO/p54nrb and PSF interact in a large multiprotein complex that may coordinately regulate transcription and mRNA processing.