p53 Negatively Regulates the PR55α Subunit of PP2A Ser/Thr Phosphatase Via FBXL20-mediated Proteasomal Degradation | Chapter 5 | Achievements and Challenges of Medicine and Medical Science Vol. 3

Protein Phosphatase 2A (PP2A) is a family of holoenzyme complexes, which constitute the major Ser/Thr phosphatase activities in human cells. Each PP2A complex consists of one catalytic subunit, one scaffold subunit, and a regulatory subunit. The latter determines the substrate specificity of the PP2A complex. PR55α is one of PP2A’s 27 possible regulatory subunits. Previous studies by us and others have defined an important role for PR55α in the support of critical oncogenic pathways required for tumorigenesis and the malignant phenotype of pancreatic cancer. Our studies subsequently reveal a novel function of the p53 tumor suppressor in inhibiting the protein stability of PR55α via FBXL20, a p53-target gene that serves as a substrate recognition subunit for the SCF (Skp1_Cullin1_F-box) E3 ubiquitin ligase complex. HPNE-E6, HPNE-E7, and HPNE-E6/E7 cell lines were established by transducing HPNE cells with retroviral vectors expressing the E6 and/or E7 proteins of the HPV 16 virus. All antibodies were purchased from Cell Signaling Technology (Danvers, MA) unless otherwise indicated. Immunoblotting (IB) and immunoprecipitation (IP) were performed. Immunofluorescence and microscopy were also performed. For statistical analysis, Student’s t-test and one-way ANOVA methods were used for the comparison of experimental groups using SigmaPlot software. The results show that inactivation of p53 by siRNA-knockdown, gene-deletion, HPV/E6-mediated degradation, or expression of the loss-of-function mutant p53R175H results in increased PR55α protein stability, accompanied by reduced protein expression of FBXL20 and decreased ubiquitination of PR55α. Likewise, knockdown of FBXL20 by siRNA mimics p53 deficiency, reducing PR55α ubiquitination and increasing PR55α protein stability. Functional tests indicate that p53R175H or PR55α overexpression results in an increase of c-Myc protein stability with concomitant dephosphorylation of c-Myc-T58, which is a PR55α substrate, whose phosphorylation otherwise promotes c-Myc degradation. A significant increase in anchorage-independent proliferation is also observed in normal human pancreatic cells expressing p53R175H or, to a greater extent, overexpressing PR55α. Consistent with the frequent loss of p53 function in pancreatic cancer, FBXL20 mRNA expression is significantly lower in pancreatic cancer tissues compared to pancreatic normal tissues and low FBXL20 levels correlate with poor patient survival. In summary, this chapter delineates an important new mechanism by which the p53/FBXL20 axis negatively regulates PR55α protein stability as a part of p53’s tumor suppression function.

 

Author (s) Details

 

Lepakshe S.V. Madduri
Department of Radiation Oncology, University of Nebraska Medical Center, 986850, Nebraska Medical Center, Omaha, NE 68198-6850, USA.

 

Alison L. Camero
Department of Radiation Oncology, University of Nebraska Medical Center, 986850, Nebraska Medical Center, Omaha, NE 68198-6850, USA and Department of Genetics, Cell Biology, and Anatomy, University of Nebraska Medical Center, Omaha, NE, USA.

 

Keith R. Johnson
Department of Genetics, Cell Biology, and Anatomy, University of Nebraska Medical Center, Omaha, NE, USA, Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA, College of Dentistry-Oral Biology, University of Nebraska Medical Center, Omaha, NE, USA and Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, USA.

 

Michel M. Ouellette
Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA and Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, USA.

 

Ying Yan
Department of Radiation Oncology, University of Nebraska Medical Center, 986850, Nebraska Medical Center, Omaha, NE 68198-6850, USA, Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA and Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, USA.

 

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