Casein Kinase II Phosphorylation-induced Conformational Switch Triggers Degradation of the Papillomavirus E2 Protein

Abstract

The major phosphorylation sites of the bovine papillomavirus E2 transactivator protein are two serine residues, 298 and 301, that are located in a flexible hinge region between the DNA binding and transactivation domains. Phosphorylation of serine residue 301 promotes ubiquitination and rapid degradation of the E2 protein by the proteasome pathway. To understand the mechanism through which phosphorylation regulates the intracellular levels of this unique papillomavirus regulatory protein, we have carried out an extensive mutational analysis of the region surrounding the phosphorylation sites of the E2 protein. Our results indicate that casein kinase II phosphorylates serine 301. However, phosphorylation of serine 301 is not a sufficient recognition motif for proteasomal degradation; other residues that directly surround the phosphorylation sites are crucial for E2 degradation. The phenotypes of E2 proteins mutated in this region indicate that phosphorylation of serine 301 induces a conformational change that leads to degradation of the E2 protein. In support of this model, circular dichroism studies of the conformational tendencies of peptides from this region indicate that phosphorylation at position 301 decreases the local thermodynamic stability of this region. Thus, this region appears to have evolved to display a marginal local thermodynamic stability that can be regulated by phosphorylation, leading to targeted degradation of the E2 protein.Papillomaviruses are small DNA viruses that infect epithelia and cause persistently infected lesions known as papillomas or warts. The viral genome is maintained as a multicopy episome in the infected, dividing basal cells. The productive stage of the viral life cycle is highly dependent on epithelial differentiation; genome amplification, late gene expression, and capsid assembly occur only in the more differentiated cells of the epithelium. A subset of human papillomaviruses are associated with development of cervical carcinoma; however, bovine papillomavirus, type 1 (BPV-1), 1 has historically served as a molecular prototype for genetic analysis of the virus.BPV-1 encodes for several E2 gene products. These proteins are crucial for the viral life cycle, participating in several functions such as transcriptional regulation, viral DNA replication, and genome maintenance and segregation. The E2 protein activates transcription by binding to enhancer sequences in the viral genome (1) and supports initiation of viral DNA replication by cooperatively binding with the E1 protein to the viral origin of replication (2). E2 mediates viral genome segregation by binding the viral genomes and tethering them to the cellular mitotic chromosomes (3-5). The full-length E2 protein contains two conserved functional domains as follows: the N-terminal transactivation domain of ϳ200 amino acids, and a C-terminal DNA binding and dimerization domain contained in the last 100 amino acids (reviewed in Ref. 6). These domains are separated by a flexible hinge region that is not well conserved am...

Temperature-sensitive Post-translational Regulation of Plant Omega-3 Fatty-acid Desaturases Is Mediated by the Endoplasmic Reticulum-associated Degradation Pathway

“…3A contains a predicted PEST signal in the N-terminal sequence, with the polypeptide sequence in this region enriched in glutamic acid residues and also containing a stretch of amino acids predicted to form a turn-type structure followed by an ␣-helix. Interestingly, these features are generally similar to the characteristics of the regulated PEST sequence found in the papillomavirus E2 protein (78). The PEST sequence in this protein is located in a marginally stable region that includes a stretch of glutamic acid residues followed by a turn region and a short ␣-helix (79).…”

“…The PEST sequence in this protein is located in a marginally stable region that includes a stretch of glutamic acid residues followed by a turn region and a short ␣-helix (79). Phosphorylation of a serine residue within the turn region destabilizes the protein structure, thereby exposing the PEST signal and rendering the E2 protein susceptible to rapid degradation (78,79). Although it is unknown whether the serine residue in the predicted turn region of some Fad3 proteins (Fig.…”

Phosphorylation of the Bovine Papillomavirus E2 Protein on Tyrosine Regulates Its Transcription and Replication Functions

“…Serine-to-alanine mutations at these sites resulted in increased copy numbers in mouse cells (28). Casein kinase II (CK2) was reported to phosphorylate these BPV-1 serines, leading to increased E2 degradation and decreased replication (29). Similar sites have been found in human papillomavirus 8 (HPV-8) at S253 (30) and in HPV-16 at S243 (31).…”

Interaction of Bovine Papillomavirus E2 Protein with Brd4 Stabilizes Its Association with Chromatin

“…To confirm the relocalization and stabilization of Brd4 by E2 in cells expressing E2 from the episomal BPV genome and to establish that it was not specific to the CV-1 line, the localization of the E2 and Brd4 proteins was also examined in C127 cells transformed with BPV-1. The 137 cell line was established from C127 cells transformed with a high-copy-number BPV-1 genome that contains mutations in the major phosphorylation sites of the E2 protein; these mutations result in higher levels of E2 protein expression because of an increased protein half-life (35,42,43). In mitotic C127 cells, Brd4 was found to diffusely coat the condensed chromosomes at all stages of mitosis ( Fig.…”