E1^E4-mediated keratin phosphorylation and ubiquitylation: a mechanism for keratin depletion in HPV16-infected epithelium

Abstract

SummaryThe keratin IF network of epidermal keratinocytes provides a protective barrier against mechanical insult, it is also a major player in absorbing stress in these cells. The human papilloma virus (HPV) type 16 E1^E4 protein accumulates in the upper layers of HPV16-infected epithelium and is known to associate with and reorganise the keratin IF network in cells in culture. Here, we show that this function is conserved amongst a number of HPV alpha-group E1^E4 proteins and that the differentiation-dependent keratins are also targeted. Using time-lapse microscopy, HPV16 E1^E4 was found to effect a dramatic cessation of keratin IF network dynamics by associating with both soluble and insoluble keratin. Network disruption was accompanied by keratin hyperphosphorylation at several sites, including K8 S73, which is typically phosphorylated in response to stress stimuli. Keratin immunoprecipitated from E1^E4-expressing cells was also found to be ubiquitylated, indicating that it is targeted for proteasomal degradation. Interestingly, the accumulation of hyperphosphorylated, ubiquitylated E1^E4-keratin structures was found to result in an impairment of proteasomal function. These observations shed new light on the mechanism of keratin IF network reorganisation mediated by HPV16 E1^E4 and provide an insight into the depletion of keratin co-incident with E1^E4 accumulation observed in HPV-infected epithelium.

Role of Calpain in the Formation of Human Papillomavirus Type 16 E1^E4 Amyloid Fibers and Reorganization of the Keratin Network

“…Analysis of both 16E1 E4 and keratin 18 patterns showed that treatment with calpain inhibitor III significantly reduced the degree of reorganization at both time points. Compared to previous reports of 16E1 E4-associated reorganization in cell culture (13,22), reorganization occurring at relatively early time points in our system, which we think is partly a function of the cell line that we are using but mostly because the necessary control addition of DMSO, which has been found to greatly increase the expression levels of 16E1 E4 in transient-transfection experiments (data not shown). At 16 h, in cells treated with DMSO, the majority of the 16E1 E4 networks are in the middle or late stages of reorganization.…”

The Interaction between Human Papillomavirus Proteins and Cytoskeletal Filaments

“…HPV16 E1^E4 was found to effect a dramatic cessation of keratin IF network dynamics by associating with both soluble and insoluble keratin. These observations shed new light on the mechanism of keratin IF network reorganisation mediated by HPV16 E1^E4 (McIntosh et al, 2010). E1^E4 also translocates to mitokondria via an N-terminal leucine-rich region and induces the detachment of mitocondria from microtubules.…”

“…HPV proteins are interacted with cell skeletal filaments. Some of these proteins are E6 (HPV16,18), E7 (HPV16,18,38), E5 (high and low risk HPV types), E1^E4 (HPV16), E4 proteins (Lee & Dominguez, 2010;McIntosh et al, 2010;Nguyen 2008;Rey et al, 2000, Safi Oz, 2010Stanley, 2001;Uribe & Jay., 2009;Yue et al, 2011).…”

“…Assembly of cytoplasmic IF proteins, such as vimentin, begins with a lateral association of dimers into tetramers and gradually into the so-called unit-length filaments (ULFs) (Strelkov et al, 2003). The molecular organisation of the intermediate filaments is specific for the cell type, the developmental stage and the type of differentiation (McIntosh et al, 2010). Major types of intermediate filament proteins in vertebrate cell are nuclear, vimentin-like, epithelial and axonal IF.…”

“…Every keratin filament is made up of type I (acidic) and type II (neutral/basic) keratin chains (Alberts et al, 2002). The keratin IF network of epidermal keratinocytes provides a protective barrier against mechanical insult, it is also a major player in absorbing stress in these cells (McIntosh et al, 2010). They contain at least 20 members, called keratin 1 (K1) to K20, which are divided into two types according to the sequence and isoelectric point (pI).…”