David Pim

Two Polymorphic Variants of Wild-Type p53 Differ Biochemically and Biologically

ABSTRACT The wild-type p53 protein exhibits a common polymorphism at amino acid 72, resulting in either a proline residue (p53Pro) or an arginine residue (p53Arg) at this position. Despite the difference that this change makes in the primary structure of the protein resulting in a difference in migration during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, no differences in the biochemical or biological characteristics of these wild-type p53 variants have been reported. We have recently shown that p53Arg is significantly more susceptible than p53Pro to the degradation induced by human papillomavirus (HPV) E6 protein. Moreover, this may result in an increased susceptibility to HPV-induced tumors in homozygous p53Argindividuals. In further investigating the characteristics of these p53 variants, we now show that both forms are morphologically wild type and do not differ in their ability to bind to DNA in a sequence-specific manner. However, there are a number of differences between the p53 variants in their abilities to bind components of the transcriptional machinery, to activate transcription, to induce apoptosis, and to repress the transformation of primary cells. These observations may have implications for the development of cancers which harbor wild-type p53 sequences and possibly for the ability of such tumors to respond to therapy, depending on their p53 genotype.

P53 POLYMORPHIC VARIANTS AT CODON 72 EXERT DIFFERENT EFFECTS ON CELL CYCLE PROGRESSION

Two common polymorphic forms of the p53 tumor suppressor protein are widely distributed throughout the human population. These encode either proline or arginine at position 72, and this difference results in a marked alteration in the primary structure of the protein. A number of previous studies have shown significant differences in the biochemical properties of the p53 protein, depending on the particular polymorphic form. There is little information, however, on their respective biologic activities. In this study, we have used an inducible switch system for expressing both polymorphic forms of p53 within Saos‐2 cells. Cell cycle analysis postinduction of p53 function reveals striking differences in how the 2 forms of p53 bring about a cessation of cell growth. Thus, the Arg72 form of p53 is significantly more efficient than the Pro72 form at inducing apoptosis. In contrast, the Pro72 form appears to induce a higher level of G1 arrest than the Arg72 form. These results demonstrate significant differences in how the codon 72 polymorphism affects the biological activity of p53.

Characterization of the human papillomavirus E2 protein: evidence of trans-activation and trans-repression in cervical keratinocytes.

The major regulator of papillomavirus transcription is encoded by the viral E2 gene. The E2 gene has been well characterized in bovine papillomavirus (BPV) where it encodes at least three different polypeptides which differentially affect viral gene expression. In human papillomaviruses (HPVs) the E2 gene product is much less well characterized. In this study we have analysed the mechanism of action of the HPV‐16, HPV‐18 and BPV‐1 E2 proteins in cervical keratinocytes. We show that the full length HPV E2 protein acts as a potent transcriptional activator of viral gene expression in both normal and immortalized keratinocytes. In contrast, the BPV‐1 E2 protein produces transcriptional repression under identical conditions. A cDNA encoding the C‐terminal half of the HPV‐16 E2 protein in these assays weakly repressed viral gene expression. Further, co‐transfection of this cDNA with the full length clone progressively abolishes the activation in trans by the full length HPV E2 protein. Gel retardation assays have defined a number of protein complexes between the long and short forms of E2 but with no evidence for preferential DNA binding. These results define two distinct activities for the HPV‐16 E2 protein, indicate functional differences with the BPV E2 protein and suggest that splicing of the HPV E2 mRNA is a critical mechanism for controlling viral gene expression.

Human papillomaviruses, cervical cancer and cell polarity

Human papillomaviruses (HPVs) are the causative agents of a number of human cancers, of which cervical cancer is the most important. This occurs following persistent infection with a limited number of viral subtypes and is characterized by continued expression of the viral E6 and E7 oncoproteins. A unique characteristic of the cancer-causing HPV types is the presence of a PDZ recognition motif on the carboxy terminus of the E6 oncoprotein. Through this motif, E6 directs the proteasome-mediated degradation of cellular proteins involved in the regulation of cell polarity and in cell proliferation control. These include components of the Scrib and Par polarity complexes, as well as a number of other PDZ domain-containing substrates. Thus, PVs are now providing novel insights into the functioning of many of these cellular proteins, and into which of these functions, in particular, are relevant for maintaining normal cellular homeostasis. In this review, we discuss the biological consequences of papillomaviral targeting of these cell polarity regulators, both with respect to the viral life cycle and, most importantly, to the development of HPV-induced malignancy.

Activation of the protein kinase B pathway by the HPV-16 E7 oncoprotein occurs through a mechanism involving interaction with PP2A

Protein kinase B (PKB) or Akt is one of several second messenger kinases that are activated by cell attachment and growth factor signaling, and that transmit signals to the cell nucleus to inhibit apoptosis and thereby increase cell survival during proliferation. Other viral proteins target this pathway by increasing PKB/Akt phosphorylation, and this pathway has been implicated in the transformation of human keratinocytes by HPV E6 and E7, together with activated notch 1. Here, we examine how HPV E7 expression affects the phosphorylation of PKB. We show that HPV-16 E7 increases the level of phosphorylation of PKB in response to serum stimulation, by a mechanism independent of downregulation of PTEN phosphatase, a known inhibitor of the PI3K (PI3 kinase) pathway. The use of specific antibodies shows that some proportion of PKB/Akt that is phosphorylated both on threonine 308 and serine 473 is maintained in the presence of E7 in a PI3 kinase-independent manner, and is activated for phosphorylation of BAD, a known downstream target of PKB/Akt. Use of E7 mutants has ruled out both an inhibition of IGFBP-3, a known E7 target and PKB/Akt modulator, and the interaction of E7 with cellular pocket proteins, as being the mechanism for the PKB/Akt stimulation. PKB binds PP2A and is a known substrate of PP2A. Here, we show that HPV E7 also binds to both the 35 kDa catalytic and 65 kDa structural subunits of PP2A, an interaction that sequesters these subunits and inhibits their interaction with PKB, thereby maintaining PKB/Akt signaling by inhibiting its dephosphorylation.

Interaction of viral oncoproteins with cellular target molecules: infection with high-risk vs low-risk human papillomaviruses

Pim D, Banks L. Interaction of viral oncoproteins with cellular target molecules: infection with high‐risk vs low‐risk human papillomaviruses. APMIS 2010; 118: 471–493.

HPV E6 and MAGUK protein interactions: determination of the molecular basis for specific protein recognition and degradation

It has recently been shown that the high-risk human papillomavirus (HPV) E6 proteins can target the PDZ-domain containing proteins, Dlg, MUPP-1, MAGI-1 and hScrib for proteasome-mediated degradation. However, the E6 proteins from HPV-16 and HPV-18 (the two most common high-risk virus types) differ in their ability to target these proteins in a manner that correlates with their malignant potential. To investigate the underlying mechanisms for this, we have mutated HPV-16 and HPV-18 E6s to give each protein the others PDZ-binding motif. Analysis of these mutants shows that the greater ability of HPV-18 E6 to bind to these proteins and to target them for degradation is indeed due to a single amino acid difference. Using a number of assays, we show that the E6 proteins interact specifically with only one of the five PDZ domains of MAGI-1, and this is the first interaction described for this particular PDZ domain. We also show that the guanylate kinase homology domain and the regions of MAGI-1 downstream of amino acid 733 are not required for the degradation of MAGI-1. Finally, in a series of comparative analyses, we show that the degradation of MAGI-1 occurs through a different mechanism from that used by the E6 protein to induce the degradation of Dlg and p53.

Viruses and the 26S proteasome: hacking into destruction

The discovery that the human papillomavirus E6 oncoprotein could direct the ubiquitination and degradation of the p53 tumour suppressor at the 26S proteasome was the beginning of a new view on virus-host interactions. A decade later, a plethora of viral proteins have been shown to direct host-cell proteins for proteolytic degradation. These activities are required for various aspects of the virus life-cycle from entry, through replication and enhanced cell survival, to viral release. As with oncogenes and cell-cycle control, the study of apparently simple viruses has provided a wealth of information on the function of a whole class of cellular proteins whose function is arguably as important as that of the kinases: the ubiquitin-protein ligases.

HPV E6 targeted degradation of the discs large protein: evidence for the involvement of a novel ubiquitin ligase.

The Discs Large (DLG) tumour suppressor protein is targeted for ubiquitin mediated degradation by the high risk human papillomavirus (HPV) E6 proteins. In this study we have used a mutational analysis of E6 in order to investigate the mechanism by which this occurs. We first show that the differences in the affinities of HPV-16 and of HPV-18 E6 proteins for binding DLG is reflected in their respective abilities to target DLG for degradation. A mutational analysis of HPV-18 E6 has enabled us to define regions within the carboxy terminal half of the protein which are essential for the ability of E6 to direct the degradation of DLG. Mutants within the amino terminal portion of E6 which have lost the ability to bind the E6-AP ubiquitin ligase, as measured by their ability to degrade p53, nonetheless retain the ability to degrade DLG. Significant levels of DLG degradation are also obtained using wheat germ extracts which lack E6-AP. Finally, we show that the transfer of the DLG binding domain onto the low risk HPV-6 E6 confers DLG binding activity to that protein and, most significantly, allows HPV-6 E6 to target DLG for degradation. These results indicate that E6 mediated degradation of DLG does not involve the E6-AP ubiquitin ligase and, in addition, shows that the high and low risk HPV E6 proteins most likely share a common cellular intermediary in the ubiquitin pathway.

Alternatively spliced HPV-18 E6* protein inhibits E6 mediated degradation of p53 and suppresses transformed cell growth

The E6 proteins originating from the tumour-associated Human Papillomavirus (HPV) types 16 and 18 have been shown to bind to and target the tumour suppressor protein, p53, for ubiquitin-mediated degradation. However, in cell lines derived from cervical neoplasias, the predominant early region transcripts are spliced and encode truncated forms of E6, termed E6*. We report here that HPV-18 E6* protein will interact both with the full-length E6 proteins from HPV-16 and HPV-18 and also with E6-AP, and subsequently blocks the association of full length E6 protein with p53. We also show that, as a result of this block, E6* can inhibit E6-mediated degradation of p53 both in vitro and in vivo. The biological consequences of this are increased transcriptional activity on p53-responsive promoters and an inhibition of cell growth in cells transfected with E6*. This is the first report of a potential biological function for this polypeptide and may represent a means by which HPV is able to modulate the activity of the full-length E6 protein with respect to p53 during viral infection.