Golgi Alkalinization by the Papillomavirus E5 Oncoprotein

Abstract

The E5 oncoprotein of bovine papillomavirus type I is a small, hydrophobic polypeptide localized predominantly in the Golgi complex. E5-mediated transformation is often associated with activation of the PDGF receptor (PDGF-R). However, some E5 mutants fail to induce PDGF-R phosphorylation yet retain transforming activity, suggesting an additional mechanism of action. Since E5 also interacts with the 16-kD pore-forming subunit of the vacuolar H+-ATPase (V-ATPase), the oncoprotein could conceivably interfere with the pH homeostasis of the Golgi complex. A pH-sensitive, fluorescent bacterial toxin was used to label this organelle and Golgi pH (pHG) was measured by ratio imaging. Whereas pHG of untreated cells was acidic (6.5), no acidification was detected in E5-transfected cells (pH ∼7.0). The Golgi buffering power and the rate of H+ leakage were found to be comparable in control and transfected cells. Instead, the E5-induced pH differential was attributed to impairment of V-ATPase activity, even though the amount of ATPase present in the Golgi complex was unaltered. Mutations that abolished binding of E5 to the 16-kD subunit or that targeted the oncoprotein to the endoplasmic reticulum abrogated Golgi alkalinization and cellular transformation. Moreover, transformation-competent E5 mutants that were defective for PDGF-R activation alkalinized the Golgi lumen. Neither transformation by sis nor src, two oncoproteins in the PDGF-R signaling pathway, affected pHG. We conclude that alkalinization of the Golgi complex represents a new biological activity of the E5 oncoprotein that correlates with cellular transformation.

Mechanisms of cell transformation by papillomavirus E5 proteins

“…It has been reported that certain E5 mutants transform cells or a ect cellular metabolism without binding to or activating the PDGF b receptor, suggesting that these mutants utilize alternative, PDGF b receptor-independent mechanisms to transform cells (Schapiro et al, 2000;Sparkowski et al, 1996;Suprynowicz et al, 2000;Vali et al, 2001). However, more recent results from our laboratory indicate that at least some of these mutants interact productively with the PDGF b receptor (CC Lai and D DiMaio, unpublished results).…”

“…A ternary complex containing the BPV E5 protein, the PDGF b receptor, and the V-ATPase subunit has been detected in cells engineered to overexpress these three proteins (Goldstein et al, 1992a). Acidi®cation of the Golgi apparatus is impaired in cells transformed by the BPV E5 protein, and it has been proposed that the BPV E5 protein directly inhibits V-ATPase activity (Schapiro et al, 2000;Suprynowicz et al, 2000). Because many important growth regulatory proteins, including the PDGF b receptor, pass through the Golgi apparatus en route to their ®nal destination in the cell, the ability of the BPV E5 protein to perturb the pH of intracellular organelles may in¯uence the activity of these proteins and contribute to transformation.…”

The HPV16 E5 oncogene inhibits endocytic trafficking

“…The E6 and E7 proteins interact with the p53 and Rb oncosuppressor genes. In contrast to E5 from bovine papillomaviruses (BPV) (Schapiro et al, 2000;Sparkowski et al, 1995) the HPV16 E5 protein is not well characterized since it has been di cult to detect and study in vitro. However, the open reading frame encodes a hydrophobic oncoprotein consisting of 83 amino acids (Bubb et al, 1988;Halbert and Galloway, 1988).…”

Presence of bovine papillomavirus type 2 DNA and expression of the viral oncoprotein E5 in naturally occurring urinary bladder tumours in cows

“…E5 is a small hydrophobic protein (44 aa) that is capable of inducing in vitro cell transformation through the activation of several kinases, from growth factor receptors to cdk cyclins (Venuti & Campo, 2002). E5 interacts physically with the cellular protein 16k ductin/ subunit c, a component of the gap junction and of the V0 sector of vacuolar H + -ATPase (Conrad et al, 1993;Faccini et al, 1996;Finbow et al, 1991;Goldstein et al, 1991), causing the downregulation of gap junction communication (Ashrafi et al, 2002;Faccini et al, 1996;Oelze et al, 1995) and the lack of acidification of endosomes and Golgi apparatus (GA) (Schapiro et al, 2000;Straight et al, 1995). However, its function in vivo is still unknown.…”