HPV-16 E5 oncoprotein upregulates lipid raft components caveolin-1 and ganglioside GM1 at the plasma membrane of cervical cells.

High-risk human papillomaviruses (HPVs), especially HPV-16, play a primary role in the pathogenesis of cervical cancer. HPV-16 encodes the E5, E6 and E7 oncoproteins. Although the biological functions of E5 are poorly understood, recent studies indicate that its expression correlates with papillomavirus oncogenicity. In this study we demonstrate that the HPV-16 E5 oncoprotein increases plasma membrane expression of caveolin-1, which is a constituent of lipid rafts and regulator of cell signaling, and that this phenotype is mediated by the C-terminal 10 amino acids of E5. Moreover, E5 (but not mutant E5) induces a 23- to 40-fold increase in the lipid raft component, ganglioside GM1, on the cell surface and mediates a dramatic increase in caveolin-1/GM1 association. Since gangliosides strongly inhibit cytotoxic T lymphocytes, block immune synapse formation and are expressed at high levels on the surface of many tumor cells, our results suggest a potential mechanism for immune evasion by the papillomaviruses. Additionally, surface gangliosides are known to enhance proliferative signaling by the epidermal growth factor (EGF) receptor, providing a possible mechanistic basis for observations that EGF signaling is enhanced in E5-expressing cells. Finally, the upregulation of caveolin-1 and ganglioside GM1 at the plasma membrane of E5-expressing cervical cells provides potential new therapeutic targets and diagnostic markers for high-risk HPV infections.

Activation of protein kinase C alters p34(cdc2) phosphorylation state and kinase activity in early sea urchin embryos by abolishing intracellular Ca2+ transients.

The p34(cdc2) protein kinase, a universal regulator of mitosis, is controlled positively and negatively by phosphorylation, and by association with B-type mitotic cyclins. In addition, activation and inactivation of p34(cdc2) are induced by Ca(2+) and prevented by Ca(2+) chelators in permeabilized cells and cell-free systems. This suggests that intracellular Ca(2+) transients may play an important physiological role in the control of p34(cdc2) kinase activity. We have found that activators of protein kinase C can be used to block cell cycle-related alterations in intracellular Ca(2+) concentration ([Ca(2+)](i)) in early sea urchin embryos without altering the normal resting level of Ca(2+). We have used this finding to investigate whether [Ca(2+)](i) transients control p34(cdc2) kinase activity in living cells via a mechanism that involves cyclin B or the phosphorylation state of p34(cdc2). In the present study we show that the elimination of [Ca(2+)](i) transients during interphase blocks p34(cdc2) activation and entry into mitosis, while the elimination of mitotic [Ca(2+)](i) transients prevents p34(cdc2) inactivation and exit from mitosis. Moreover, we find that [Ca(2+)](i) transients are not required for the synthesis of cyclin B, its binding to p34(cdc2) or its destruction during anaphase. However, in the absence of interphase [Ca(2+)](i) transients p34(cdc2) does not undergo the tyrosine dephosphorylation that is required for activation, and in the absence of mitotic [Ca(2+)](i) transients p34(cdc2) does not undergo threonine dephosphorylation that is normally associated with inactivation. These results provide evidence that intracellular [Ca(2+)](i) transients trigger the dephosphorylation of p34(cdc2) at key regulatory sites, thereby controlling the timing of mitosis entry and exit.

E5 oncoprotein mutants activate phosphoinositide 3-kinase independently of platelet-derived growth factor receptor activation.

The E5 oncoprotein of bovine papillomavirus type 1 is a Golgi-resident, 44-amino acid polypeptide that can transform fibroblast cell lines by activating endogenous platelet-derived growth factor receptor beta (PDGF-R). However, the recent discovery of E5 mutants that exhibit strong transforming activity but minimal PDGF-R tyrosine phosphorylation indicates that E5 can potentially use additional signal transduction pathway(s) to transform cells. We now show that two classes of E5 mutants, despite poorly activating the PDGF-R, induce tyrosine phosphorylation and activation of phosphoinositide 3-kinase (PI 3-K) and that this activation is resistant to a selective inhibitor of PDGF-R kinase activity, tyrphostin AG1296. Consistent with this independence from PDGF-R signaling, the E5 mutants fail to induce significant cell proliferation in the absence of PDGF, unlike wild-type E5 or the sis oncoprotein. Despite differences in growth factor requirements, however, both wild-type E5 and mutant E5 cell lines form colonies in agarose. Interestingly, activation of PI 3-K occurs without concomitant activation of the ras-dependent mitogen-activated protein kinase pathway. The known ability of constitutively activated PI 3-K to induce anchorage-independent cell proliferation suggests a mechanism by which the mutant E5 proteins transform cells.

Golgi alkalinization by the papillomavirus E5 oncoprotein.

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 (pH(G)) was measured by ratio imaging. Whereas pH(G) of untreated cells was acidic (6.5), no acidification was detected in E5-transfected cells (pH approximately 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 pH(G). We conclude that alkalinization of the Golgi complex represents a new biological activity of the E5 oncoprotein that correlates with cellular transformation.

Ca2+ triggers premature inactivation of the cdc2 protein kinase in permeabilized sea urchin embryos.

Exit from mitosis requires inactivation of the cyclin B-p34cdc2 protein kinase complex. Since increased cytosolic Ca2+ has been implicated as a potential trigger of mitotic progression, we directly tested the possibility that Ca2+ triggers the pathway responsible for inactivating the cdc2 kinase, using sea urchin embryos permeabilized at various stages of the cell cycle. In cells permeabilized during late interphase and prophase, micromolar Ca2+ induced premature inactivation of the cdc2 kinase without affecting the absolute amount of p34cdc2 protein. Inactivation was selective for the cdc2 kinase, as elevated Ca2+ had no effect on cAMP-dependent protein kinase activity. Premature cdc2 kinase inactivation did not require cyclin B destruction, but did coincide with the dissociation of cyclin B-p34cdc2 complexes. In cells permeabilized during prometaphase and metaphase, cdc2 kinase inactivation was Ca(2+)-independent, presumably because at these later times the inactivating pathway had been enabled prior to permeabilization. This work provides evidence that Ca2+ is the physiological trigger enabling cdc2 kinase inactivation during mitosis.

Inactivation of cdc2 kinase during mitosis requires regulated and constitutive proteins in a cell-free system.

Inactivation of the cyclin-p34cdc2 protein kinase complex is a major requirement for anaphase onset and exit from mitosis. To facilitate identification of specific molecules that regulate this event in mammalian cells, I have developed a cell-free assay in which cdc2 kinase associated with a chromosomal fraction from metaphase tissue culture cells is inactivated by a cell-cycle-regulated cytosolic system. In vitro kinase inactivation requires ATP, Mg2+ and the dephosphorylation of one or more sites in the chromosomal fraction by protein phosphatase 1 and/or 2A. Cyclin B is destroyed during inactivation, while the level of p34cdc2 remains constant. Ammonium sulfate fractionation resolves the cytosolic inactivating system into at least two distinct protein components that are both required for inactivation and are differentially regulated during mitosis.

Identification of mitosis-specific p65 dimer as a component of human M phase-promoting factor.

Antisera raised against two mitosis-specific protein kinases from human cells recognized a single 65-kDa polypeptide (p65) that is present in similar amounts in interphase and mitotic cell extracts. Immunoblot analysis of reduced and unreduced extracts revealed that p65 exists as a 65-kDa monomer during interphase but forms a 130-kDa disulfide-linked homodimer during mitosis. Several different antibodies recognizing the p34cdc2 protein kinase and cyclin B components of M phase-promoting factor (MPF) coprecipitated p65 from mitotic but not from interphase extracts. In addition, an anti-p65 immunoaffinity column substantially depleted mitotic extracts of histon H1 kinase activity assayed under conditions diagnostic for MPF. These results suggest that active human MPF may be a complex of p34cdc2, cyclin B, and dimeric p65. A sulfhydryl cycle, proposed in the earlier literature on the biochemistry of mitosis, might underlie the dimerization of p65 and formation of active MPF.

A fractionated cell-free system for analysis of prophase nuclear disassembly.

We describe a cell-free system in which a postribosomal supernatant (s140) from metaphase Chinese hamster ovary (CHO) cells induces prophase-like changes in isolated CHO cell nuclei, including chromatin condensation, and nuclear envelope and lamina disassembly. These events are strongly promoted by gamma-S-ATP and an ATP-regenerating system, and do not take place with an s140 derived from G2-phase cells. The metaphase cell s140 also induces disassembly of an isolated nuclear lamina fraction that is depleted of membranes, chromatin, and nuclear pore complexes. Disassembly of the isolated lamina is accompanied by phosphorylation of the major lamina proteins (lamins A, B, and C) to levels characteristic of metaphase cells. Kinetic analysis of lamina depolymerization indicates that cooperativity may be involved in this process. The biochemical properties of in vitro lamina disassembly suggest that the activity that depolymerizes the lamina during mitosis is soluble in metaphase cells, and support the notion that this activity is a lamin protein kinase.

Fluctuation of the Ca-sequestering activity of permeabilized sea urchin embryos during the cell cycle.

We have followed the sequestration of Ca(2+) by intracellular compartments in sea urchin embryos through the first cell cycles. To gain biochemical access to these compartments, the embryos were permeabilized by brief exposure to an intense electric field. Sequestration was determined as the retention of tracer, (45)Ca, after filtration of aliquots on Millipore filters. The permeabilized cells sequester Ca(2+) at a constant rate for at least 20 min, with the following characteristics: (i) ATP is required. (ii) Sequestration occurs at Ca(2+) levels corresponding to those estimated in vivo. (iii) The Ca(2+) concentration dependence of sequestration and its insensitivity to mitochondrial poisons imply that the activity derives from a single, nonmitochondrial transport system. The Ca(2+)-sequestering activities of embryos that are permeabiized at successive stages of the first cell cycle (one-cell stage) progressively increase to 5 times the initial level. The rate of sequestration is maximal during telophase and, in some populations of zygotes, is nearly as great throughout prophase. Over the course of the second cell cycle (two-cell stage), the activity undergoes a 2-fold oscillation that bears the same temporal relationship to mitosis as the previous fluctuation.

Cytoplasmic dynein-like ATPase cross-links microtubules in an ATP-sensitive manner.

We have prepared dynein-like ATPase from the eggs of the sea urchin Strongylocentrotus purpuratus using differential centrifugation and column chromatography. This ATPase preparation is inhibited by vanadate and erythro-9-(3-[2-hydroxynonyl]) adenine (EHNA) at concentrations similar to those that inhibit reactivated flagellar beating and spindle elongation in lysed cell models. Using microtubule affinity and ATP-induced release, we can purify this ATPase activity to a composition on SDS PAGE of four peptides ranging in molecular weight from 180,000-300,000. When viewed in darkfield optics, this affinity-purified ATPase caused extensive parallel bundling of microtubule-associated protein-free microtubules. These bundles were dispersed by 1 mM ATP but not by ATP gamma S or AMP-5'-adenylimidodiphosphate. The reformation of microtubule bundles after dispersal by ATP required ATP hydrolysis; bundles did not reform in the presence of 10 microM vanadate. Negative stain electron microscopy of these bundled microtubules revealed that they are arranged in parallel networks with extensive close lateral association.

Regulation of neutral protease activity through the life cycles of Tetrahymena pyriformis.

Substantial fluctuations in the intracellular specific activity of neutral proteases, as assayed at pH 8 with azocasein as substrate, occur during the life cycle of the protozoan Tetrahymena pyriformis. Specific activity increases during growth in 2% proteose peptone, despite slow secretion into the medium. The most rapid increase occurs during late stationary phase and appears to be a response to one or more low molecular weight (less than 10 000), heat-stable, trypsin-insensitive, polar molecules secreted into the medium. In contrast, intracellular specific activity drops by a factor of 2--5 within the first 2--3 h after transfer to non-nutritive medium. The decrease in activity under these conditions results from an enhanced rate of secretion and the cessation of net synthesis. Its kinetics are unaffected by cycloheximide and concanavalin A.