Phosphatidylinositol metabolism and polyoma-mediated transformation.

Abstract

The effect of polyoma middle-sized tumor antigen (MTAg) on phosphatidylinositol metabolism has been characterized in vivo and in vitro using polyoma-transformed and polyoma-infected cells. Cells infected with transformationcompetent polyoma virus exhibit increased levels of inositol phospholipids and the second messenger inositol trisphosphate. MTAg or pp6Ocsrc Immunoprecipitates from MTAg-transformed cells contain an activity that phosphorylates phosphatidylinositol and phosphatidylinositol 4-phosphate. This activity is induced in parallel with MTAg when the MTAg synthesis is regulated by hormonal or heavy metal inducers. Immunoprecipitates from one class of polyoma mutants defective in transformation have a reduced level of associated phosphatidylinositol kinase activity in vitro yet are capable of tyrosine phosphorylation on exogenous protein substrates at rates comparable to wild-type virus. Thus, for these mutants, phosphatidylinositol kinase activity is more tightly correlated with transformation than is protein kinase activity. These results suggest that alterations in phosphatidylinositol metabolism by MTAg play a role in transformation by polyoma virus.A possible mechanism for generating some of the phenotypic changes associated with transformation may involve altered phosphorylation and turnover of the membrane phospholipid phosphatidylinositol (PtdIns). Experiments with pp60v-src and pp68v-rs, the transforming gene products of the Rous sarcoma virus and UR2 viruses, respectively, suggest that PtdIns phosphorylation is regulated by viral oncogene products. These studies have identified PtdIns kinase activities associated with pp6Ovsrc and pp6ov-ros proteins and increased PtdIns turnover in vivo in cells transformed with these oncogenes (1, 2). Increased turnover of PtdIns and its phosphorylated derivatives, phosphatidylinositol 4-phosphate (PtdInsP) and phosphatidylinositol 4,5-bis(phosphate) (PtdInsP2), has been implicated in cellular responses to a wide variety of stimuli, including response to some growth factors (3, 4). PtdInsP2 breakdown generates two second messengers, diacylglycerol and inositol tris(phosphate) (InsP3). Diacylglycerol activates the calcium-and phospholipid-dependent protein kinase, which is the major cellular receptor for tumor promoting phorbol esters (5). InsP3 mobilizes calcium from internal stores (6).The roles oftyrosine and PtdIns phosphorylation in cellular growth regulation are relevant to the mechanism of transformation by the DNA tumor virus polyoma. Central to polyoma viral-mediated transformation is the activity of one of the polyoma virus early gene products, the middle-sized tumor antigen (MTAg) (7,8). Mutations that alter MTAg but do not affect the other viral proteins can either abolish or drastically diminish the ability of the virus to transform (7, 9). In established lines, MTAg alone is sufficient to cause transformation (10). MTAg is a membrane-bound protein anchored by a stretch of hydrophobic amino acids at its carboxyl terminus (11,12). A subpopul...

The Catalytic Subunit of Phosphoinositide 3-Kinase: Requirements for Oncogenicity

“…These lipid kinases affect many biological functions such as cell proliferation, differentiation, apoptosis, and glucose transport (1)(2)(3)(4)(5)(6)(7)(8). A possible role of PI 3-kinases in oncogenic transformation was first suggested by the observation that PI 3-kinase activity was associated, via the regulatory subunit p85, with oncogene products such as polyoma middle T antigen (9,10) or v-Src (11,12). The catalytic subunit p110␣ can also be activated by a direct interaction with GTP-loaded Ras, another oncoprotein (13,14).…”

Mapping of polyomavirus middle T domain that is responsible for AP-1 activation

“…MT overexpressing cell lines have been shown to display increased pp60 c-src kinase activity (Bolen et al, 1994) high phosphatidylinositol turnover and levels of phosphorylated derivatives (Kaplan et al, 1986;Serunian et al, 1990;Ulug et al, 1990) and activated Ras (bound to GTP) (Srinivas et al, 1994). Although some correlation between activation of these signal pathways and MT cell transformation can be traced, none of them have been shown to be su cient for malignant cell transformation, since transformationdefective MT mutants that display associated tyrosine kinase, phosphatidylinositol 3-kinase and protein phosphatase activities at wild type levels, were described (Druker and Roberts, 1991;Druker et al, 1990).…”

Thrombin-dependent association of phosphatidylinositol-3 kinase with p60c-src and p59fyn in human platelets.

“…This enzyme has been implicated in the mitogenic response to polypeptide growth factors through its association with the intracellular domain of ligand-bound growth factor receptors (1,2). PI-3 kinase kinase activity also physically associates with transforming but not with nontransforming versions of the polyomavirus middle T-p60csrc complex (8,9,15,(20)(21)(22) and with the protein product of the v-src oncogene (4). In this study, we have investigated the association of PI-3 kinase with normal nonreceptor protein-tyrosine kinases expressed in their natural settings.…”

“…These include the c-raf proto-oncogene product, p7Oraf (16), phospholipase Cy (13), GTPase-activating protein (14), and PI-3 kinase (1, 2). PI-3 kinase has also been shown to associate with p60f-src when activated by polyomavirus middle T antigen (8,9,15,(20)(21)(22) and with p60vsrc (8), an oncogenic version of the src translational product. We reasoned that assays for associated substrates may be a more sensitive means of detecting the activation of nonreceptor kinases PtdinsP -_ Immunoprecipitates were washed three times with cold phosphate-buffered saline containing 1 mM Na4VO3 and 1% Nonidet P-40, twice with cold 0.5 M LiCl-100 mM Tris (pH 7.6), once with cold 100 mM NaCl-1 mM EDTA-10 mM Tris (pH 7.6), and once more with 20 mM HEPES (pH 7.6).…”