Cellular expression of retroviral Gag proteins can induce significant alterations in cell behavior which may be involved in pathogenesis. For example, mouse AIDS is induced by a mutated virus expressing a truncated Gag protein. The PI and colleagues have isolated, characterized, cloned, and sequenced a retroviral Gag protein p58gag, the product of an expressed endogenous retroviral sequence, from a highly malignant 13762 rat mammary ascites tumor. Sublines expressing p58gag have highly stabilized cell surfaces; cell surface stability correlates with xenotransplantability of these rat tumor sublines into mice. Perhaps p58gag modifies the cell surface through stabilizing interactions between microfilaments (MF) and the plasma membrane. Preliminary studies reveal that p58gag is a membrane associated complex that binds the cytoskeleton and contains signaling molecules. Purified p58 binds to mf, phospholipid and a membrane glycoprotein of a mf-associated signal transduction particle (STP). The STP contains the receptor tyrosine kinase p185neu and all of the components of the Ras to MAPK mitogenic pathway, as well as Src and Abl. p58gag binds Src and Abl directly in vitro, and activates the kinases. It is also phosphorylated by them. Binding to the kinases is proposed to occur through specific polyproline motifs, which have been shown to bind to SH3 domains of proteins involved in many signaling pathways. Further, several of the characterized SH3 domain-containing proteins associate with mf. These have been implicated in transmission of extracellular signal to the cytoskeleton. Polyproline sequences occur ubiquitously in retroviral Gag proteins, including those of MAIDS and HIV. The hypothesis is that interaction of p58gag with SH3 domain-containing proteins and with mfs affect the location of signaling proteins and perturb cell structure. To test this hypothesis, the investigators will analyze the interaction of p58gag, the p60gag MAIDS Gag, and HIV Gag with tyrosine kinases and other signaling proteins using recombinant fusion proteins, transfection studies, cell perturbation analyses and binding assays. 1) Deletion and site-directed mutagenesis will be used to characterize the domains of p58 which bind Src, ABl and mf. 2) The effects of p58 and these mutants on kinase localization, mf organization, cell proliferation, and membrane receptor mobility will be examined by cell transfections and analyses using COS 7, MDCK, Jurkat, and 13762 tumor cells. 3) GST fusion proteins of p58, its mutants, and phosphorylated p58 will be used to delineate other signaling proteins which bind p58 and tyr-phosphorylated p58 for correlation with biological effects. 4) The association of MAIDs and HIV Gag proteins with SH3 domain-containing signaling and membrane skeletal proteins will be investigated using the appropriate fusion and native proteins. These studies should provide new insights into the behavior of retroviral Gag proteins and their potential for a role in the pathogenesis of retroviruses.
| Li, Y; Hua, F; Carraway, K L et al. (1999) The p185(neu)-containing glycoprotein complex of a microfilament-associated signal transduction particle. Purification, reconstitution, and molecular associations with p58(gag) and actin. J Biol Chem 274:25651-8 |
| Huang, J; Zhang, B T; Li, Y et al. (1999) c-Src association with and phosphorylation of p58gag, a membrane- and microfilament-associated retroviral Gag-like protein in a xenotransplantable rat mammary tumor. Oncogene 18:4099-107 |
