The genital tract human papilloma viruses( HPVs) can be separated in to two groups based on their clinical associations and risk for malignant progression. The high risk HPVs (such as HPV-16 and HPV-18th)a t are associated with specific anogenital cancers encode two oncoproteins E6 and E7, which are expressed in the? HPV positive cancers. The E7 protein functions in cellular transformation, at least in part, through interactions with the product of the retinoblastoma susceptibility gene, pRB, andt he other pRB related """"""""pocket proteins"""""""". The major target of the E6 oncoprotein encoded by the genital tract, cancer associated human papillomaviruses isth e p53 tumor suppressor protein. The binding of E6 to p53 is mediated by a cellular protein that we have previously identified and characterized, called the E6 associated protein? (E6AP). In the presence of E6, E6AP catalyses the ubiquitination and proteolysis of p53. E6AP is an E3 ubiquitin protein ligase that is expressed in all tissues. In the absence of E6, p53 is not a substrate for EGAP, but HPV16 E6 promotes the ubiquitination and degradation of p53 by forming a complex with EGAP.? Other proteins have also been shown to be targeted for degradation by E6 including PDZ domain containing proteins such as hDlg and hScrib. A number of E6-independent substrates of E6AP have now been identified, including the human homologue of the yeast Rad23, hHR23th, e Src-family kinase Blk, MCM7 subunit of replication licensing factor and, from our recent work, C -terminal Src kinase, Csk. We have found that HPV16 E6 promotes E6AP auto-ubiquitination resulting in its enhanced turnover. We are exploring how E6 affects E6APf unction and the consequent effects of E6 on the E6-independents substrates of E6AP. In addition our studies have identified a class of related ubl domain containing proteins, that includes the hPLlC and hHR23 proteins, that participate in ubiquitin mediated proteolysis by directly linking the ubiquitination? machinery to the proteasomes. This grant will further explore the regulation and functions of EGAP and its cellular partners. Furthermore we will establish chemical screens to identify small molecules that target the interaction of E6 with EGAP, as well as the ability of EGAP to participate in the E6 dependent ubiquitination of? p53.? ? ? AIMS? The specific aims of this grant have been to ? (1) evaluate the role of E6AP in the ubiquitylation of hHR23A and? related proteins( i.e. the PLIC proteins)? (2 ) evaluate the roleo f E6AP in the regulation of the Src family of? tyrosine kinases; and? ( 3) determine the consequences of HPV E6 binding to E6AP on E6AP and on the E 6-independent substrates of E6AP.?
