Recent studies have revealed that posttranslational modification of the transcription factor c-Jun plays a functional and necessary role in the mechanism of transformation by activated c-Ha-Ras. We have extended these studies to show that a similar mechanism is operating in v-sis transformed cells and that multiple transcription factors such as other Jun and Fos family members as well as unrelated transcription factors like Egr-1 also exhibit substantially increased phosphorylation. Preliminary studies have been carried out in model cells which exhibit inducible, complete and reversible transformation by an inducible sis construct. Phosphorylation of these factors does not occur in normally growing cells but is constant in sis-transformed cells and is quantitatively reversible suggesting transformation-specific mode of regulation. Preliminary studies using transdominant negative mutations show that c-raf-1 and c-ras, both reported to activated by PDGF, are required for transformation by v-sis.
Specific Aim #1 is to test whether the model cell system exhibits an autocrine mechanism of transformation.
Specific Aim #2 is to use these cells to test the hypothesis that phosphorylation of c-Jun and family members is transformation-specific, mediates transformation by v-sis, requires, and is necessary for, the action of c-ras and c-raf-1 according to the general signal transduction scheme:PDGF-activated receptor, C ras, c raf-1, c- Jun(ser63,73)-(PO4)2, AP-1 activity and other transforming events. The phosphorylation sites will be further characterized by two-dimensional phosphopeptide mapping in combination with microsequencing.
Specific Aim #3 is to test whether phosphorylation may be important in human tumors by determining whether phosphorylated transcription factors occur in human tumor lines and fresh tumor tissue of breast, ovary, prostate, osteosarcomas and glioblastomas where PDGF/sis autocrine mechanism has been implicated. Knowledge of transformation-specific regulatory events such as phosphorylations may provide targets of specific intervention which will not alter normal growth and function.
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