Project #1. Interferon and Cytokine Activation of Early Response Genes: We have developed a cell free system to study Both IFN alpha and IFN gamma activation of the assembly of several transcription complexes. Using these systems we have been able to demonstrate that activation of these complexes required a combination of a membrane associated tyrosine phosphatase(s) and tyrosine kinase(s). Using a combination of antibodies to known components needed for either IFN alpha or IFN gamma induced gene expression, we have been able to demonstrate that two SH2 domain containing tyrosine phosphatases (PTP1C and PTP1D) are regulators of IFN alpha signaling. We also have been able to demonstrate a role for MAP Kinase in IFN alpha stimulated early response genes. In addition, we have recently obtained evidence that several other cytokines such as GM-CSF, IL-3, IL-5, IL-10, EPO prolactin and growth hormone are functioning by similar mechanisms as the IFNs to activate the expression of early response genes. The domains in the growth hormone receptor required for Jak/Stat activation have been mapped using cell lines which express truncations of the receptor. Recently we have obtained evidence of tyrosine phosphorylated DNA-binding proteins in Drosophila. Efforts are now underway to purify and clone this protein from Drosophila to proceed with developmental and genetic studies. Project #2: Modulation of IFN Signaling by Growth Factors and transformation. Phorbol Esters, expression of adenovirus E1A or human papilloma virus E6/7 inhibit IFN formation of the ISGF3 transcription complex. We have now characterized several cell lines which are defective in IFN activation of ISGF3 and contain within their cytoplasm a factor actually disrupts the formation of ISGF3. This factor (TKO) is present in human cell lines derived from cervical, hematopoietic, ovarian, colon and small cell lung cancers. TKO has been enriched about 5000 fold and we have purified the protein to near homogeneity. The purified protein is now being sequenced and we hope to have it cloned in the near future.
