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 contining tyrosine phosphatases (PTP1C and PTP1D) are regulators of IFN alpha signaling. We also have been able to demonstate a role for MAP Kinase in IFN alpha stimulated early response genes. Experiments done in the past year demonstrate that IFN alpha and gamma also activate RAF-1 but not Ras. RAF-1 is upstream of MAPK and its activation appears to require the Jak1 tyrosine kinase. 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 and inactivation have been mapped using cell lines which express truncations of the receptor. The tyrosine phosphatase PTP1C is required for downregulation of Jak2 activation by growth hormone. Recent efforts in the lab have also defined a role for the T cell receptor in the antiproliferative actions, but not the antiviral activity of IFN alpha. Jurkat T cell lines which do not express either the tyrosine phosphatase CD45 or the tyrosine kinase ZAP-70, both of which are required for engagment of the T cell receptor, are not responsive to the antigrowth effects of IFN. Project #2: Modulation of IFN Signaling by Growth Factors and transformation. Phorbol Esters, expression of the viral 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, hematopoetic, 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.
NIH ExPorter
NIH ExPorter