The long-range goal of this research project is to understand how HTLV-I Tax protein deregulates the cellular NF-kappaB transcription factor pathway. This knowledge is critical for understanding how HTLV-I induces human T-cell transformation and, thus, is important for rational development of anti-HTLV therapies. Our studies performed during the current granting period demonstrate that Tax activates NF-kappaB by inducing phosphorylation and subsequent degradation of the NF-kappaB inhibitor IkappaB. Tax triggers IkappaB phosphorylation by stimulating the activity of a cellular IkappaB kinase (IKK), which is assembled in a large (greater than 700kD), multicomponent complex named IKK signalsome. The overall objective of the studies proposed in this continuation application is to elucidate the mechanisms by which Tax persistently activates the IKK signaling machinery. As will be presented under Progress Report, we have recently demonstrated that Tax physically interacts with the IKK complex via direct binding to the IKK regulatory subunit, IKKgamma. The IKKgamma- directed Tax/IKK association occurs in both transfected and HTLV- I infected T cells and is correlated with persistent activation of IKK. Additional studies demonstrate that Tax activation of IKK also requires upstream kinases. Interestingly, one of the putative IKK upstream kinases, Cot, physically interacts with Tax. These findings indicate that Tax may stimulate the chronic activity of IKK by forming stable complexes with both IKK and its upstream activators. We have also found that Tax-stimulated IKK activation is associated with phosphorylation of the IKK subunits, a modification that often triggers the catalytic activity of protein kinases. Thus, the central hypothesis to be tested is that Tax stimulates persistent activity of IKK via a mechanism involving its physical interaction with IKK and upstream activators and phosphorylation-dependent IKK activation. To accomplish the objective of this application, we will pursue four specific aims: (1) determination of the biochemical mechanism and functional significance of Tax/IKKgamma physical interaction in Tax activation of IKK; (2) investigation of the role of Cot and other upstream kinases in Tax-mediated IKK activation; (3) dissecting the molecular components of the IKK signalsomes in normal versus HTLV1-infected T cells; and (4) determination of the functional significance of IKK subunit phosphorylations in Tax-stimulated IKK activation.
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