The differentiation of antibody producing B-cells from pluripotent stem cells has been a paradigm for studying molecular aspects of mammalian development. To understand the mechanisms that regulate this developmental pathway, we have focused our attention on two important regulators of transcription in B-cells, the transcription factor NF- kappaB and its inhibitor, IkappaB. Aberrant expression of the genes, through translocations have been linked to human lymphomas, highlighting their importance in regulating normal growth of cells. In addition NF- kappaB plays a very important role in the activation of HIV from a latent state in infected T-cells. It is therefore clear that understanding how the activity and expression of NF-kappaB and IkappaB are regulated will not only provide insights into fundamental mechanisms of B-cell development but also into diseases such as lymphomagenesis and immunodeficiency. The primary objective of our proposal is to characterize the family of IkappaB proteins including IkappaB-alpha, IkappaB-beta and pp40. Both IkappaB-alpha and pp40 have been cloned and we intend to clone the cDNA encoding IkappaB-beta. this will be necessary to carry out a comprehensive study on the regulation and expression of IkappaB activity in cells including mature B-cells. We will compare the sequence and the structure of the different IkappaB forms and use the most conserved regions to search for related family members. To analyze the function of the IkappaB proteins we will produce large quantities of the recombinant protein for use both as immunogens and for functional studies. We have previously characterized phosphorylation of IkappaB as an important event in the induction of NF-kappaB activity. We propose to use recombinant IkappaB proteins and antibodies against IkappaB to determine the sites of phosphorylation used both in vivo and in vitro. We will alter the in vitro sites of phosphorylation and introduce the mutant proteins into the cell. Such studies will help to determine if the sites used for in vitro phosphorylation are necessary for in vivo activation of NF-kappaB. We will also attempt to isolate the in vivo inactive form of IkappaB and characterize the modification that prevents it from interacting with NF-kappaB. Finally, we wish to determine how NF-kappaB changes from an inducible cytosolic protein in preB cells to a nuclear, constitutively active protein in mature B cells. We will explore options of either transcriptional shut-off of IkappaB synthesis or inactivation of IkappaB through modification of the protein in mature B cells.
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