CD40 is a cell surface transmembrane 45kDa glycoprotein receptor expressed on B- lymphocytes. CD40 activation is critical for B-cell proliferation, immunoglobulin class switching. NfkB activation and rescue of germinal center B-cells from apoptosis following somatic mutation. Resistance to apoptosis is conferred by the induction of the anti-death gene product, Bcl-x. CD40 is a member of the tumor necrosis factor receptor family and, like other members, it appears to possess no intrinsic signalling capacity (e.g. kinase activity), suggesting that signal transduction is likely mediated by associating molecules. To identify such molecules, the yeast two hybrid system was used to clone cDNAs encoding proteins that bind to the CD40 cytoplasmic domain. One such interacting protein, designated CD40-binding protein (CD40bp), has a N- terminal RING finger motif and a prominent central coiled-coil segment that may allow homo- or hetero-oligomerization. Significantly, the C- terminus possesses substantial homology to the tumor necrosis factor receptor-associated factor (TRAF) domain that is found in two proteins (TRAF1 and TRAF2) that associate with the cytoplasmic domain of the related 75kD tumor necrosis factor receptor. Dominant-negative CD40bp inhibited the induction of Bcl-x in response to CD40 activation suggesting an important role in signaling. The second CD40 interacting protein identified was TRAF2, over- expression of which resulted in the activation of NF-kB. Notably, dominant- negative TRAF2 completely abrogated the activation of NfkB by CD40, confirming that TRAF2 couples the receptor to the NF-k B pathway. It is the intention of this grant application to capitalize on these findings. The following Specific Aims will be undertaken.
Specific Aim 1 : Confirm that endogenous CD40bp and TRAF2 bind CD40 in a ligand dependent fashion and that the TRAF domain is indeed responsible for receptor association.
Specific Aim 2 : Use the yeast two-hybrid and complementary biochemical approaches to clone and characterize CD40bp and TRAF2 interacting proteins.
Specific Aim 3 : Determine if CD40bp and TRAF2 modulate other CD40 signaling events including activation of cytoplasmic src-like kinases and PI3 kinase.
| Zetoune, F S; Murthy, A R; Shao, Z et al. (2001) A20 inhibits NF-kappa B activation downstream of multiple Map3 kinases and interacts with the I kappa B signalosome. Cytokine 15:282-98 |
