Role of AKT/ PKB and 14-3-3 in the regulation of B cell receptor signaling and signalosome assembly

AKT/PKB is an oncogenic serine/threonine kinase regulated via the PI3K pathways. 14-3-3s represent a large group of adaptor proteins that are known to interact with a plethora of signaling proteins and regulate diverse signal transduction pathways. The B-cell antigen receptor (BCR) activation and signalosome assembly are dynamic processes controlled by protein phosphorylation. The signaling events share their functions in controlling cell proliferation, differentiation, and/or apoptosis. In Paper I, the first characterized protein is 14-3-3ζ, which was found to be a new regulator of BTK. Two 14-3-3ζ binding-sites were found to be phosphorylated by AKT/PKB and mapped to phospho-serine pSer51 in the PH domain and to phospho-threonine pThr495 in the kinase-domain. The PI3K inhibitor LY294002 abolished S51/T495 phosphorylation and disrupted the interaction. Moreover, inhibitors targeting 14-3-3 (BV02) and BTK (Ibrutinib) compromised interaction between the two proteins. Nuclear translocation of BTK was promoted following down regulation of 14-3-3ζ. Furthermore, the loss-of-function mutant S51A/T495A displayed reduced tyrosine-phosphorylation and inability to bind to 14-3-3ζ. Conversely, the gain-of-function mutant S51D/T495D exhibited intense phosphorylation, enhancing interaction of BTK with 14-3-3 ζ. Phosphorylation of this mutant was associated with ubiquitination and degradation of the protein, presumably, contributing to the termination of the B-cell receptor signaling. In Paper II, we identified a new BTK-partner, ankyrin repeat domain 54 protein (ANKRD54) that binds to the BTK SH3 domain. Our results suggest that ANKRD54 specifically mediates nuclear export of both BTK and another TEC family kinase member, TXK/RLK. The interaction site was mapped to the Cterminus of the BTK SH3 domain, since a synthetic peptide covering this region, ARDKNGQEGYIPSNYVTEAEDS, was sufficient for mediating this interaction. ANKRD54 is the first protein reported to specifically influence nucleo-cytoplasmic shuttling of BTK. ANKRD54 ...