Modulation of DRAK2 autophosphorylation by antigen receptor signaling in primary lymphocytes.

Death-associated protein-related apoptotic kinase-2 (DRAK2), a member of the death-associated protein-like family of serine/threonine kinases, is highly expressed in lymphoid organs and is a negative regulator of T cell activation. To investigate the regulation of DRAK2 activity in primary lymphocytes, we employed mass spectrometry to identify sites of autophosphorylation on DRAK2. These studies have revealed a key site of autophosphorylation on serine 12. Using a phospho-specific antibody to detect Ser(12) phosphorylation, we found that autophosphorylation is induced by antigen receptor stimulation in T and B cells. In Jurkat T cells, resting B cells and thymocytes, DRAK2 was hypophosphorylated on Ser(12) but rapidly phosphorylated with antigen receptor ligation. This increase in phosphorylation was dependent on intracellular calcium mobilization, because BAPTA-AM blocked DRAK2 kinase activity, whereas the SERCA inhibitor thapsigargin promoted Ser(12) phosphorylation. Our results show that DRAK2 kinase activity is regulated in a calcium-dependent manner and that Ser(12) phosphorylation is necessary for optimal suppression of T cell activation by this kinase, suggesting a potential feedback loop may act to modulate the activity of this kinase following antigen receptor signaling.