Oxygen Generation Influx and Mitochondrial Reactive 2+of DRAK2 in Response to TCR-Induced Ca Protein Kinase D Orchestrates the Activation

DRAK2 is a serine/threonine kinase highly enriched in lymphocytes that raises the threshold for T cell activation and maintains T cell survival following productive activation. T cells lacking DRAK2 are prone to activation under suboptimal conditions and exhibit enhanced calcium responses to AgR stimulation. Despite this, mice lacking DRAK2 are resistant to organ-specific autoimmune diseases due to defective autoreactive T cell survival. DRAK2 kinase activity is induced by AgR signaling, and in this study we show that the induction of DRAK2 activity requires Ca 2+ influx through the Ca 2+ release-activated Ca 2+ channel formed from Orai1 subunits. Blockade of DRAK2 activity with the protein kinase D (PKD) inhibitor Go¨6976 or expression of a kinase-dead PKD mutant prevented activation of DRAK2, whereas a constitutively active PKD mutant promoted DRAK2 function. Knockdown of PKD in T cells strongly blocked endogenous DRAK2 activation following TCR ligation, implicating PKD as an essential intermediate in the activation of DRAK2 by Ca 2+ influx. Furthermore, we identify DRAK2 as a novel substrate of PKD, and demonstrate that DRAK2 and PKD physically interact under conditions that activate PKD. Mitochondrial generation of reactive oxygen intermediates was necessary and sufficient for DRAK2 activation in response to Ca 2+ influx. Taken together, DRAK2 and PKD form a novel signaling module that controls calcium homeostasis following T cell activation. The Journal of Immunology , 2011, 186: 940–950. 12 on WT-DRAK2 in the presence of CA-PKD1. The 293T cells were transfected with pEGFP-DRAK2 or pEGFP-DRAK2-K62A in combination with pcDNA3.1 or pEGFP-PKD1-S738/742E (CA-PKD1). Thirty minutes before lysis, cells were left untreated or treated with 12 mM LNAC. E , LNAC blocks DRAK2 and PKD coimmunoprecipitation. Mitochondrial fractions were obtained from D10 T cells stimulated with 1 m M thapsigargin for 5 min, or with vehicle, in the presence or absence of 12 mM LNAC. Anti-PKD1 was used to immunoprecipitate DRAK2 and PKD from mitochondrial lysate. F , ROS generation through inhibition of mitochondrial complex I using CCCP is sufficient to activate DRAK2. D10 T cells were stimulated with 200 ng/ml PMA, 1 m M thapsigargin or DMSO for 5 min, or CCCP at the indicated times and concentrations. Lysates were resolved by SDS-PAGE and probed with the indicated Abs. G , Model for PKD-dependent DRAK2 activation. TCR stimulation leads to activation of PLC g , which leads to production of IP 3 , and subsequent release of calcium from the ER through IP 3 receptors. ER calcium release allows Orai1 to form functional CRAC channels in the plasma membrane, resulting in a large influx of extracellular calcium, which accelerates mitochondrial respiration and the production of ROS. ROS positively feed back on PLC g activation and, through Src and Abl, can activate PKD. PKD, following activation through potentially one of these pathways, is then able to activate DRAK2, which serves to limit calcium influx.