Modulation of DRAK2 Autophosphorylation by Antigen Receptor Signaling in Primary Lymphocytes

Abstract

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 Ser12 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 Ser12 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 Ser12 phosphorylation. Our results show that DRAK2 kinase activity is regulated in a calcium-dependent manner and that Ser12 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. 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 Ser12 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 Ser12 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 Ser12 phosphorylation. Our results show that DRAK2 kinase activity is regulated in a calcium-dependent manner and that Ser12 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. DRAK2, 4The abbreviations used are: DRAK2, death-associated protein-related apoptotic kinase-2; DAP, death-associated protein; DAPK, DAP kinase; DRP-1, DAP-related protein kinase-1; ZIP, zipper-interacting protein kinase; TCR, T cell antigen receptor; Ab, antibody; GST, glutathione S-transferase; GFP, green fluorescent protein; EGFP, enhanced GFP; IP, immunoprecipiation; LC, liquid chromatography; MS/MS, tandem mass spectrometry; IL, interleukin; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl ester); XIC, extracted ion chromatogram; ERK, extracellular signal-regulated kinase. a member of the death-associated protein (DAP) kinase family, has recently emerged as a regulator of immune function. The DAP family of pro-apoptotic serine/threonine kinases consists of five members; all are capable of inducing apoptosis upon overexpression in cell culture (1.Kogel D. Prehn J.H. Scheidtmann K.H. BioEssays. 2001; 23: 352-358Crossref PubMed Scopus (88) Google Scholar). Indeed, the prototype DAP kinase (DAPK1) was identified in a screen for positive mediators of apoptosis. Sequence homology between the five family members is restricted to the N-terminal kinase domain, whereas the C-terminal regions are unique and are required for the regulatory functions of each kinase, linking individual family members to specific signal transduction pathways. The fifth member, DRAK2, is expressed primarily in hematopoietic tissues. DRAK2-deficient mice have no defects in apoptosis, and we have shown that retroviral expression of DRAK2 in NIH3T3 cells does not induce overt apoptosis. Rather, recent data have demonstrated an important role for DRAK2 in negative regulation of T cell activation (2.McGargill M.A. Wen B.G. Walsh C.M. Hedrick S.M. Immunity. 2004; 21: 781-791Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 3.Friedrich M.L. Wen B.G. Bain G. Kee B.L. Katayama C. Murre C. Hedrick S.M. Walsh C.M. Int. Immunol. 2005; 17: 1379-1390Crossref PubMed Scopus (22) Google Scholar). In response to suboptimal T cell antigen receptor (TCR) stimulation, T cells from mice deficient in DRAK2 hyperproliferate, produce higher levels of cytokines, and mobilize greater levels of calcium when compared with wild-type T cells. DRAK2 is developmentally regulated, with low levels of expression in immature thymocytes and high levels in mature thymocytes, and analysis of DRAK2 knock-out mice has shown that this kinase negatively regulates the T cell activation threshold in developing thymocytes (3.Friedrich M.L. Wen B.G. Bain G. Kee B.L. Katayama C. Murre C. Hedrick S.M. Walsh C.M. Int. Immunol. 2005; 17: 1379-1390Crossref PubMed Scopus (22) Google Scholar). Although double positive (immature) thymocytes from Drak2-/- mice mobilize calcium in a manner similar to thymocytes from wild-type mice, mature, single positive thymocytes from Drak2-/- mice have enhanced calcium responses to suboptimal TCR stimulation. Drak2-/- mice have also been reported to possess B cells with a similarly diminished activation threshold (2.McGargill M.A. Wen B.G. Walsh C.M. Hedrick S.M. Immunity. 2004; 21: 781-791Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Recent data have also shown that Drak2-/- mice have defective germinal center formation, demonstrating a critical role for DRAK2 in adaptive humoral immunity. 5A. Al-Qahtani and P. Casali, personal communication. Like other members of the DAP kinase family, DRAK2 is capable of both auto- and trans-phosphorylation (4.Sanjo H. Kawai T. Akira S. J. Biol. Chem. 1998; 273: 29066-29071Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Kinase activity is abolished in a C-terminal truncation mutant and when the critical lysine of the ATP-binding site is mutated (4.Sanjo H. Kawai T. Akira S. J. Biol. Chem. 1998; 273: 29066-29071Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). The first two members of the DAP kinase family, DAPK1 and DRP-1 kinase, both have autophosphorylation sites at Ser308 (Refs. 5.Shohat G. Spivak-Kroizman T. Cohen O. Bialik S. Shani G. Berrisi H. Eisenstein M. Kimchi A. J. Biol. Chem. 2001; 276: 47460-47467Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar and 6.Shani G. Henis-Korenblit S. Jona G. Gileadi O. Eisenstein M. Ziv T. Admon A. Kimchi A. EMBO J. 2001; 20: 1099-1113Crossref PubMed Scopus (63) Google Scholar, respectively). These autophosphorylation sites exist within their calmodulin regulatory domains, a domain absent from DRAK2. Additionally, six autophosphorylation sites have been defined in ZIP kinase (7.Graves P.R. Winkfield K.M. Haystead T.A. J. Biol. Chem. 2005; 280: 9363-9374Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), the third member of the DAP kinase family. In many cases, these sites of autophosphorylation have been shown to play key roles in modulating the function of these DAP kinases (8.Bialik S. Kimchi A. Annu. Rev. Biochem. 2006; 75: 189-210Crossref PubMed Scopus (356) Google Scholar). Engagement of the TCR or B cell antigen receptor results in rapid recruitment of various signaling molecules to the receptor complex to transmit signals generated at the cell a of intracellular activation results in calcium Annu. Rev. Immunol. 2001; PubMed Scopus Google Scholar). is generated to TCR and B cell antigen receptor stimulation, to in the and that the Annu. Rev. Immunol. 2001; PubMed Scopus Google Scholar). intracellular have been calcium in the calcium in calcium the this has been shown to the of the protein and the protein T cell activation J. 2005; PubMed Scopus Google Scholar, O. 2006; PubMed Scopus Google Scholar, M. S. S. A. 2006; PubMed Scopus Google Scholar, S. M. A. J. 2005; PubMed Scopus Google Scholar). calcium a role in and because DRAK2 for calcium in T cells and thymocytes, we to the and the that regulates DRAK2 function. In an to the regulation of DRAK2 kinase activity we have employed mass spectrometry to identify sites of autophosphorylation within the of this kinase. two sites have been and has been in primary a phospho-specific In the we show that DRAK2 activity is induced rapidly following antigen receptor and that phosphorylation is regulated by intracellular calcium that of of these sites to autophosphorylation the activity of DRAK2 in primary T cells. was the from at serine to was the and to acid the The to at was the was from was as (3.Friedrich M.L. Wen B.G. Bain G. Kee B.L. Katayama C. Murre C. Hedrick S.M. Walsh C.M. Int. Immunol. 2005; 17: 1379-1390Crossref PubMed Scopus (22) Google Scholar). protein was from cells by in by in The was for at and to with The with was by the for in glutathione in In Kinase was at with kinase and of of as a Kinase by the of the by and by analysis of DRAK2 kinase activity in cells, cells with DRAK2 or by the calcium cells in high and was from cell antibody for in in the of to in kinase as in with for mass by and by The and by and the to analysis as C. C. P. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). the by liquid to a tandem mass The was in an in each was by the for that tandem mass for data and protein the of C. C. P. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). The mass for and as and The was as by and phosphorylation of and was as The within the of P.R. M. 2005; Full Text Full Text PDF PubMed Scopus Google was used to and the results from The identified phosphorylated by of the from the at to the and of each phosphorylated an by and PubMed Scopus Google Scholar, C. S. D. PubMed Scopus Google Scholar). by with a to Ser12 of DRAK2. The by protein and and was as (3.Friedrich M.L. Wen B.G. Bain G. Kee B.L. Katayama C. Murre C. Hedrick S.M. Walsh C.M. Int. Immunol. 2005; 17: 1379-1390Crossref PubMed Scopus (22) Google Scholar). protein by and to antibody was at in antibody was for at in as Walsh C.M. J. Immunol. 2005; PubMed Scopus Google Scholar). and from mice with or for in low by with and for at To the of the Ab, from cells with or of was with or of or phosphorylated Ser12 for to and with to mice have been (2.McGargill M.A. Wen B.G. Walsh C.M. Hedrick S.M. Immunity. 2004; 21: 781-791Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Drak2-/- and mice and in with the regulation of the and at the of used between and of and T cells with for the by and from to mice and with by with for the and mature B cells from to cells was between and was for the in with and of as in the or of BAPTA-AM thapsigargin with or calcium stimulation was following a and and cells, and retroviral as (3.Friedrich M.L. Wen B.G. Bain G. Kee B.L. Katayama C. Murre C. Hedrick S.M. Walsh C.M. Int. Immunol. 2005; 17: 1379-1390Crossref PubMed Scopus (22) Google Scholar). T cells from Drak2-/- mice for and at and following primary stimulation retroviral in the of in each and a calcium as (3.Friedrich M.L. Wen B.G. Bain G. Kee B.L. Katayama C. Murre C. Hedrick S.M. Walsh C.M. Int. Immunol. 2005; 17: 1379-1390Crossref PubMed Scopus (22) Google Scholar), that cells with and with of in an the regulation of DRAK2 kinase activity following antigen receptor stimulation, we to sites of Our was that in the of to the phosphorylation of DRAK2 in primary the autophosphorylation sites of DAPK1 and DRP-1 are not in the DRAK2 we employed mass spectrometry to unique sites of autophosphorylation in DRAK2. first demonstrated activity for DRAK2 by in kinase DRAK2 kinase the C-terminal truncation of acid and the mutant from and activity was to in kinase in the of Although the wild-type kinase was highly this activity upon the of the C-terminal regulatory domain and the ATP-binding lysine in the ATP-binding loop of the kinase These results with data protein from cells (4.Sanjo H. Kawai T. Akira S. J. Biol. Chem. 1998; 273: 29066-29071Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, M. M. H. D. C. H. H. J. Biochem. 2001; PubMed Scopus Google Scholar). To the sites of autophosphorylation a mass wild-type and DRAK2 and to in kinase The protein by by and liquid The identified phosphorylated are in phosphorylated identified in the of two phosphorylation sites in the regions of the protein and the whereas the other sites in the DRAK2 of the mass spectrometry is shown in ion from the of DRAK2 was and was as a phosphorylated of the on the in the of was the identified as phosphorylated higher the and in a In a of to the of was for the of phosphorylated serine or These results that the phosphorylation on at from the of this to in the DRAK2 of autophosphorylation sites in wild-type and mutant of are shown at the for from and the acid within the in each wild-type and mutant the of phosphorylation within the for each each the phosphorylated serine is by within the identified by found in found in DRAK2 not for this not for this in a In we have two phosphorylated of the The was as whereas the two phosphorylated with the as are in respectively). shown in a of and and in the of the the as This the of the DRAK2 the in the and the as the whereas in these suggesting that the from not and phosphorylation to the In for the and higher their shown in that these The of a of to the of their phosphorylated the phosphorylation of at from The unique ion and ion the In all of the higher the in The ion in was higher the in and suggesting that phosphorylation at from In the of and the ion in that the at the of the at from was the phosphorylation of two and Ser12 in the of in the was in a of phosphorylation sites found in the of and To these analysis of a mutant was an in kinase and of the not phosphorylated To that was no phosphorylation in the mutant the in we to both and the from the and the extracted ion of each ion in both wild-type and mutant no phosphorylation in the mutant the but not the analysis on and Ser12 to potential phosphorylation in the mutant The of the DRAK2 first generated from the of both to and to the of with the that two in the analysis shown in identified that the with at at was the is in both To or Ser12 phosphorylated in the we compared the of the from both and Although these two have the at These the and two on the and the and Ser12 and Although the was from the no phosphorylated in the or and we that Ser12 phosphorylated in the In no other phosphorylation was in this mutant in phosphorylation, that this mutant kinase no phosphorylation was found in the truncation mutant not To investigate the potential function of two of the identified phosphorylation of to was at Ser12 and The single and as was in the two and mass analysis was used to identify phosphorylation phosphorylation sites identified for the and the a phosphorylation site at was in to the phosphorylation of and phosphorylation of and was not in The for phosphorylation at these two in the is autophosphorylation is necessary for protein kinase activity, of Ser12 or to to kinase activity of both Ser12 and in also to DRAK2 autophosphorylation to phosphorylation at other sites in the kinase. at these autophosphorylation sites not the of DRAK2 to these results phosphorylation of Ser12 and is for DRAK2 auto- and of in in phosphorylation of DRAK2, to wild-type DRAK2 or the mutant in cells, by a Although we activity of wild-type DRAK2 in kinase with the mutant was with reported results (4.Sanjo H. Kawai T. Akira S. J. Biol. Chem. 1998; 273: 29066-29071Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, M. M. H. D. C. H. H. J. Biochem. 2001; PubMed Scopus Google Scholar). The and mutant to in kinase and the by and both and Ser12 phosphorylation identified in wild-type DRAK2 by tandem mass To phosphorylation in the we extracted the of the or in the of from both the mutant and wild-type DRAK2 the of the two higher in DRAK2 in suggesting that DRAK2 on these two that a of phosphorylation on and Ser12 was for the mutant in cells that expression of DRAK2, is that or other kinases may also DRAK2 on the two sites in To this cells with the inhibitor for to to potential in with in a for both wild-type and mutant DRAK2, suggesting that other kinases within cells are capable of DRAK2 in To and Ser12 may to trans-phosphorylation by other kinases in cells, these DRAK2 with and in phosphorylation. of and Ser12 demonstrated that both in the two following the of the two higher for wild-type DRAK2 as compared with of the mutant This that or other kinases may these two in DRAK2 in cells. we to detect or Ser12 in cells with the demonstrating that of in DRAK2 phosphorylation in the of on the activity of the kinase. the of the and Ser12 no other phosphorylation in wild-type DRAK2 or in the is that the other phosphorylation may to or that the high of in may have to phosphorylation in kinase as of DRAK2 to of the sites of we the of the identified by of DRAK2 various Our was that autophosphorylation site Indeed, all sites identified by are in and DRAK2 of these sites are within the kinase domain, but are within an acid N-terminal domain and in the acid C-terminal regulatory domain of the kinase Although the identified sites are not to phosphorylated in other DAP family kinases with defined autophosphorylation sites and Ser12 on DRAK2 with DAP kinase are two of DAPK1 identified by at J.H. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and at S. 2004; PubMed Scopus Google Scholar). Additionally, ZIP kinase, the member of the DAP family of kinases, was found to phosphorylated by DAPK1 in and in upon in cells G. D. Bialik S. G. Kimchi A. Biol. 2004; PubMed Scopus Google Scholar). of the the DAP kinase phosphorylation sites of all revealed a Ser12 on DRAK2 The Ser12 and the phosphorylation sites of ZIP kinase, and are each by a serine and or for and or a for ZIP This to that autophosphorylation at Ser12 is Although this is from an of the these results that DRAK2 has at an for other DAP kinase phosphorylation of a DRAK2 of the autophosphorylation sites phospho-specific to Ser12 and to these phosphorylation To that both phospho-specific specific for DRAK2, we from the B cell and of and Drak2-/- mice by the and DRAK2 in wild-type and cells but to detect these in Drak2-/- demonstrating that both are highly specific for DRAK2. of wild-type and cells with induced a as in This enhanced phosphorylation also on the Ser12 because the signal to the with this To the of these cells with or with and a DRAK2 that to the The antibody to detect DRAK2 in cells the demonstrating that this antibody is highly specific for the antibody not to because was capable of to the mutant DRAK2. an to the of the Ab, we a of or used to the of the with to cell blocked of by the at all when at a of to the to of by the a of the of this Ab, we with of to or the by the of the of by antibody of the antibody that the both the and higher in this in following is that of both by the but the higher by the This Ser12 phosphorylation in also to phosphorylation at a of other has not been to the activation of of DRAK2 in DRAK2 has been found to modulate T and B cell activation in response to stimulation, we to DRAK2 autophosphorylation is by antigen receptor stimulation. we phosphorylation in Jurkat T cells following TCR Jurkat cells a low of DRAK2, whereas with enhanced DRAK2 Ser12 autophosphorylation a for T cell the was and with the to detect Although we found that primary T cells a high of DRAK2 phosphorylation, T cells phosphorylation stimulation of these thymocytes with to of DRAK2 phosphorylation. these with to TCR signaling. Although the of DRAK2 phosphorylation in Jurkat cells and thymocytes we that the activation in these are potential in the and of activation in T cells, these data that DRAK2 phosphorylation on Ser12 is induced by TCR stimulation. DRAK2 is also highly expressed in B cells (2.McGargill M.A. Wen B.G. Walsh C.M. Hedrick S.M. Immunity. 2004; 21: 781-791Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 3.Friedrich M.L. Wen B.G. Bain G. Kee B.L. Katayama C. Murre C. Hedrick S.M. Walsh C.M. Int. Immunol. 2005; 17: 1379-1390Crossref PubMed Scopus (22) Google Scholar), we to phosphorylation B cell activation. B cell antigen receptor with DRAK2 was phosphorylated at this with levels of stimulation DRAK2 autophosphorylation to levels in resting B cells. In phosphorylation was of stimulation. These results that DRAK2 is rapidly in B cells following antigen receptor this of activation for the protein kinase of for DRAK2 in activity has been shown to regulated by the B protein H. J. M. H. H. J. Biochem. PubMed Scopus Google Scholar). is that DRAK2 regulates calcium in T cells (2.McGargill M.A. Wen B.G. Walsh C.M. Hedrick S.M. Immunity. 2004; 21: 781-791Abstract Full Text Full Text PDF PubMed Scopus (55) Google and thymocytes (3.Friedrich M.L. Wen B.G. Bain G. Kee B.L. Katayama C. Murre C. Hedrick S.M. Walsh C.M. Int. Immunol. 2005; 17: 1379-1390Crossref PubMed Scopus (22) Google Scholar), we to autophosphorylation by the high intracellular calcium levels that following activation. we to the of following with a inhibitor of calcium J. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Although T cells a high of with thapsigargin enhanced DRAK2 phosphorylation This also enhanced phosphorylation, with results demonstrating that of protein kinase activation J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). In B cells, thapsigargin similarly to high DRAK2 phosphorylation on Ser12 In both cell the of thapsigargin was dependent on of because the intracellular BAPTA-AM blocked the of DRAK2 and phosphorylation. To of intracellular was necessary for DRAK2 phosphorylation induced by antigen receptor B cells with in the or of the and BAPTA-AM extracellular whereas BAPTA-AM in intracellular these data that DRAK2 activation on intracellular of DRAK2 investigate the role of Ser12 autophosphorylation in modulating DRAK2 activity in lymphocytes, we to Drak2-/- T cells with retroviral wild-type and mutant of DRAK2. similar has been employed to investigate the role of auto- and trans-phosphorylation of the kinase in primary T cells J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (63) Google Scholar). activation and cell we first to Drak2-/- T cells to TCR upon following the activation and necessary for retroviral Indeed, suboptimal stimulation of Drak2-/- T cells following activation for retroviral to enhanced expression when compared with wild-type cells DRAK2 signals from the TCR in both and T cells. Drak2-/- T cells with and to induce with the retroviral P. Biochem. 2001; PubMed Scopus Google Scholar), and following a the cells for calcium T cells with the calcium and with and and with a suboptimal of to calcium with the no on Drak2-/- T cells, as by between and with to compared with cells with the retroviral Drak2-/- cells with the levels of suggesting that phosphorylation at Ser12 is necessary for activity of DRAK2. cells in the expression found to possess similar demonstrating that the diminished in cells was a of that similar results have been in wild-type primary T cells and Jurkat cells wild-type and DRAK2 suggesting that DRAK2 or with signaling in T cells. Although a role for DRAK2 in negatively suboptimal signals that TCR has been the that DRAK2 function to Using mass we have identified sites of in phosphorylation, two of on the kinase activity of DRAK2. have used a phospho-specific antibody to autophosphorylation in and data show that DRAK2 autophosphorylation is dependent on of following antigen receptor in we have demonstrated that phosphorylation at Ser12 has important because a at this site the of DRAK2 to with following TCR stimulation. data that DRAK2 is by intracellular mobilization, and because DRAK2 in T cells and thymocytes, data a in this kinase in a negative feedback In a data that DRAK2 as a to TCR signaling is from intracellular Although we to sites of DRAK2 autophosphorylation by of from cells, to we to first these sites Although this was for an of various that a of sites identified in DRAK2 kinase that found to To the of we of these data to of each the sites found within and Ser12 phosphorylated when expressed in cells of these sites are in all DRAK2 suggesting that both play key roles in the activity of the kinase. Although revealed of DRAK2 in we to identify these specific sites to the and of mass in the of phosphorylated and in the and of analysis by mass spectrometry are required to the in phosphorylation of DRAK2. phosphorylation, by other regulatory kinases, may modulate the activity of DRAK2 following antigen receptor stimulation, or other results that DRAK2 is to autophosphorylation on and because a mutant kinase activity to these sites in In resting Jurkat T cells, primary thymocytes and B cells, DRAK2 is phosphorylated at low levels of these with antigen receptor to rapid phosphorylation on as by the T cells demonstrated a high of Ser12 phosphorylation is this is to an or may a of activation in resting mature T cells from lymphoid of this is that DRAK2 Ser12 phosphorylation is in by stimulation of To the regulation of DRAK2 we found that calcium from intracellular was for inducing phosphorylation at Ser12 in primary B and T cells. of with calcium calcium in the and results in of calcium the to an increase in Ser12 phosphorylation, whereas BAPTA-AM this Ser12 phosphorylation following with thapsigargin in the of the not we extracellular calcium does not DRAK2 phosphorylation, intracellular calcium is both necessary and for inducing DRAK2 autophosphorylation on Ser12 in primary In studies with DRAK2 have demonstrated that protein kinase activity in response to a key in activity in T cells H. J. M. H. H. J. Biochem. PubMed Scopus Google Scholar, S. A. PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). on analysis we that to DRAK2 autophosphorylation in protein negatively DRAK2 activity in response to mobilization, and this regulation is in lymphocytes, to DRAK2 is to phosphorylation on at two phosphorylation of Ser12 has important The diminished the of DRAK2 to following TCR This that this site may for the of DRAK2 to following T cell activation. These defects in DRAK2 function not to diminished kinase activity, because the mutant activity as by in kinase that phosphorylation at and at other may necessary for DRAK2 to in following antigen receptor Ser12 not with kinase activity, is that this site to in of potential to the kinase or may in of the of DRAK2 autophosphorylation at Ser12 of in lymphoid cells. Like other DAP kinase family DRAK2 is to on data this autophosphorylation is regulated in with other kinases in this family, we have demonstrated that autophosphorylation the function of DRAK2. Although we have found in the of phosphorylation in B cells T cells, of to the of DRAK2 activity in B and T cell is that other immune may the activity of DRAK2, to important DRAK2 an important role in T cell the of regulation in primary in modulating immune