Redox Regulation of the Calcium/Calmodulin-dependent Protein Kinases

Reactive oxygen intermediates (ROI) have been viewed traditionally as damaging to the cell. However, a predominance of evidence has shown that ROI can also function as important activators of key cellular processes, and ROI have been shown to play a vital role in cell signaling networks. The calcium/calmodulin-dependent protein kinases (CaM kinases) are a family of related kinases that are activated in response to increased intracellular calcium concentrations. In this report we demonstrate that hydrogen peroxide treatment results in the activation of both CaM kinase II and IV in Jurkat T lymphocytes. Surprisingly, this activation occurs in the absence of any detectable calcium flux, suggesting a novel means for the activation of these kinases. Treatment of Jurkat cells with phorbol 12-myristate 13-acetate (PMA), which does not cause a calcium flux, also activated the CaM kinases. The addition of catalase to the cultures inhibited PMA-induced activation of the CaM kinases, suggesting that similar to hydrogen peroxide, PMA also activates the CaM kinases via the production of ROI. One mechanism by which this likely occurs is through oxidation and consequential inactivation of cellular phosphatases. In support of this concept, okadaic acid and microcystin-LR, which are inhibitors of protein phosphatase 2A (PP2A), induced CaM kinase II and IV activity in these cells. Overall, these results demonstrate a novel mechanism by which ROI can induce CaM kinase activation in T lymphocytes. Reactive oxygen intermediates (ROI) have been viewed traditionally as damaging to the cell. However, a predominance of evidence has shown that ROI can also function as important activators of key cellular processes, and ROI have been shown to play a vital role in cell signaling networks. The calcium/calmodulin-dependent protein kinases (CaM kinases) are a family of related kinases that are activated in response to increased intracellular calcium concentrations. In this report we demonstrate that hydrogen peroxide treatment results in the activation of both CaM kinase II and IV in Jurkat T lymphocytes. Surprisingly, this activation occurs in the absence of any detectable calcium flux, suggesting a novel means for the activation of these kinases. Treatment of Jurkat cells with phorbol 12-myristate 13-acetate (PMA), which does not cause a calcium flux, also activated the CaM kinases. The addition of catalase to the cultures inhibited PMA-induced activation of the CaM kinases, suggesting that similar to hydrogen peroxide, PMA also activates the CaM kinases via the production of ROI. One mechanism by which this likely occurs is through oxidation and consequential inactivation of cellular phosphatases. In support of this concept, okadaic acid and microcystin-LR, which are inhibitors of protein phosphatase 2A (PP2A), induced CaM kinase II and IV activity in these cells. Overall, these results demonstrate a novel mechanism by which ROI can induce CaM kinase activation in T lymphocytes. The CaM kinases 1The abbreviations used are: CaM kinase or CaM-K, calcium/calmodulin-dependent protein kinase; CaM, calmodulin; CaM-KK, CaM kinase; PP, protein phosphatase; PMA, phorbol 12-myristate 13-acetate; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; BAPTA-AM, BAPTA-acetoxymethyl ester; ROI, reactive oxygen intermediates; H2O2, hydrogen peroxide; AP, alkaline phosphatase; BCIP, 5-bromo-4-chloro-3-indolyl phosphate; NBT, nitro blue tetrazolium; MPB, Nα-(3-maleimidylpropionyl)biocytin. are a family of related kinases activated in response to increased calcium levels (for review, see Refs. 1Hook S.S. Means A.R. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 471-505Crossref PubMed Scopus (415) Google Scholar, 2Nairn A.C. Picciotto M.R. Semin. Cancer Biol. 1994; 5: 295-303Crossref PubMed Scopus (21) Google Scholar, 3Soderling T.R. Trends Biochem. Sci. 1999; 24: 232-236Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar). The primary members of this family that demonstrate broad substrate specificity are CaM kinase I, II, and IV. Other family members are dedicated to the phosphorylation of a specific substrate (i.e. myosin light chain kinase, phosphorylase kinase, and elongation factor 2 kinase/CaM kinase III). All members of the CaM kinase family are activated in response to the binding of calcium/calmodulin, which causes a conformational change revealing the substrate binding domain of the kinase (1Hook S.S. Means A.R. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 471-505Crossref PubMed Scopus (415) Google Scholar). However, there are differences in the way each kinase is regulated. CaM-KII is a multimeric enzyme composed of 10–12 catalytic subunits (4Bulleit R.F. Bennett M.K. Molloy S.S. Hurley J.B. Kennedy M.B. Neuron. 1988; 1: 63-72Abstract Full Text PDF PubMed Scopus (107) Google Scholar, 5Soderling T.R. Fukunaga K. Brickey D.A. Fong Y.L. Rich D.P. Smith K. Colbran R.J. Prog. Brain Res. 1991; 89: 169-183Crossref PubMed Scopus (10) Google Scholar, 6Soderling T.R. Stull J.T. Chem. Rev. 2001; 101: 2341-2352Crossref PubMed Scopus (167) Google Scholar). Upon calcium/calmodulin binding, CaM-KII undergoes autophosphorylation in a region of the protein that prevents the autoinhibitory domain from interacting with the kinase domain. Once phosphorylated in this manner, CaM-KII activity becomes independent of calcium (7Colbran R.J. Schworer C.M. Hashimoto Y. Fong Y.L. Rich D.P. Smith M.K. Soderling T.R. Biochem. J. 1989; 258: 313-325Crossref PubMed Scopus (231) Google Scholar, 8Fong Y.L. Soderling T.R. J. Biol. 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Kemp B.E. J. Biol. Chem. 1996; 271: 10806-10810Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). CaM-KK is activated by calcium/calmodulin complexes as well, but the potential for other means of regulation has also been suggested (13Edelman A.M. Mitchelhill K.I. Selbert M.A. Anderson K.A. Hook S.S. Stapleton D. Goldstein E.G. Means A.R. Kemp B.E. J. Biol. Chem. 1996; 271: 10806-10810Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 14Anderson K.A. Means R.L. Huang Q.H. Kemp B.E. Goldstein E.G. Selbert M.A. Edelman A.M. Fremeau R.T. Means A.R. J. Biol. Chem. 1998; 273: 31880-31889Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 15Haribabu B. Hook S.S. Selbert M.A. Goldstein E.G. Tomhave E.D. Edelman A.M. Snyderman R. Means A.R. EMBO J. 1995; 14: 3679-3686Crossref PubMed Scopus (167) Google Scholar). Phosphorylation of CaM-KIV by CaM-KK occurs in the pseudosubstrate domain that interacts with the catalytic domain of the kinase. The phosphatase PP2A has been shown to form a complex with CaM-KIV and at least in vitro can cleave the phosphate group off CaM-KIV (16Park I.K. Soderling T.R. J. Biol. Chem. 1995; 270: 30464-30469Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 17Westphal R.S. Anderson K.A. Means A.R. Wadzinski B.E. Science. 1998; 280: 1258-1261Crossref PubMed Scopus (223) Google Scholar). This interaction is also believed to be important in vivo because transfection of cells with the PP2A inhibitor, SV40 small T antigen, is able to potentiate CaM-KIV of protein R.S. Anderson K.A. Means A.R. Wadzinski B.E. Science. 1998; 280: 1258-1261Crossref PubMed Scopus (223) Google Scholar). inhibitors of the phosphatases as okadaic acid and can both PP2A and through binding to the catalytic domain Y. Trends Biochem. Sci. 1990; Full Text PDF PubMed Scopus Google Scholar, K.A. S. 1990; 264: PubMed Scopus Google Scholar). hydrogen peroxide has also been shown to these this is believed to via the oxidation of a reactive in the catalytic domain of these phosphatases Biochem. Res. PubMed Scopus Google Scholar, R.L. M.A. Biochem. 1995; PubMed Scopus Google Scholar). of this phosphatase in the activation of the CaM kinases in the absence of an in intracellular The CaM kinases have been in T cell hydrogen and PMA-induced kinase activation and phosphorylation in Jurkat T lymphocytes. Treatment of T with hydrogen peroxide calcium in these cells similar to by to the T cell J.T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). PMA treatment of T is not to induce a calcium Surprisingly, PMA-induced activation is to be to and CaM kinase inhibitors K. S. T. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, K. T. 1998; PubMed Scopus Google Scholar). In we have that hydrogen phosphorylation not by of the cells with and CaM kinase inhibitors this response J.T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). results that activation of the CaM kinases is In this report we demonstrate the activation of both CaM-KII and CaM-KIV in the absence of in intracellular This activation is induced by ROI as as by we report that phosphatase inactivation can also to the activation of the CaM kinases. that both ROI and PMA can induce CaM kinase activation through the inactivation of phosphatases by and Jurkat T cell from and in with 2 and PMA, and from and in kinase protein and okadaic acid also from and protein kinase from kinase II, kinase and from from and in also from for in and in to and at for at least 1 to to the cells to to with hydrogen peroxide or PMA for the with or okadaic acid for In the catalase for to cells and the The cell in of 2 2 2 1 and each of the inhibitor, and to and for for at in a The to a and the of protein to of the from with for 2 at and and the in a for the kinase IV or II to the which a for 2 at to the and for an 1 at at and the in the The in of CaM kinase 1 of the used to of the kinase To with 2 of for and in The used in a kinase In of to the specificity of as by (16Park I.K. Soderling T.R. J. Biol. Chem. 1995; 270: 30464-30469Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). of the for CaM kinase II or IV activity in of a 2 protein kinase inhibitor, 2 protein kinase 1 microcystin-LR, of and substrate kinase activity and 1 activity in the of for each as as kinase the to for autophosphorylation The activity from the to the kinase The for at and by the addition of of of the kinase The in and a In of hydrogen peroxide to the CaM-KII or CaM-KIV to the of hydrogen peroxide the CaM kinases. of calcium by the cells with as J.T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). cells in and at in and at for and at 1 in and in a as in the and the a The for and at for and in One cells to and at for at least 1 to the of the with The in a for and the in of 1 and for with for at in a and the and with of and used in the through to to at in the for 2 with the primary in kinase II, kinase and in with alkaline phosphatase in for 1 at The in and with the and in and in to and at for at least 1 to with hydrogen peroxide or for in of 1 activity the phosphatase from This the of phosphate in a by the of a complex Biochem. PubMed Scopus Google Scholar, Biochem. PubMed Scopus Google Scholar). phosphate from the a for at the with of The phosphatase in a of PP2A 1 of of of the and of The at for and the by of the The at for to for and of the a with a with a and are as of of Res. 1989; PubMed Scopus Google Scholar, Biochem. PubMed Scopus Google Scholar). in and in to and at for at least 1 to with hydrogen peroxide or PMA for in of 1 of the by the protein the at a of of and at in the as of the in and in of with of and and to The at in with in for 1 The in and with the and and have suggested the for the activation of the CaM kinases in the absence of a calcium J.T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, K. S. T. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). To this we Jurkat cells in the and absence of 2 have shown that this of is to any calcium the cells and also calcium from the the of calcium J.T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the of of CaM kinase II and IV from these cells to the CaM substrate This kinase in the of calcium and to activity but also in the of to The of in the cultures to the role of calcium in the activation of the kinases. The of in the kinase the activity of the kinase; the kinase has activated and is of any calcium that CaM-KII and CaM-KIV activity increased with hydrogen peroxide both in the and absence of and in CaM kinase activity similar to the levels induced by a calcium However, hydrogen CaM kinase activity by the addition of to the hydrogen peroxide induce CaM kinase activity both in the or absence of a calcium flux, induced CaM kinase activity in the of a calcium and also the CaM kinase activity in these and the addition of calcium and to the kinase The levels of activity of both CaM kinase II and CaM kinase IV increased by the addition of and that the kinase activity in the can be to the CaM kinases. The addition of to the activation of CaM kinase II because of autophosphorylation Y.L. Soderling T.R. J. Biol. Chem. 1990; 265: 11091-11097Abstract Full Text PDF PubMed Google Scholar, R.J. Smith M.K. Schworer C.M. Fong Y.L. Soderling T.R. J. Biol. 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PMA does not cause a calcium in these cells as of the cells with PMA results in the activation of both CaM kinase II and IV and PMA does not cause a calcium flux, this activation must also be in a independent of in intracellular PMA also able to the CaM kinases in the of and as CaM kinase activity by the addition of to the cultures and suggesting that PMA-induced activation of the CaM kinases also occurs in the absence of in intracellular To this activity the of oxygen we Jurkat cells with PMA in the of shown to the activation of both CaM kinase II and IV by PMA and has cells that catalase is specific in results that PMA is via the production of oxygen In support of that causes the production of peroxide, is also able to both CaM kinase II and IV that both CaM kinase II and IV can be activated in a in response to activates the CaM kinases by a mechanism in the absence of a calcium Jurkat cells and in at a of each treatment of cells to cells with 2 for and or of catalase for and with PMA or for CaM kinase II and or CaM kinase IV and from cell and used in a kinase to the phosphorylation of the The of the a are as shown are the of activates the CaM kinases. Jurkat cells and in at a of each treatment of cells to with or for CaM kinase II or CaM kinase IV from cell and used in a kinase to the phosphorylation of the The of the a are as shown are the of of to potential mechanism for the in CaM kinase activity is the inactivation of phosphatases. the inhibitors okadaic acid or to the activation of both CaM kinase II and IV and of these inhibitors induce a calcium in these cells that can be by is an the T cell and as a for an intracellular calcium in these cells. to the results with hydrogen peroxide, has the of these inhibitors to induce kinase activity suggesting that activation of the CaM kinases by phosphatase inhibitors also occurs in a and have shown that hydrogen peroxide can cause the phosphorylation of the kinase J.T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). This phosphorylation inhibited by CaM kinase inhibitors but to the calcium and can also that the phosphatase inhibitors okadaic acid and induce the phosphorylation of results that of phosphatase activity is in to induce the of hydrogen peroxide and with the we have shown that phosphatase can the CaM kinases in a similar to treatment with hydrogen peroxide or CaM kinase are also phosphorylated phosphatase has been shown that hydrogen peroxide can phosphatase activity in other cell we this occurs in the Jurkat cell treatment with hydrogen peroxide, we the of to cleave the phosphate from a in phosphatase phosphate be by the of a and in a that treatment with hydrogen peroxide, the phosphatase activity in these this activity similar to the activity treatment with the phosphatase at a with of PP2A have shown that PP2A form a complex with CaM-KIV and can be with CaM-KIV R.S. Anderson K.A. Means A.R. Wadzinski B.E. 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PubMed Scopus Google Scholar). of the PP2A results in CaM-KIV and an to demonstrate that PP2A does with CaM-KIV also that an of PP2A with CaM-KIV of the treatment be that the PP2A in the in and the PP2A with CaM-KIV and likely a small of the PP2A in the cell. an to PP2A that hydrogen peroxide PP2A is PMA treatment also results in the oxidation of PP2A results catalase of PMA-induced CaM kinase This oxidation of PP2A can be both in the of PP2A that with CaM-KIV as as the cellular PP2A that CaM-KIV is also these To this change in oxidation CaM-KIV we hydrogen peroxide to CaM-KIV or CaM-KII kinase these a in kinase activity be that the oxidation of CaM-KIV or CaM-KII not the in activity both the phosphatase as as we can demonstrate that hydrogen peroxide causes the oxidation and inactivation of the phosphatase of this phosphatase is a potential mechanism for CaM kinase activation by oxidation of the CaM kinases does not in in Jurkat cells and in at a of each of cells to with in or for The cells. and cells used to CaM kinase II or CaM kinase IV. of CaM kinase II or IV from cells with hydrogen peroxide in for used in a kinase in the absence of calcium to the phosphorylation of the and have suggested the of a mechanism that activates the CaM kinases in the absence of a calcium J.T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, K. S. T. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). In this we this novel form of regulation of the CaM kinases. in vitro kinase we able to demonstrate that hydrogen peroxide can both CaM kinase II and IV Jurkat T cells To the of has that the CaM kinases are to in the cell. can cause a calcium in these we that this calcium with has the activation of the CaM kinases results are novel as an in intracellular calcium has been to be for the activation of these kinases. K. S. T. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google that PMA of activation in Jurkat cells and that this response inhibited by the addition of CaM kinase In this we demonstrate that PMA treatment activates both CaM kinase II and IV has been shown that PMA treatment of T cells in an in intracellular hydrogen peroxide as a Biol. 2001; PubMed Scopus Google Scholar). In treatment of cells with an R. 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In phosphatase and kinase activity in a cell. is that inactivation of phosphatases results in an in phosphorylation a cell. in kinase activity in increased protein phosphorylation in the absence of any change in phosphatase also be that a in phosphatase activity in the absence of any change in kinase activity also in the increased phosphorylation of a of phosphatases by reactive oxygen is an way for cells to the for the activation of signaling In reactive oxygen are J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, R. B. R. S. J. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. J. PubMed Scopus Google Scholar, J. S. Biol. PubMed Scopus Google Scholar). results in the phosphorylation of kinases, phosphatases are by reactive oxygen the for the activation of a is The of the activation be for the of and can be a of kinases have been of the T cell to the production of both and and both play a role in the activation of the T cell S. J. 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In PMA activation of the CaM kinases is also peroxide production and also occurs in the absence of a calcium This activation of the CaM kinases is likely because of the oxidation and inactivation of phosphatases the cell. results demonstrate a novel mechanism by which the CaM kinases can be results be for the activation of T in the any cellular in the production of oxygen can the CaM kinases in the absence of any calcium