title: Regulation of the tyrosine kinase Pyk2 by calcium is through production of reactive oxygen species in cytotoxic T lymphocytes.
abstract: Pyk2 was identified as a Ca(2+)-dependent kinase, however, the regulation of Pyk2 by Ca(2+) in T cells remains controversial. We found that Ca(2+) mobilization preferentially induced Pyk2 phosphorylation in cytotoxic T lymphocytes (CTL). Furthermore, Pyk2 phosphorylation in CTL was not absolutely Ca(2+) dependent but relied on the strength of T cell receptor stimulation. Ionomycin-stimulated Pyk2 phosphorylation did not require calmodulin activity, because phosphorylation was not inhibited by the calmodulin inhibitor W7, and we detected no Ca(2+)-regulated association between Pyk2 and calmodulin. Ca(2+)-stimulated Pyk2 phosphorylation was dependent on Src-family kinase activity, even at the Pyk2 autophosphorylation site. We sought to identify a Ca(2+)-regulated pathway that could trigger Pyk2 phosphorylation in T cells and found that ionomycin stimulated the production of reactive oxygen species and an H(2)O(2) scavenger inhibited ionomycin-induced Pyk2 phosphorylation. Additionally, H(2)O(2) induced strong Erk activation and ionomycin-stimulated Pyk2 phosphorylation was Erk dependent. These data support the conclusion that Ca(2+) mobilization induces the production of reactive oxygen species, which in turn activate the Erk pathway, leading to Src-family kinase-dependent Pyk2 phosphorylation. Our data demonstrate that Pyk2 is not a Ca(2+)-dependent kinase in T cells but instead, increased intracellular Ca(2+) induces Pyk2 phosphorylation through production of reactive oxygen species. These findings are consistent with the possibility that Pyk2 acts as an early sensor of numerous extracellular signals that trigger a Ca(2+) flux and/or reactive oxygen species to amplify tyrosine phosphorylation signaling events.
authors.affiliation: Department of Medical Microbiology and Immunology, University of Alberta, Edmonton T6G 2S2, Canada.
authors.forename: Tara L
authors.initials: TL
authors.lastname: Lysechko
authors.forename: Samuel M S
authors.initials: SM
authors.lastname: Cheung
authors.forename: Hanne L
authors.initials: HL
authors.lastname: Ostergaard
mesh_terms: D000818:Animals; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects* / Q000502:physiology; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D051418:Focal Adhesion Kinase 2 / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology*; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D019169:Jurkat Cells; D051379:Mice; D016877:Oxidants / Q000378:metabolism / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D051381:Rats; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: D007476:Ionophores; D016877:Oxidants; D015759:Ionomycin; D006861:Hydrogen Peroxide; D051418:Focal Adhesion Kinase 2; C103547:PTK2B protein, human; C496053:Ptk2b protein, mouse; C496054:Ptk2b protein, rat; D019061:src-Family Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D002118:Calcium