Regulation of the Tyrosine Kinase Pyk2 by Calcium Is through Production of Reactive Oxygen Species in Cytotoxic T Lymphocytes

Pyk2 was identified as a Ca²⁺-dependent kinase, however, the regulation of Pyk2 by Ca²⁺ in T cells remains controversial. We found that Ca²⁺ mobilization preferentially induced Pyk2 phosphorylation in cytotoxic T lymphocytes (CTL). Furthermore, Pyk2 phosphorylation in CTL was not absolutely Ca²⁺ 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 Ca²⁺-regulated association between Pyk2 and calmodulin. Ca²⁺-stimulated Pyk2 phosphorylation was dependent on Src-family kinase activity, even at the Pyk2 autophosphorylation site. We sought to identify a Ca²⁺-regulated pathway that could trigger Pyk2 phosphorylation in T cells and found that ionomycin stimulated the production of reactive oxygen species and an H₂O₂ scavenger inhibited ionomycin-induced Pyk2 phosphorylation. Additionally, H₂O₂ induced strong Erk activation and ionomycin-stimulated Pyk2 phosphorylation was Erk dependent. These data support the conclusion that Ca²⁺ 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 Ca²⁺-dependent kinase in T cells but instead, increased intracellular Ca²⁺ 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 Ca²⁺ flux and/or reactive oxygen species to amplify tyrosine phosphorylation signaling events.