Calmodulin and PI(3,4,5)P₃ cooperatively bind to the Itk pleckstrin homology domain to promote efficient calcium signaling and IL-17A production

Precise regulation of the kinetics and magnitude of Ca²⁺ signaling enables this signal to mediate diverse responses, such as cell migration, differentiation, vesicular trafficking, and cell death. We showed that the Ca²⁺-binding protein calmodulin (CaM) acted in a positive feedback loop to potentiate Ca²⁺ signaling downstream of the Tec kinase family member Itk. Using NMR (nuclear magnetic resonance), we mapped CaM binding to two loops adjacent to the lipid-binding pocket within the Itk pleckstrin homology (PH) domain. The Itk PH domain bound synergistically to Ca²⁺/CaM and the lipid phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P₃], such that binding to Ca²⁺/CaM enhanced the binding to PI(3,4,5)P₃ and vice versa. Disruption of CaM binding attenuated Itk recruitment to the membrane and diminished release of Ca²⁺ from the endoplasmic reticulum. Moreover, disruption of this feedback loop abrogated Itkdependent production of the proinflammatory cytokine IL-17A (interleukin-17A) by CD4⁺ T cells. Additionally, we found that CaM associated with PH domains from other proteins, indicating that CaM may regulate other PH domain-containing proteins.