Leukocyte transmigration is modulated by chemokine‐mediated PI3Kγ‐dependent phosphorylation of vimentin

Phosphoinositide 3‐kinase γ (PI3Kγ) plays a fundamental role in mediating leukocyte migration to inflammation sites. However, the downstream cytoplasmic events triggered by its signaling activity are still largely obscure. To address this issue, tyrosine and serine/threonine phosphorylated proteins of chemokine‐stimulated WT or PI3Kγ‐null macrophages were investigated. Among the proteins analyzed, the intermediate filament vimentin was found as a downstream effector of the PI3Kγ signaling pathway. Specific analysis of the phosphorylation state of vimentin in macrophages showed that this protein becomes rapidly phosphorylated in both tyrosine and serine residues upon chemokine stimulation. In the absence of PI3Kγ or the kinase activity of PI3Kγ (PI3Kγ^KD/KD), phosphorylation of vimentin was reduced. PI3Kγ‐null macrophages displayed impaired chemokine‐driven vimentin fiber disassembly as well as reduced ability to transmigrate across endothelial cells. While WT macrophages infected with a vimentin mutant resistant to N‐terminal serine phosphorylation showed a reduction in transendothelial migration, infection of PI3Kγ‐null macrophages with a vimentin mutant mimicking serine phosphorylation of N‐terminal residues rescued the transendothelial migration defect. These results define vimentin N‐terminal phosphorylation and fiber reorganization as a target of chemokine‐dependent PI3Kγ signaling in leukocytes.