Different modes and qualities of tyrosine phosphorylation of Fak and Pyk2 during epithelial-mesenchymal transdifferentiation and cell migration: analysis of specific phosphorylation events using site-directed antibodies

Integrin signaling is activated during epithelial-mesenchymal transdifferen tiation (EMT) and cell migration, processes serving as models for carcinoge nesis, We have shown that paxillin and p130Cas become highly tyrosine phosp horylated during these processes in NMuMG cells. Here, we examined the regu lation of Fak and Pyk2, kinases implicated in this phosphorylation, Pyk2 be came phosphorylated at the major autophosphorylation site (Tyr-402) and the potential Grb2-binding site (Tyr-881) during EMT, In contrast, phosphoryla tion of Fak at the corresponding autophosphorylation site (Tyr-397) occurre d even in sedentary epithelial cells, whereas phosphorylation at Tyr-407 an d Tyr-861 was induced during EMT. During cell migration, these phosphorylat ion events, except Fak Tyr-397, were augmented further, and phosphorylation of Fak Tyr-577 and the corresponding Pyk2 Tyr-580, both within the kinase activation loops, was also induced. In all cases, phosphorylation of the pu tative Grb2-binding site in Fak (Tyr-925) was almost undetectable, Although Fak and Pyk2 have several phosphorylation sites in common, Tyr-407 and Tyr -861 are unique to Fak, Our results revealed that Fak and Pyk2 are non-equi valent in the tyrosine phosphorylation events and thereby likely to evoke d ifferent downstream signaling cascades during EMT and cell migration of NMu MG cells, We also show that Fak Tyr-397 phosphorylation occurs exclusively at the cytoplasm, but not at focal contacts, in the sedentary epithelial ce lls, In contrast, all other tyrosine phosphorylated forms of Fak and Pyk2 a re predominantly localized to focal adhesions and the cell periphery in mot ile cells, all colocalized with paxillin and p130Cas.