LCK PHOSPHORYLATES THE ACTIVATION LOOP TYROSINE OF THE ITK KINASE DOMAIN AND ACTIVATES ITK KINASE-ACTIVITY

The Tec family tyrosine kinase Itk has been implicated in T cell recep tor (TCR) signaling, yet its precise role and mechanism of activation remain undefined. To investigate these issues, we examined the biochem ical response of Itk to TCR stimulation. We found that Itk is tyrosine -phosphorylated after TCR cross-linking and that this phosphorylation depends on the presence of functional Lck. To determine if this Lck de pendence results from direct phosphorylation of Itk by Lck, we generat ed recombinant Itk and Lck using a baculovirus expression system and u sed these proteins in subsequent biochemical analyses, We found that L ck phosphorylates Itk upon co-expression in insect cells and, further, that this phosphorylation of Itk results in increased Itk in vitro ki nase activity. The major site of Lck phosphorylation on Itk was mapped to the conserved tyrosine (Tyr(511)) in the activation loop of the It k kinase domain. Substitution of this tyrosine with phenylalanine abol ishes Itk kinase activity in insect cells, indicating that phosphoryla tion at this site plays a critical role in regulating Itk function.