Identification of Phosphorylation Sites for Bruton's Tyrosine Kinase within the Transcriptional Regulator BAP/TFII-I

Bruton's tyrosine kinase (Btk), a member of the Tec family of cytosolic kinases, is essential for B cell development and function. BAP/TFII-I, a protein implicated in transcriptional regulation, is associated with Btk in B cells and is transiently phosphorylated on tyrosine following B cell receptor engagement. BAP/TFII-I is a substrate for Btk in vitro and is hyperphosphorylated on tyrosine upon coexpression with Btk in mammalian cells. In an effort to understand the physiologic consequences of BAP/TFII-I tyrosine phosphorylation following B cell receptor stimulation, site-directed mutagenesis and phosphopeptide mapping were used to locate the predominant sites of BAP/TFII-I phosphorylation by Btk in vitro. These residues, Tyr ²⁴⁸, Tyr³⁵⁷, and Tyr⁴⁶², were also found to be the major sites for Btk-dependent phosphorylation of BAP/TFII-I in vivo. Residues Tyr³⁵⁷ and Tyr⁴⁶² are contained within the loop regions of adjacent helix-loop-helix-like repeats within BAP/TFII-I. Mutation of either Tyr²⁴⁸, Tyr³⁵⁷, or Tyr⁴⁶² to phenylalanine reduced transcription from a c-fos promoter relative to wild-type BAP/TFII-I in transfected COS-7 cells, consistent with the interpretation that phosphorylation at these sites contributes to transcriptional activation. Phosphorylation of BAP/TFII-I by Btk may link engagement of receptors such as surface immunoglobulin to modulation of gene expression.