Abstract
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, Tyr248, 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.
Highlights
Bruton’s tyrosine kinase (Btk),1 which is expressed in B cells and cells of the myeloid lineage, was initially identified as the target of mutations responsible for X-linked agammaglobulinemia in humans [1, 2]
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
Expression and Purification of BAP/TFII-I and BAP/TFII-I Fragments—The overall strategy used in these phosphorylation site mapping studies was 1) to determine which portions of BAP/TFII-I are phosphorylated by Btk in vitro; 2) to define the specific tyrosine residues phosphorylated by Btk in vitro by a combination of phosphopeptide mapping and site-directed mutagenesis; and 3) to verify that the same tyrosine residues are hyperphosphorylated in vivo upon coexpression of BAP/TFII-I with active Btk
Summary
Bruton’s tyrosine kinase (Btk), which is expressed in B cells and cells of the myeloid lineage, was initially identified as the target of mutations responsible for X-linked agammaglobulinemia in humans [1, 2]. Btk null mice exhibit a phenotype resembling xid, suggesting that B cell development has a more stringent requirement for Btk in humans than in mice [7,8,9,10]. Upon engagement of the B cell receptor, Btk is phosphorylated by the tyrosine kinase Lyn at residue Tyr551. This permits Btk to undergo autophosphorylation at residue Tyr223, after which kinase activity is increased [22]. BAP is identical to the putative transcription factor TFII-I [27], which was identified by its ability to stimulate transcription from initiator elements [27] and its synergy with Phox I and serum response factor in enhancing transcription from the c-fos promoter [28]
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