Phosphatidylinositol 3-kinase-dependent membrane association of the Bruton's tyrosine kinase pleckstrin homology domain visualized in single living cells

Phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P-3) has been proposed t o act as a second messenger to recruit regulatory proteins to the plasma me mbrane via their pleckstrin homology (PH) domains. The PH domain of Bruton' s tyrosine kinase (Btk), which is mutated in the human disease X-linked aga mmaglobulinemia, has been shown to interact with PI(3,4,5)P-3 in vitro. In this study, a fusion protein containing the PH domain of Btk and the enhanc ed green fluorescent protein (BtkPH-GFP) was constructed and utilized to st udy the ability of this PH domain to interact with membrane inositol phosph olipids inside living cells. The localization of expressed BtkPH-GFP in qui escent NIH 3T3 cells was indistinguishable from that of GFP alone, both bei ng cytosolic as assessed by confocal microscopy. In NIH 3T3 cells coexpress ing BtkPH-GFP and the epidermal growth factor receptor, activation of epide rmal growth factor or endogenous platelet-derived growth factor receptors c aused a rapid (<3 min) translocation of the cytosolic fluorescence to ruffl e-like membrane structures. This response was not observed in cells express ing GFP only and was completely inhibited by treatment with the PI 3-kinase inhibitors wortmannin and LY 292004, Membrane-targeted PI 3-kinase also ca used membrane localization of BtkPH-GFP that was slowly reversed by wortman nin. When the R28C mutation of the Btk PH domain, which causes X-linked aga mmaglobulinemia, was introduced into the fluorescent construct, no transloc ation was observed after stimulation. Tn contrast, the E41K mutation, which confers transforming activity to native Btk, caused significant membrane l ocalization of BtkPH-GFP with characteristics indicating its possible bindi ng to PI(4,5)P-2. This mutant, but not wild-type BtkPH-GFP, interfered with agonist-induced PI(4,5)P-2 hydrolysis in COS-7 cells. These results show i n intact cells that the PH domain of Btk binds selectively to 3-phosphoryla ted lipids after activation of PI 3-kinase enzymes and that losing such bin ding ability or specificity results in gross abnormalities in the function of the enzyme. Therefore, the interaction with PI(3,4,5)P-3 is likely to be an important determinant of the physiological regulation of Btk and can be utilized to visualize the dynamics and spatiotemporal organization of chan ges in this phospholipid in living cells.