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

P. Várnai, Kristina I. Rother, Tamas Balla

Research output: Contribution to journalArticle

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Abstract

Phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) has been proposed to act as a second messenger to recruit regulatory proteins to the plasma membrane via their pleckstrin homology (PH) domains. The PH domain of Bruton's tyrosine kinase (Btk), which is mutated in the human disease X- linked agammaglobulinemia, has been shown to interact with PI(3,4,5)P3 in vitro. In this study, a fusion protein containing the PH domain of Btk and the enhanced green fluorescent protein (BtkPH-GFP) was constructed and utilized to study the ability of this PH domain to interact with membrane inositol phospholipids inside living cells. The localization of expressed BtkPH-GFP in quiescent NIH 3T3 cells was indistinguishable from that of GFP alone, both being cytosolic as assessed by confocal microscopy. In NIH 3T3 cells coexpressing BtkPH-GFP and the epidermal growth factor receptor, activation of epidermal growth factor or endogenous platelet-derived growth factor receptors caused a rapid (2. This mutant, but not wild- type BtkPH-GFP, interfered with agonist-induced PI(4,5)P2 hydrolysis in COS- 7 cells. These results show in intact cells that the PH domain of Btk binds selectively to 3-phosphorylated lipids after activation of PI 3-kinase enzymes and that losing such binding ability or specificity results in gross abnormalities in the function of the enzyme. Therefore, the interaction with PI(3,4,5) P3 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 changes in this phospholipid in living cells.

Original languageEnglish
Pages (from-to)10983-10989
Number of pages7
JournalJournal of Biological Chemistry
Volume274
Issue number16
DOIs
Publication statusPublished - Apr 16 1999

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Phosphatidylinositol 3-Kinase
Cells
Association reactions
Membranes
NIH 3T3 Cells
Chemical activation
Platelet-Derived Growth Factor Receptors
Confocal microscopy
COS Cells
Second Messenger Systems
Enzymes
Cell membranes
Phosphatidylinositols
Phosphatidylinositol 3-Kinases
Epidermal Growth Factor Receptor
Epidermal Growth Factor
Confocal Microscopy
Hydrolysis
Phospholipids
Proteins

ASJC Scopus subject areas

  • Biochemistry

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Phosphatidylinositol 3-kinase-dependent membrane association of the Bruton's tyrosine kinase pleckstrin homology domain visualized in single living cells. / Várnai, P.; Rother, Kristina I.; Balla, Tamas.

In: Journal of Biological Chemistry, Vol. 274, No. 16, 16.04.1999, p. 10983-10989.

Research output: Contribution to journalArticle

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