It has been proposed that phosphoinositides and inositol phosphates serve as general ligands for members of the structurally related pleckstrin homology (PH) domain family. The N-terminal PH domain of pleckstrin (N-PH), in contrast with other PH domains, does not bind to any of these ligands with the high affinity expected for a physiological interaction. To examine whether N-PH might instead mediate protein-protein interaction, a fusion protein with glutathione S-transferase (GST) expressing N-PH (GST-N-PH) was used to screen [35S]methionine metabolically labelled HL-60 and Bac1.2F5 cell lysates for potential binding partners. A 30 kDa binding protein was identified in both cell lines. Binding to N-PH demonstrated specificity, because binding was approx. 10-fold higher than when an equimolar amount of pleckstrin C-terminal PH domain (GST-C-PH) was used as probe. The 30 kDa protein could also be metabolically labelled with [32P]Pi and proved to be a tyrosine-phosphorylated protein. Binding to N-PH could be specifically inhibited with phosphotyrosine but not with phosphothreonine; the inhibition was concentration-dependent. Site-directed mutagenesis indicated that a positively charged region previously identified as the phosphoinositide-binding site in N-PH and other PH domains, rather than a putative phosphotyrosine-binding region previously identified in structurally similar phosphotyrosine-binding (PTB) domains, served as the binding site. These results suggest that the positively charged region of N-PH has the potential to interact with a protein ligand that contains phosphotyrosine.
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Research Article|
August 24 1999
Phosphotyrosine protein of molecular mass 30 kDa binds specifically to the positively charged region of the pleckstrin N-terminal pleckstrin homology domain
Limin LIU;
Limin LIU
1Department of Biochemistry, State University of New York Health Science Center at Brooklyn, 450 Clarkson, Brooklyn, NY 11203, U.S.A.
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Mary MAKOWSKE
Mary MAKOWSKE
1
1Department of Biochemistry, State University of New York Health Science Center at Brooklyn, 450 Clarkson, Brooklyn, NY 11203, U.S.A.
1To whom correspondence should be addressed (mmakowske@netmail.hscbklyn.edu).
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Publisher: Portland Press Ltd
Received:
April 14 1999
Revision Received:
May 26 1999
Accepted:
June 25 1999
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1999
1999
Biochem J (1999) 342 (2): 423–430.
Article history
Received:
April 14 1999
Revision Received:
May 26 1999
Accepted:
June 25 1999
Citation
Limin LIU, Mary MAKOWSKE; Phosphotyrosine protein of molecular mass 30 kDa binds specifically to the positively charged region of the pleckstrin N-terminal pleckstrin homology domain. Biochem J 1 September 1999; 342 (2): 423–430. doi: https://doi.org/10.1042/bj3420423
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