Phosphotyrosine protein of molecular mass 30 kDa binds specifically to the positively charged region of the pleckstrin N-terminal pleckstrin homology domain.
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 [(35)S]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 [(32)P]P(i) 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.
Liu
L
L
Department of Biochemistry, State University of New York Health Science Center at Brooklyn, 450 Clarkson, Brooklyn, NY 11203, USA.
Makowske
M
M
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001798:Blood Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D018922:HL-60 Cells; D006801:Humans; D066298:In Vitro Techniques; D008024:Ligands; D008239:Lysine / Q000737:chemistry; D051379:Mice; D008958:Models, Molecular; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019000:Phosphotyrosine / Q000378:metabolism*; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D001798:Blood Proteins; D017931:DNA Primers; D008024:Ligands; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C039077:platelet protein P47; D019000:Phosphotyrosine; D008239:Lysine