Binding of phosphatidic acid to the protein-tyrosine phosphatase SHP-1 as a basis for activity modulation

Activation of the SH2 domain-possessing protein-tyrosine phosphatase SHP-1 by acidic phospholipids as phosphatidic acid (PA) has been described earlie r and suggested to participate in regulation of SHP-1 activity toward cellu lar substrates. The mechanism of this activation is poorly understood. Dire ct binding of phosphatidic acid to recombinant SHP-1 could be demonstrated by measuring the extent of [C-14]PA binding in a chromatographic assay, by measuring the extent of binding of SHP-1 to PA-coated ELISA plates or silic a beads (TRANSIL), and by spectroscopic assays employing fluorescently labe led PA liposomes. In addition to PA, phosphatidylinositol 3,4,5-trisphospha te (PIP3), dipalmitoylphosphatidylglycerol, phosphatidylinositol 4,5-bispho sphate, and phosphatidylserine (PS) were found to bind to SHP-1, albeit to a lesser extent. A high-affinity binding site for PA and PIP3 was mapped to the 41 C-terminal amino acids of SHP-1. This site was absent from the rela ted protein-tyrosine phosphatase SHP-2 and conferred activation of SHP-1 by PA toward two different substrates at low lipid concentrations. A SHP-1 mu tant missing this binding site could, however, still be activated toward ph osphorylated myelin basic protein as a substrate at high PA concentrations. This activation is likely to be mediated by a second, low-affinity binding site for PA in the N-terminal part of SHP-1 within the SH2 domains. High-a ffinity phospholipid binding to the C-terminus of SHP-1 may present a speci fic mechanism of regulating activity and/or cellular localization.