ABSTRACT
Signalling through the leptin receptor has been shown to activate the SH2 domain-containing tyrosine phosphatase SHP-2 through tyrosine phosphorylation. The human leptin receptor contains five tyrosine residues in the cytoplasmic domain that may become phosphorylated. We show here using BIAcore studies, wherein binding of peptides to SHP-2 was detected, that peptides corresponding to sequences containing phosphotyrosines 974 and 986 (LR974P and LR986P, respectively) from the leptin receptor cytoplasmic domain were the only two peptides that bound to the enzyme. Binding of LR974P to SHP-2 was inhibited in a dose-dependent fashion by orthovanadate, whereas binding of LY986P was not, indicating that the enzyme binds to these peptides through different sites. Only the leptin receptor-derived peptide corresponding to tyrosine 974 was dephosphorylated by recombinant purified SHP-2. Time courses of the reaction were complex, and fitted a two exponent rate equation. Preincubation of SHP-2 with LR986P markedly activated the enzyme at early time points and time courses of the activated enzyme fitted a single exponential first order rate equation. We propose that LR974P binds to the active site of SHP-2, whereas LR986P may bind to the N- and C-terminal SH2 domains of SHP-2, thus activating the phosphatase activity. These data support a model in which SHP-2 binds to phosphotyrosine 986 in the activated leptin receptor and is activated to dephosphorylate phosphotyrosine 974, downregulating signalling events emanating from SH2 domain-containing proteins that bind here.
Subject(s)
Carrier Proteins/physiology , Protein Processing, Post-Translational , Protein Tyrosine Phosphatases/metabolism , Receptors, Cell Surface , Binding Sites , Carrier Proteins/chemistry , Catalytic Domain , Enzyme Activation , Escherichia coli , Humans , Intracellular Signaling Peptides and Proteins , Kinetics , Models, Chemical , Neuroblastoma/pathology , Peptide Fragments/metabolism , Phosphorylation , Phosphotyrosine/chemistry , Phosphotyrosine/pharmacology , Protein Binding/drug effects , Protein Conformation/drug effects , Protein Structure, Tertiary , Protein Tyrosine Phosphatase, Non-Receptor Type 11 , Protein Tyrosine Phosphatase, Non-Receptor Type 6 , Receptors, Leptin , Recombinant Fusion Proteins/metabolism , SH2 Domain-Containing Protein Tyrosine Phosphatases , Signal Transduction , Tumor Cells, Cultured , Vanadates/pharmacology , src Homology DomainsABSTRACT
Platelet-derived growth factor-stimulated actin rearrangement and edge ruffle formation have previously been shown to be dependent on activation of phosphatidylinositol 3'-kinase, the activity of which also is important for directed migration of cells. This lipid kinase binds to phosphorylated tyrosine residues Y740 and Y751 in the kinase insert of the human platelet-derived growth factor ss-receptor. We examined the role of two other tyrosine residues in the kinase insert of this receptor, Y775 and Y778, for ligand-induced actin rearrangement. Both were shown to be phosphorylation sites; Y775 was only marginally phosphorylated in cells expressing the wild-type ss-receptor, whereas Y778 was phosphorylated at higher stoichiometry. Mutant receptors Y775F, Y778F and Y775/778F were active kinases and mediated proliferative responses when expressed in porcine aortic endothelial cells. Fluorescence staining of actin in platelet-derived growth factor-stimulated PAE cells revealed that Y778 is involved in regulation of the actin cytoskeleton since the cells contained, apart from edge ruffles and circular ruffles, a novel type of giant ruffle on the dorsal side of the cell, which consisted of irregular multilayered actin structures. Mutation at Y778 had no effect on activation of phosphatidylinositol 3'-kinase, nor on the GTPase activating protein of Ras and phospholipase C(gamma), and the extent of directed migration towards platelet-derived growth factor of these cells was not changed. We conclude that actin rearrangement is regulated in part by Y778 in the platelet-derived growth factor ss-receptor, potentially through binding of a novel signaling molecule to this site.
