title: Src family kinases mediate receptor-stimulated, phosphoinositide 3-kinase-dependent, tyrosine phosphorylation of dual adaptor for phosphotyrosine and 3-phosphoinositides-1 in endothelial and B cell lines.
abstract: DAPP-1 (dual-adaptor for phosphotyrosine and 3-phosphoinositides-1) is a broadly distributed pleckstrin homology (PH) and Src homology 2 domain containing protein that can bind phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and can be phosphorylated on tyrosine 139 and internalised in response to activation of type I phosphoinositide 3-kinases (PI3K). Tyrosine phosphorylation of DAPP-1 appears important for appropriate intracellular targeting and creates a potential binding site for Src homology 2 domain-containing proteins. In endothelial cells overexpressing wild-type platelet-derived growth factor beta (PDGFbeta) receptors, which express Bmx and Src as their major Btk (Bruton's tyrosine kinase) family and Src family tyrosine kinases, respectively, PDGF can stimulate PI3K-dependent tyrosine phosphorylation of DAPP-1. Transient overexpression of Src most effectively, compared with Bmx and Syk, augments basal and PDGF-stimulated tyrosine phosphorylation of DAPP-1, whereas overexpression of dominant-negative Src, but not dominant-negative Bmx, inhibits PDGF-stimulated phosphorylation of DAPP-1. Cells expressing mutant PDGFbeta (Y579F/Y581F) receptors (which fail to bind and activate Src-type kinases) fail to tyrosine phosphorylate DAPP-1 in response to PDGF. We show that in DT40 chicken B cell lines, antibody stimulation leads to PI3K-dependent tyrosine phosphorylation of DAPP-1 that is lost in Lyn- or Syk-deficient cell lines but not Btk-deficient cell lines. PI3K-dependent activation of PKB is only lost in Syk-deficient lines. Finally, in vitro we find lipid-modified Src to be the most effective DAPP-1 tyrosine kinase (versus Syk, Lyn, Btk, and Bmx); phosphorylation of DAPP-1 but not Src autophosphorylation is stimulated approximately 10-fold by PtdIns(3,4,5)P(3) (IC(50) = 150 nm) and phosphatidylinositol 3,4-bisphosphate but not by their nonbiological diastereoisomers and depends on PH domain mediated binding of DAPP-1 to PtdIns(3,4,5)P(3)-containing membranes. We conclude that Src family kinases are responsible for tyrosine phosphorylation of DAPP-1 in vivo and that PI3K regulation is at the level of PH domain-mediated translocation of DAPP-1 to PI3K products in the membrane.
authors.affiliation: Inositide Laboratory, Signalling Programme, The Babraham Institute, Babraham, Cambridge CB2 4AT, United Kingdom. len.stephens@bbsrc.ac.uk
authors.forename: L R
authors.initials: LR
authors.lastname: Stephens
authors.forename: K E
authors.initials: KE
authors.lastname: Anderson
authors.forename: P T
authors.initials: PT
authors.lastname: Hawkins
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism; D001665:Binding Sites; D001798:Blood Proteins / Q000378:metabolism; D002460:Cell Line; D002645:Chickens; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005227:Fatty Acids / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D020128:Inhibitory Concentration 50; D007313:Insecta; D008074:Lipoproteins; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D017479:Receptors, Platelet-Derived Growth Factor / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism; D018909:src Homology Domains; D019061:src-Family Kinases / Q000378:metabolism* / Q000502:physiology*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D001798:Blood Proteins; C486555:DAPP1 protein, human; D005227:Fatty Acids; D008074:Lipoproteins; D010716:Phosphatidylinositols; D010982:Platelet-Derived Growth Factor; D011994:Recombinant Proteins; D014443:Tyrosine; D004247:DNA; D005982:Glutathione Transferase; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; D017479:Receptors, Platelet-Derived Growth Factor; D019061:src-Family Kinases