title: Evidence that the tandem-pleckstrin-homology-domain-containing protein TAPP1 interacts with Ptd(3,4)P2 and the multi-PDZ-domain-containing protein MUPP1 in vivo.
abstract: PtdIns(3,4,5)P3 is an established second messenger of growth-factor and insulin-induced signalling pathways. There is increasing evidence that one of the immediate breakdown products of PtdIns(3,4,5)P3, namely PtdIns(3,4)P2, whose levels are elevated by numerous extracellular agonists, might also function as a signalling molecule. Recently, we identified two related pleckstrin-homology (PH)-domain-containing proteins, termed 'tandem-PH-domain-containing protein-1' (TAPP1) and TAPP2, which interacted in vitro with high affinity with PtdIns(3,4)P2, but did not bind PtdIns(3,4,5)P3 or other phosphoinositides. In the present study we demonstrate that stimulation of Swiss 3T3 or 293 cells with agonists that stimulate PtdIns(3,4)P2 production results in the marked translocation of TAPP1 to the plasma membrane. This recruitment is dependent on a functional PtdIns(3,4)P2-binding PH domain and is inhibited by wortmannin, a phosphoinositide 3-kinase inhibitor that prevents PtdIns(3,4)P2 generation. A search for proteins that interact with TAPP1 identified the multi-PDZ-containing protein termed 'MUPP1', a protein possessing 13 PDZ domains and no other known modular or catalytic domains [PDZ is postsynaptic density protein (PSD-95)/Drosophila disc large tumour suppressor (dlg)/tight junction protein (ZO1)]. We demonstrate that immunoprecipitation of endogenously expressed TAPP1 from 293-cell lysates results in the co-immunoprecipitation of endogenous MUPP1, indicating that these proteins are likely to interact with each other physiologically. We show that TAPP1 and TAPP2 interact with the 10th and 13th PDZ domain of MUPP1 through their C-terminal amino acids. The results of the present study suggest that TAPP1 and TAPP2 could function in cells as adapter proteins to recruit MUPP1, or other proteins that they may interact with, to the plasma membrane in response to signals that elevate PtdIns(3,4)P2.
authors.affiliation: MRC Protein Phosphorylation Unit, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, UK. w.a.kimber@dundee.ac.uk
authors.forename: Wendy A
authors.initials: WA
authors.lastname: Kimber
authors.forename: Laura
authors.initials: L
authors.lastname: Trinkle-Mulcahy
authors.forename: Peter C F
authors.initials: PC
authors.lastname: Cheung
authors.forename: Maria
authors.initials: M
authors.lastname: Deak
authors.forename: Louisa J
authors.initials: LJ
authors.lastname: Marsden
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Kieloch
authors.forename: Stephen
authors.initials: S
authors.lastname: Watt
authors.forename: Ronald T
authors.initials: RT
authors.lastname: Javier
authors.forename: Alex
authors.initials: A
authors.lastname: Gray
authors.forename: C Peter
authors.initials: CP
authors.lastname: Downes
authors.forename: John M
authors.initials: JM
authors.lastname: Lucocq
authors.forename: Dario R
authors.initials: DR
authors.lastname: Alessi
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D001798:Blood Proteins / Q000737:chemistry*; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D005347:Fibroblasts / Q000378:metabolism; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D015151:Immunoblotting; D007150:Immunohistochemistry; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins; D051379:Mice; D016253:Microscopy, Immunoelectron; D018384:Oxidative Stress; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry*; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors; D020798:Two-Hybrid System Techniques
chemical_list: D001426:Bacterial Proteins; D001798:Blood Proteins; D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; C111442:MPDZ protein, human; D008565:Membrane Proteins; C491750:Mpdz protein, mouse; C435927:PLEKHA1 protein, human; D018129:Phosphatidylinositol Phosphates; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C060980:phosphatidylinositol 3,4-diphosphate; C039077:platelet protein P47; C054562:yellow fluorescent protein, Bacteria; D006861:Hydrogen Peroxide; D005982:Glutathione Transferase; D019869:Phosphatidylinositol 3-Kinases