title: The phosphatidylinositol transfer protein RdgBβ binds 14-3-3 via its unstructured C-terminus, whereas its lipid-binding domain interacts with the integral membrane protein ATRAP (angiotensin II type I receptor-associated protein).
abstract: PITPs [PI (phosphatidylinositol) transfer proteins] bind and transfer PI between intracellular membranes and participate in many cellular processes including signalling, lipid metabolism and membrane traffic. The largely uncharacterized PITP RdgBβ (PITPNC1; retinal degeneration type B β), contains a long C-terminal disordered region following its defining N-terminal PITP domain. In the present study we report that the C-terminus contains two tandem phosphorylated binding sites (Ser(274) and Ser(299)) for 14-3-3. The C-terminus also contains PEST sequences which are shielded by 14-3-3 binding. Like many proteins containing PEST sequences, the levels of RdgBβ are regulated by proteolysis. RdgBβ is degraded with a half-life of 4 h following ubiquitination via the proteasome. A mutant RdgBβ which is unable to bind 14-3-3 is degraded even faster with a half-life of 2 h. In vitro, RdgBβ is 100-fold less active than PITPα for PI transfer, and RdgBβ proteins (wild-type and a mutant that cannot bind 14-3-3) expressed in COS-7 cells or endogenous proteins from heart cytosol do not exhibit transfer activity. When cells are treated with PMA, the PITP domain of RdgBβ interacts with the integral membrane protein ATRAP (angiotensin II type I receptor-associated protein; also known as AGTRAP) causing membrane recruitment. We suggest that RdgBβ executes its function following recruitment to membranes via its PITP domain and the C-terminal end of the protein could regulate entry to the hydrophobic cavity.
authors: Department of Neuroscience, Physiology and Pharmacology, University College London, London WC1E 6JJ, UK.
authors: Kathryn
authors: K
authors: Garner
authors: Michelle
authors: M
authors: Li
authors: Natalie
authors: N
authors: Ugwuanya
authors: Shamshad
authors: S
authors: Cockcroft
mesh_terms: D048948:14-3-3 Proteins / Q000235:genetics / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D026481:Blotting, Far-Western; D019556:COS Cells; D002522:Chlorocebus aethiops; D005455:Fluorescent Antibody Technique; D006801:Humans; D047468:Immunoprecipitation; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D048948:14-3-3 Proteins; C449676:AGTRAP protein, human; D048868:Adaptor Proteins, Signal Transducing; D026901:Membrane Transport Proteins; C107847:PITPNC1 protein, human; D013755:Tetradecanoylphorbol Acetate