Heterotrimeric G-proteins, as well as small GTPases of the Rho and ADP-ribosylation factor (ARF) family, are implicated in the regulation of lipid kinases, including PtdIns 4-kinases and PtdIns(4)P 5-kinases. Here, we describe a PtdIns 4-kinase activity on immature secretory granules (ISGs), regulated secretory organelles formed from the trans-Golgi network (TGN), and investigate the regulation of PtdIns4P levels on these membranes. Over 50% of the PtdIns 4-kinase activity on ISGs is inhibited by both a low concentration of adenosine and the monoclonal antibody 4C5G, a specific inhibitor of the type II PtdIns 4-kinase. Treatment of ISGs with mastoparan 7 (M7) stimulates the type II PtdIns 4-kinase via pertussis-toxin-sensitive Gi/G0 proteins, which, in contrast with previous results obtained with chromaffin granules [Gasman, Chasserot-Golaz, Hubert, Aunis and Bader (1998) J. Biol. Chem. 273, 16913–16920], does not require Rho A, B or C. M7 treatment also leads to an inhibition in the recruitment of ARF to ISG membranes: this inhibition is not dependent on Gi/G0 activation, and is not linked to the stimulation of PtdIns 4-kinase observed with M7. PtdIns 4-kinase activity on ISGs is not regulated by myristoylated ARF1—GTP, in contrast with results obtained with Golgi membranes [Godi, Pertile, Meyers, Marra, Di Tullio, Iurisci, Luini, Corda and De Matteis (1999) Nat. Cell Biol. 1, 280–287; Jones, Morris, Morgan, Kondo, Irvine and Cockcroft (2000) J. Biol. Chem. 275, 13962–13170], whereas ARF1—GTP does regulate the production of PtdIns(4,5)P2. Our results suggest that the regulation of PtdIns 4-kinase on the ISGs differs in comparison with that on the TGN, and might be related to a specific requirement of ISG maturation.
Abbreviations used: AP-1, adaptor protein-1; ARF, ADP-ribosylation factor; BAMC, bovine adrenal medulla cytosol; GDP[βS], guanosine [β-thio]diphosphate; GTP[γS], guanosine [γ-thio]triphosphate; HB, homogenization buffer; (I/M)SGs, (immature/mature) secretory granules; mAb, monoclonal antibody; M7/M17, mastoparan 7 or 17 respectively; mARF-1, myristoylated ARF-1; PT, pertussis toxin; SCRM7, scrambled M7; TGN, trans-Golgi network.
Present address: The Bloomsbury BioSeed Fund, Brooke House, 2–16 Torrington Place, London, WC1E 7HN, U.K.
Cancer Research UK London Research Institute comprises the Lincoln's Inn Fields and Clare Hall Laboratories of the former Imperial Cancer Research Fund (ICRF), following the merger of the ICRF with the Cancer Research Campaign in February 2002.