title: Effect of protein kinase A on accumulation of brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1) in HepG2 cell nuclei.
abstract: Brefeldin A-inhibited guanine nucleotide-exchange proteins, BIG1 and BIG2, are activators of ADP-ribosylation factor GTPases that are essential for regulating vesicular traffic among intracellular organelles. Biochemical analyses and immunofluorescence microscopy demonstrated BIG1 in nuclei as well as membranes and cytosol of serum-starved HepG2 cells. Within 20 min after addition of 8-Br-cAMP, BIG1 accumulated in nuclei, and this effect was blocked by protein kinase A (PKA) inhibitors H-89 and PKI, suggesting a dependence on PKA-catalyzed phosphorylation. BIG2 localization was not altered by cAMP, nor did BIG2 small interfering RNA influence nuclear accumulation of BIG1 induced by cAMP. Mutant BIG1 (S883A) in which Ala replaced Ser-883, a putative PKA phosphorylation site, did not move to the nucleus with cAMP addition, whereas replacement with Asp (S883D) resulted in nuclear accumulation of BIG1 without or with cAMP exposure, consistent with the mechanistic importance of a negative charge at that site. Mutation (712KPK714) of the nuclear localization signal inhibited BIG1 accumulation in nuclei, and PKA-catalyzed phosphorylation of S883, although necessary, was not sufficient for nuclear accumulation, as shown by the double mutation S883D/nuclear localization signal. A role for microtubules in cAMP-induced translocation of BIG1 is inferred from its inhibition by nocodazole. Thus, two more critical elements of BIG1 molecular structure were identified, as well as the potential function of microtubules in a novel PKA effect on BIG1 translocation.
authors.affiliation: Pulmonary-Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA. citteric@nhlbi.nih.gov
authors.forename: Carmen
authors.initials: C
authors.lastname: Citterio
authors.forename: Heather D
authors.initials: HD
authors.lastname: Jones
authors.forename: Gustavo
authors.initials: G
authors.lastname: Pacheco-Rodriguez
authors.forename: Aminul
authors.initials: A
authors.lastname: Islam
authors.forename: Joel
authors.initials: J
authors.lastname: Moss
authors.forename: Martha
authors.initials: M
authors.lastname: Vaughan
mesh_terms: D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D019943:Amino Acid Substitution; D002462:Cell Membrane / Q000737:chemistry; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism*; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D017868:Cyclic AMP-Dependent Protein Kinases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003600:Cytosol / Q000737:chemistry; D020662:Guanine Nucleotide Exchange Factors / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D009154:Mutation; D015739:Nocodazole / Q000494:pharmacology; D019913:Nuclear Localization Signals / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology; D014407:Tumor Cells, Cultured
chemical_list: C119087:ARFGEF1 protein, human; C119088:ARFGEF2 protein, human; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D020662:Guanine Nucleotide Exchange Factors; D019913:Nuclear Localization Signals; C517834:PRKAR1A protein, human; D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; D015124:8-Bromo Cyclic Adenosine Monophosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D015739:Nocodazole