Regulation Of GTP-binding Proteins

Heart, Lung, And Blood Institute

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Abstract

ADP-ribosylation factors (ARFs) are GTP-binding proteins that regulate numerous vesicular trafficking pathways. ARF function requires the regulated alternation between GTP-bound active and GDP-bound inactive forms. GTP binding is catalyzed by guanine nucleotide-exchange proteins (GEPs), some of which are inhibited by brefeldin A (BFA, a drug that inhibits protein sectretion and causes reversible disintegration of Golgi cisternae) and some of which are BFA-resistant. Two BFA-inhibited GEPs (BIG1 and BIG2) had been purified and cloned by the group. Endogeneous BIG1 and BIG2 often precipitated together from cultured cells with specific antibodies, but they behaved quite differently in yeast two-hybrid experiments, where BIG1 interacted with FKBP13 and BIG2 with RIalpha, a regulatory subunit of cyclic AMP-activated protein kinase (PKA). Three sequences characteristic of A kinase-anchoring proteins (AKAPs), were identified in the BIG2 molecule. Work in progress revealed that both BIG1 and BIG2, and ARF accumulate in nuclei of cells incubated with a cAMP analogue, and these effects were prevented by PKA inhibition. These findings are consistent with a role for BIG2 in coordinating cAMP and ARF regulatory pathways. FKBP13 is an immunophilin of the FK506-binding protein family that does not bind or inhibit calcineurin. Incubation of Jurkat cells with FK506, a widely used immunosuppresant drug, increased binding of BIG1, BIG2, and ARF to Golgi and other membranes. Endogenous BIG1 in HepG2 cells accumulated in nuclei of cells incubated for 18h without serum and was further increased (and largely absent from Golgi) after 30 min exposure to FK506, which also caused movement of FKBP13 into nuclei, but not colocalized with BIG1. In serum-starved cells, BIG1 was identified in nucleoli, nuclear matrix, and pore structures. Alterations in BIG1 location, and probably functions, appear to be associated with cell cycle events, as well as exogenous signals. Characterization of RNAs associated with BIG1 in nucleoli is in progress.