Regulation Of GTP-binding Proteins

Heart, Lung, And Blood Institute

Investigators

Linked publications & trials

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 secretion and causes reversible disintegration of Golgi cisternae. Two BFA-inhibited GEPs (BIG1 and BIG2) that were purified and cloned earler by the group often precipitated together from cultured cells with specific antibodies, but they behaved quite differently in yeast two-hybrid screens, where BIG1 interacted with FKBP13 and BIG2 with RIalpha, a regulatory subunit of cyclic AMP-activated protein kinase (PKA). FKBP13 is an immunophilin of the FK506-binding protein family that does not bind or inhibit calcineurin. Incubation of Jurkat cells with FK506 resulted in nuclear accumulation of both FKBP13 and BIG1, but they were not colocalized. In addition, BIG1 moved into nuclei in cells deprived of serum, where it was demonstrated in nuclear matrix and nucleoli as well as associated with nucleoporin. Characterization of RNAs associated with BIG1 in nuclei is continuing after interruption caused by unexpected departure of the responsible post-doctoral fellow before arrival of his replacement. Investigation of the RIalpha-BIG2 interaction led to identification of sequences corresponding to those in A kinase-anchoring proteins in the BIG2 molecules, as reported last year. A manuscript in preparation characterizes protein kinase A-dependent effects of cyclic AMP on nuclear accumulation of endogenous BIG1 and other proteins. Exploring the interaction of BIG2 with exocyst protein Exo70 (detected in the yeast two-hybrid system) led to demonstration of the two proteins in trans-Golgi network and centrosomes, as well as in exocyst structures or complexes that move along microtubules to the plasma membrane. All findings are consistent with their functional association in both early and late stages of vesicular transport.