Effect of 8‐Bromo‐cAMP on accumulation of brefeldin A‐inhibited guanine nucleotide‐exchange protein 1 (BIG1) in the HepG2 cell nucleus

Brefeldin A‐inhibited guanine nucleotide‐exchange proteins, BIG1 and BIG2, are activators of ADP‐ribosylation factor (ARF) GTPases that are essential in the molecular machinery regulating vesicular traffic among intracellular compartments. Biochemical analyses and immunofluorescence microscopy demonstrated BIG1 in nuclei as well as membranes and cytosol of serum‐starved cells. Within 20 min after addition of 8‐Br‐cAMP, BIG1 accumulated in HepG2 cell nuclei and this 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 siRNA influence nuclear accumulation of BIG1 induced by cAMP. Mutant BIG1 in which Ser883, a putative PKA phosphorylation site, was replaced with Ala (S883A) did not move to the nucleus with cAMP stimulation, whereas the 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 of the nuclear localization signal (NLS) prevented BIG1 accumulation, and PKA‐catalyzed phosphorylation of S883 was necessary, but not sufficient as shown by the double mutation S883D/NLS. 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 with consequences for cell function that remain to be described.