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 S883D͞ 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.ADP-ribosylation factor ͉ protein trafficking ͉ A kinase-anchoring protein M ammalian ADP-ribosylation factors (ARFs) comprise six 20-kDa GTP-binding proteins with essential regulatory roles in the formation of membrane trafficking vesicles. These vesicles bud from a donor membrane and fuse with a target membrane to deliver cargo molecules (1-3). Conversion of ARF from a cytosolic GDP-bound inactive state to a GTP-bound active form that is tightly membrane-associated is accelerated by guanine nucleotide-exchange factors (GEFs) (4). All known ARF GEFs contain a Sec7 domain of Ϸ200 aa that catalyzes ARF activation (5-7). The large-molecular-weight BIG1, BIG2, and GBF1, which localize in Golgi regions, are, with different sensitivities, inhibited by the fungal fatty acid metabolite brefeldin A (8-10). The Ϸ200-kDa BIG1 and Ϸ190-kDa BIG2 were first purified together from bovine brain cytosol on the basis of their BFA-inhibited GEF activities (11) and appeared to exist as parts of the same macromolecular complex(es) in HepG2 cells (12). When these cells were growing in medium with serum, BIG1 was primarily cytosolic and Golgi-associated. After overnight incubation without serum, a large fraction of endogenous BIG1 was found in the nuclei localized with nucleoporin p62 at the nuclear envelope (probably in transit between nucleus and cytoplasm), as well as in the nuclear matrix and nucleoli (13).The cAMP-dependent protein kinase A (PKA) is a tetrameric protein comprising two ca...

Interaction of phosphodiesterase 3A with brefeldin A-inhibited guanine nucleotide-exchange proteins BIG1 and BIG2 and effect on ARF1 activity

“…In other experiments, amounts of BIG1 and BIG2 in membrane fractions of cells incubated with okadaic acid to inhibit phosphatases were similarly decreased, without changes in nuclear fractions (15). As shown here, effective inhibition of PDE3A activity via its depletion with specific siRNA resulted in lesser amounts of BIG1 and BIG2 associated with membranes, most apparent at Golgi structures, consistent with reported effects of cAMP elevation (14,15). It seems likely that phosphorylation of BIG1 and BIG2 by PKA was enhanced as cAMP accumulated in the vicinity when hydrolysis by PDE3A diminished.…”

“…However, incubation of cells with 8-Br-cAMP resulted in nuclear accumulation of BIG1, (Fig. S4) as had been reported in HepG2 cells (14). This effect clearly did not resemble the wide cytoplasmic dispersion of BIG1 (and BIG2) seen after PDE3A depletion (Fig.…”

“…Our laboratory had found that incubation of HepG2 cells with 8-Br-cAMP resulted in partial dissociation of BIG1 and BIG2 from membranes and accumulation of BIG1 in nuclei (14). In other experiments, amounts of BIG1 and BIG2 in membrane fractions of cells incubated with okadaic acid to inhibit phosphatases were similarly decreased, without changes in nuclear fractions (15).…”

“…PKA-catalyzed phosphorylation of serine 883 in BIG1, was required for nuclear accumulation of the protein in HepG2 cells incubated with 8-Br-cAMP (14). The absence of nuclear accumulation of BIG2 in those cells was not surprising, because the BIG2 molecule contains no serine corresponding to BIG1 S883.…”

“…Consistent with the presence of AKAP sequences for each of the 4 R subunits in BIG1 and BIG2, both proteins were coimmunoprecipitated from HepG2 cell cytosol with antibodies against RI or RII, although direct interaction of the molecules was not established (13). PKAcatalyzed phosphorylation of endogenous BIG1 in HepG2 cells resulted in its rapid nuclear accumulation (14). Differences between mechanisms, and perhaps functions, of BIG1 accumulation in nuclei after cAMP elevation and after serum deprivation remain to be determined.…”