Gßγ regulates mitotic Golgi fragmentation and G2/M cell cycle progression.

Heterotrimeric G proteins (αßγ) function at the cytoplasmic surface of a cell's plasma membrane to transduce extracellular signals into cellular responses. However, numerous studies indicate that G proteins also play noncanonical roles at unique intracellular locations. Previous work has established that G protein ßγ subunits (Gßγ) regulate a signaling pathway on the cytoplasmic surface of Golgi membranes that controls the exit of select protein cargo. Now, we demonstrate a novel role for Gßγ in regulating mitotic Golgi fragmentation, a key checkpoint of the cell cycle that occurs in the late G2 phase. We show that small interfering RNA-mediated depletion of Gß1 and Gß2 in synchronized cells causes a decrease in the number of cells with fragmented Golgi in late G2 and a delay of entry into mitosis and progression through G2/M. We also demonstrate that during G2/M Gßγ acts upstream of protein kinase D and regulates the phosphorylation of the Golgi structural protein GRASP55. Expression of Golgi-targeted GRK2ct, a Gßγ-sequestering protein used to inhibit Gßγ signaling, also causes a decrease in Golgi fragmentation and a delay in mitotic progression. These results highlight a novel role for Gßγ in regulation of Golgi structure.

Inducible Inhibition of Gßγ Reveals Localization-dependent Functions at the Plasma Membrane and Golgi.

Heterotrimeric G proteins signal at a variety of endomembrane locations, in addition to their canonical function at the cytoplasmic surface of the plasma membrane (PM), where they are activated by cell surface G protein-coupled receptors. Here we focus on ßγ signaling at the Golgi, where ßγ activates a signaling cascade, ultimately resulting in vesicle fission from the trans-Golgi network (TGN). To develop a novel molecular tool for inhibiting endogenous ßγ in a spatial-temporal manner, we take advantage of a lipid association mutant of the widely used ßγ inhibitor GRK2ct (GRK2ct-KERE) and the FRB/FKBP heterodimerization system. We show that GRK2ct-KERE cannot inhibit ßγ function when expressed in cells, but recruitment to a specific membrane location recovers the ability of GRK2ct-KERE to inhibit ßγ signaling. PM-recruited GRK2ct-KERE inhibits lysophosphatidic acid-induced phosphorylation of Akt, whereas Golgi-recruited GRK2ct-KERE inhibits cargo transport from the TGN to the PM. Moreover, we show that Golgi-recruited GRK2ct-KERE inhibits model basolaterally targeted but not apically targeted cargo delivery, for both PM-destined and secretory cargo, providing the first evidence of selectivity in terms of cargo transport regulated by ßγ. Last, we show that Golgi fragmentation induced by ilimaquinone and nocodazole is blocked by ßγ inhibition, demonstrating that ßγ is a key regulator of multiple pathways that impact Golgi morphology. Thus, we have developed a new molecular tool, recruitable GRK2ct-KERE, to modulate ßγ signaling at specific subcellular locations, and we demonstrate novel cargo selectivity for ßγ regulation of TGN to PM transport and a novel role for ßγ in mediating Golgi fragmentation.