Cell Cycle-dependent Subcellular Localization of Exchange Factor Directly Activated by cAMP

Vsevolod L. Popov,Jingbo Qiao,Xiaodong Cheng,Fang C. Mei,Leoncio A. Vergara

doi:10.1074/jbc.m203571200

Vsevolod L. Popov, Jingbo Qiao + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m203571200

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Abstract

Epac belongs to a new family of proteins that can directly mediate the action of the intracellular second messenger cAMP by activating a downstream small GTPase Rap1. The Epac/Rap1 pathway represents a novel cAMP-signaling cascade that is independent of the cAMP-dependent protein kinase (PKA). In this study, we have used fluorescence microscopy to probe the intracellular targeting of Epac during different stages of the cell division cycle and the structural features that are important for Epac localization. Our results suggest Epac, endogenous or expressed as a green fluorescent protein fusion protein, is mainly localized to the nuclear membrane and mitochondria during interphase in COS-7 cells. Deletion mutagenesis analysis reveals that whereas the DEP domain is responsible for membrane association, the mitochondrial-targeting sequence is located at the N terminus. Although Epac predominantly exhibits perinuclear localization in interphase, the subcellular localization of Epac is cell cycle-dependent. Epac disassociates from the nuclear membrane and localizes to the mitotic spindle and centrosomes in metaphase. At the end of the cell cycle, Epac is observed to reassociate with the nuclear envelope and concentrate around the contractile ring. Furthermore, overexpression of Epac in COS-7 cells leads to an increase in multinuclear cell populations. These results suggest that Epac may play an important role in mitosis.

Highlights

Epac belongs to a new family of proteins that can directly mediate the action of the intracellular second messenger cAMP by activating a downstream small GTPase Rap1
The search for a new cAMP receptor has recently revealed a novel class of signaling molecules, Epacs or cAMP-GEFs, which can directly mediate the effects of cAMP intracellularly [11, 12]
Unlike PKA, in which regulatory and catalytic components are encoded by two separate genes, the cAMP-binding domain (CBD) of Epac is directly fused with the catalytic component, the GEF domain as a single polypeptide chain, in a manner similar to that of cGMP-dependent protein kinase

Summary

EXPERIMENTAL PROCEDURES

Constructs and Reagents—Human Epac cDNA was a gift from Dr J. C-terminal, GFP-tagged Epac (Epac-EGFP) was constructed by PCR using a 5Ј primer (5Ј-ccatatgctagcATGGTGTTGAGAAGGATGCAC-3Ј) and a 3Ј primer (5Ј-tcatatagagctcCTGGCTCCAGCTCTCGGGAGAG3Ј.) The amplified cDNA fragment was subcloned into the NheI-SacI sites of eukaryotic expression vector pEGFP-N3 (CLONTECH), in which the Epac gene was fused in-frame and upstream from the GFP gene. The day before transfection, cells were subcultured into a 6-well tissue culture plate, grown to 50 –75% confluency overnight, and transfected with purified plasmid DNA at 1 ␮g/well using the LipofectAMINE PlusTM reagent (Invitrogen). After incubation with the affinity-purified primary Epac antibody (2.5 ␮g/ml) for 1 h at room temperature, the cells were incubated with Alexa Fluor 488 goat anti-rabbit IgG secondary antibodies (5 ␮g/ml) for Epac. Fluorescence Microscopy—To detect the subcellular localization of Epac-EGFP, cells were subcultured after transfection with an appropriate plasmid in 6-well plates with a poly(L-lysine)-coated coverslip in each well and grown for 16 –24 h. Confocal images were recorded using an OZ Intervision video rate confocal laser-scanning microscope (Noran Instruments, Middleton, WI) with krypton/argon ion laser excitation at 488 nM and emission filter 525/52 BP

RESULTS

TABLE I Effects of Epac expression on cytokinesis

Total cellsa

DISCUSSION