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J. Biol. Chem., Vol. 277, Issue 29, 26581-26586, July 19, 2002

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

Jingbo Qiao, Fang C. Mei, Vsevolod L. Popov^§, Leoncio A. Vergara^¶, and Xiaodong Cheng

From the  Department of Pharmacology and Toxicology, Sealy Center for Structural Biology, the ^§ Department of Pathology, World Health Organization Collaborating Center for Tropical Diseases, and the ^¶ Department of Physiology and Biophysics, School of Medicine, The University of Texas Medical Branch, Galveston, Texas 77555

Received for publication, April 14, 2002, and in revised form, May 8, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cyclic adenosine 3',5'-monophosphate (cAMP) is the first intracellular second messenger to be discovered and plays an important role in mediating the actions of extracellular signals (1). cAMP has been implicated in regulating a myriad of cellular functions such as metabolism, proliferation, differentiation, secretion, and gene expression. For many years, the consensus was that cAMP-mediated signaling in eukaryotic cells existed as a linear pathway that involves the sequential activation of a series of signaling molecules. The cAMP signaling pathway consists of both plasma membrane and intracellular components. Upon binding of ligand, the membrane receptor at the cell surface transduces an extracellular signal across the cell membrane via stimulatory or inhibitory heterotrimeric G-proteins that interact with the membrane-bound adenylyl cyclase to regulate cAMP production inside the cell. It was believed until recently that all known effects of cAMP in mammalian cells, with the exception of cyclic nucleotide-gated channels in photoreceptor cells and olfactory sensory neurons (2), were mediated intracellularly by cAMP-dependent protein kinase (PKA).¹

The effects of cAMP on different cellular functions are often described as cell type-specific and dependent on biological response (3-10). These differential effects of cAMP on various cell functions suggest that more than one cAMP receptor may exist in mammalian cells. The search for a new cAMP receptor has recently revealed a novel class of signaling molecules, Epacs (exchange proteins directly activated by cAMP) or cAMP-GEFs (cAMP-regulated guanine nucleotide exchange factors), which can directly mediate the effects of cAMP intracellularly (11, 12). Like PKA, Epacs contain a regulatory cAMP-binding domain (CBD) that is homologous to the evolutionarily conserved CBD of Escherichia coli cAMP receptor protein. However, unlike PKA, in which regulatory and catalytic components are encoded by two separate genes, the 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. It is most likely that the CBD folds upon the GEF domain and prevents its interaction with downstream effectors. Binding of cAMP to Epac leads to a conformational change exposing the GEF domain, which in turn activates downstream targets. Although activated by the common second messenger cAMP, Epac and PKA have been shown to mediate opposing effects on protein kinase B (PKB) activation. The net outcome of cAMP signaling on PKB activation may be dependent upon the dynamic abundance and distribution of intracellular Epac and PKA (13). These results suggest the observed cell type-specific effects of cAMP can be partially explained by differential expression/distribution of Epac and PKA in different tissues/cells.

Epac/cAMP-GEF was initially cloned as a guanine nucleotide exchange factor for Rap1 (Ras-proximate), a small GTPase that shares greater than 50% identity with Ras (11, 12, 14). Rap1 and Ras are identical in the regions of the GTP-binding domain and the putative "effector" domain, suggesting Rap1 can interact with the same set of downstream effectors as Ras (15). Moreover, Rap1 can act as a suppressor of the Ras oncogene (16), leading to the hypothesis that Rap1 functions in vivo as a Ras antagonist (17, 18). However, more recent evidence points to an alternative situation in which Rap1 assumes distinct intracellular localizations and affects different signaling pathways (10, 19). Although the physiological functions of Epac are not clear at present, as one of the upstream regulators of Rap1 Epac may also be targeted to specific subcellular locations for its biological functions. Therefore, one of the keys to unlocking the mystery of Epac functions may lie in its specific cellular localization. In this study, we have probed the subcellular localization of Epac using fluorescence microscopy to address several fundamental questions regarding the physiological functions of Epac. These include the following. Where is Epac localized inside the cell for its cellular functions? What structural elements are responsible for the specific subcellular targeting of Epac? What are the roles of Epac in mediating the diverse cellular functions of cAMP in vivo?

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Constructs and Reagents-- Human Epac cDNA was a gift from Dr. J. L. Bos (University Medical Center, Utrecht, The Netherlands). C-terminal, GFP-tagged Epac (Epac-EGFP) was constructed by PCR using a 5' primer (5'-ccatatgctagcATGGTGTTGAGAAGGATGCAC-3') and a 3' primer (5'-tcatatagagctcCTGGCTCCAGCTCTCGGGAGAG-3'.) 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. By the same method, we constructed (1-148)Epac-EGFP, which lacks the first 148 N-terminal Epac amino acid residues. Mutant (72-148)Epac-EGFP lacking amino acids 72-148, which span the DEP domain, was constructed by inserting 1-71 Epac PCR fragments into the NheI site upstream from the (1-148)Epac-EGFP mutant. The specific oligonucleotide primers used to construct these deletion mutants were as follows: (1-148)Epac-EGFP, 5' primer (5'-catgctagcATGGTGGGAACTCATGAGATGGAG-3') and 3' primer (5'-tcatatagagctc -CTGGCTCCAGCTCTCGGGAGAG-3'); and (72-148)Epac-EGFP, 5' primer (5'-ccatatgctagcATGGTGTTGAGAAGGATGCAC-3') and 3' primer (5'-ccatatgctagcGAGGTTTGGGCAGGTGGCCA-3'). Cterminal, FLAG-tagged Epac (Epac-FLAG) was subcloned into the NheI-EcoRI sites of pcDNA3.1 mammalian expression vector by PCR using a 5' primer (5'-ccatatgctaGCATGGTGTTGAGAAGGATGCAC-3') and a 3' primer (5'-ttcggaattcTTATTTGTCGTCGTCTTTGTAGTCTGGCTCCAGCTCTCGGGAGA-3'). The uppercase roman and italic nucleotides represent coding sequences for Epac and FLAG-tag, respectively, and restriction sites are underlined. All DNA constructs were confirmed by DNA sequencing. Monoclonal anti--tubulin Cy3-conjugated clone TUB2.1-purified mouse IgG was from Sigma. Alexa Fluor 488 goat anti-rabbit IgG (H+L) and MitoTracker Red were obtained from Molecular Probes. All chemicals were reagent grade.

Cell Culture and Transfection-- COS-7 and HEK293 cells were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. The cultures were maintained at 37 °C in a humidified chamber supplemented with 5% CO_2. 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 µg/well using the LipofectAMINE Plus^TM reagent (Invitrogen). Stable transfectants were selected using G418.

Epac Antibody Production and Affinity Purification-- Specific polyclonal antibodies against Epac were generated by Alpha Diagnostic International Inc. (San Antonio, Texas) using the synthetic Epac peptide spanning residues 41-60, (C)DFSESLEQASTERVLRAGR. The immune sera were initially purified using an ImmunoPure IgG purification kit (Pierce) and further affinity-purified by immobilized Epac peptide using the SulfoLink antibody purification kit (Pierce). Antibody elution fractions were dialyzed in PBS and stored at 20 °C.

Immunofluorescent Staining-- Cells were grown on cover slips in 6-well plates overnight. The cells were fixed in 2% formaldehyde and 0.2% glutaraldehyde in PBS and permeabilized by 0.1% Triton X-100. After incubation with the affinity-purified primary Epac antibody (2.5 µg/ml) for 1 h at room temperature, the cells were incubated with Alexa Fluor 488 goat anti-rabbit IgG secondary antibodies (5 µg/ml) for Epac. The samples were examined by fluorescence microscopy.

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. Then the cells were fixed in 2% paraformaldehyde in PBS. The samples were rinsed with PBS, mounted on glass slides, and sealed in 70% glycerol. Fluorescent signals were revealed under the fluorescence microscope (Olympus BX51), using a FITC/GFP filter with maximum excitation at 488 nm and maximum emission at 525 nm. Fluorescence images were recorded using a Hamamatsu digital camera (C4742-95). 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

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Subcellular Targeting of Epac-- Epac/cAMP-GEF is a newly discovered intracellular cAMP receptor that is capable of transmitting signals directly from cAMP to downstream effector molecules. To probe the site of the cellular function of the Epac protein, we constructed an Epac-GFP fusion protein using the pEGFP-N3 vector. With the GFP tag at its C-terminal, the specific subcellular localization of Epac was monitored by first transfecting COS-7 cells with pEGFP-Epac plasmid and then observing the transfected cells fixed or live under a confocal fluorescence microscope. In Epac-GFP-expressing COS-7 cells, Epac-GFP protein displayed distinct intracellular localizations (Fig. 1A), whereas GFP alone showed a diffused expression pattern across the entire cell (data not shown). Two populations of interphase COS-7 cells with different Epac subcellular localizations were observed. In one subpopulation, Epac-GFP was mainly associated with the nuclear membranes, inside and around the nucleus (Fig. 1A, left panel), whereas in the other subpopulation Epac localized mostly in distinct puncture structures (Fig. 1A, right panel). The percentage of one particular subpopulation of cells varied from 30 to 70% between experiments.

View larger version (81K): [in this window] [in a new window]   Fig. 1.   Cellular localization of Epac-GFP and endogenous Epac in COS-7 cells. A, confocal images of full-length chimeric Epac protein with GFP fusion at the C terminus. B, immunofluorescence of endogenous Epac probed by affinity-purified Epac antibody.

Although GFP fusion proteins have been extensively used as a tool to probe intracellular localization of proteins, we are aware of the potential pitfalls associated with using a heterologously expressed recombinant fusion protein to define the intracellular location of Epac. The addition of the GFP tag could interfere with a native targeting signal, and the excess of recombinant fusion protein might mask the correct targeting of the protein. Therefore, we confirmed our results generated by the Epac-GFP fusion protein by immunofluorescence staining of the endogenously expressed Epac in COS-7 cells, using an affinity-purified Epac antibody. Subcellular localization results similar to that of Epac-GFP were obtained (Fig. 1B). These results suggest that the C-terminal GFP tag in the Epac-GFP fusion protein does not significantly affect the subcellular targeting of Epac.

Using distinct identification markers of specific cellular compartments, we determined that the puncture structures observed in Fig. 1 were mitochondria, because green fluorescence signals from Epac-GFP were shown to co-localize with MitoTracker, a mitochondrial-specific fluorescent dye (Fig. 2). This is the first report that Epac is targeted to mitochondria in vivo.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Localization of Epac in mitochondria. Co-localization of Epac-GFP with MitoTracker in mitochondria.

Sequence Motifs That Are Responsible for Specific Intracellular Epac Targeting-- To determine the sequence/structural elements important for in vivo Epac localization, we constructed N-terminal deletion Epac fusion proteins with a C-terminal GFP tag. Individual Epac-GFP fusion constructs were transfected into COS-7 cells and observed by fluorescence microscopy to determine the effect of specific structural alteration on the subcellular targeting of Epac. As shown in Fig. 3A, deletion of the first 148 amino acid residues at the N terminus of Epac completely abolished the specific cellular localization pattern observed for the full-length Epac protein. The (1-148)Epac-GFP was diffused across the entire cell body and was never observed to localize in mitochondria as was seen with the full-length Epac. To further dissect the specific sequence or motif within the first 148 amino acid residues that is important for Epac targeting, we constructed an additional Epac deletion mutant, (72-148)Epac-GFP. This mutant was used to determine the function of the Disheveled, Egl-10, Pleckstrin (DEP) domain, which has been implicated in membrane association (20). Although in many (72-148)Epac-GFP-expressing cells, fluorescent signals of the GFP fusion deletion mutant were diffused across the entire cell compartment similar to that of (1-148)Epac-GFP, (72-148)Epac-GFP retained mitochondrial localization ability in a small fraction of the cell population (Fig. 3B). These data suggest that the DEP domain is important for membrane association of Epac but not for mitochondrial targeting (Fig. 3B).

View larger version (31K): [in this window] [in a new window]   Fig. 3.   Localization of deletion Epac-GFP proteins. Subcellular localization of deletion Epac-GFP fusion proteins monitored by fluorescence microscopy in COS-7 cells. A, diffused cellular localization of (1-148)Epac-GFP. B, mitochondrial localization of (72-148)Epac-GFP. C, schematic representation of various Epac fusion proteins. D, the N-terminal sequence of the first 37 amino acid residues of Epac with positively charged (Arg) and hydroxylated (Ser and Thr) residues highlighted in blue and red, respectively. The predicted cleavage site for the mitochondrial presequence is marked by an arrow.

Proper targeting of Epac has been shown to be essential for activating the downstream effector Rap1 (13). Among the observed cellular effects associated with Epac are changes in cell morphology. Epac-transfected cells were more flattened and adhesive compared with the parental cells. The observed effects of the N-terminal sequence of Epac on its subcellular localization is physiologically relevant because removal of the N-terminal targeting sequence in (1-148)Epac generates a deletion Epac protein that is incapable of changing cell morphology as does the full-length Epac protein (Fig. 4). These results suggest that specific subcellular localization of Epac is essential for its biological functions.

View larger version (118K): [in this window] [in a new window]   Fig. 4.   Change of cell morphology in Epac expression HEK293 cells. Cell morphology of parental and Epac-transfected HEK293 cells observed under a phase-contrast microscope.

Intracellular Targeting of Epac and Cell Division Cycle-- Cyclic AMP has been implicated in cell cycle regulation (21-23). Because we found that in interphase Epac is predominantly localized on the nuclear envelope that undergoes dynamic disassembling/reassembling during mitosis, we examined the specific cellular localization of Epac at different stages of the mitotic cell cycle. As shown in Fig. 5, Epac underwent dramatic redistribution during the mitotic cell division cycle. At prophase/prometaphase, Epac started to dissociate from the nuclear envelope (Fig. 5A) and was observed to associate with the mitotic spindle and the centrosomes in metaphase (Fig. 5B). At the later stages of mitosis, Epac was seen again to concentrate around the chromosomes during anaphase and reassociate the newly formed nuclear envelope in telophase (Fig. 5, C and D). Most interestingly, a strong fluorescent signal was observed at the middle of the cell where cleavage occurs to form two separate daughter cells (Fig. 5, C and D) and at the middle bodies between the two daughter cells. Identical results were obtained when signals from heterologously-expressed Epac-GFP or endogenous Epac in COS-7 cells were probed by GFP fluorescence or by immunofluorescence using affinity-purified Epac antibody, respectively. Furthermore, stable expression of Epac in COS-7 cells led to a significant increase of the population of cells that contain two or more nuclei (Table I). Taken together, these data suggest that Epac play a physiological role in mitotic cell division by assuming cell cycle-dependent localizations.

View larger version (33K): [in this window] [in a new window]   Fig. 5.   Subcellular targeting of Epac and cell division cycle. Cellular localization of Epac at different stages of the cell division cycle in COS-7 cells. A, prophase; B, metaphase; C, anaphase; D, telophase.

Effects of cAMP on Epac Subcellular Localization-- Intracellular cAMP concentration fluctuates not only in response to external stimuli but also at different stages of the cell cycle. It has been observed that cAMP concentration decreases at the onset of mitosis and increases at the transition between mitosis and interphase (21, 22). The observation that subcellular localization of Epac is cell cycle-dependent suggests a potential regulatory role of cAMP in controlling the cellular targeting of Epac. To test the effect of cAMP on subcellular localization of Epac, the localizations of Epac-GFP in live COS-7 cells at the basal state were compared with that of the stimulated state. An apparent increase in nuclear membrane localization of Epac was observed (data not shown). Since it is unlikely that a change in the level of Epac-GFP protein was induced by the forskolin treatment within the time frame of our experiment, this observation suggests there is a net shift of Epac-GFP protein to the nuclear membrane fraction following cAMP stimulation. This is consistent with the cell cycle-dependent subcellular localization observation: Epac is mainly associated with membrane fractions in interphase cells where intracellular cAMP concentration is high, and Epac binds to the mitotic spindle in M-phase where cAMP concentration is low. To further test the effect of cAMP we introduced an Epac mutant, EpacR279E, which has been shown to be defective in cAMP binding, into COS-7 cells. In about 10-20% of EpacR279E-GFP-expressing COS-7 cells, green fluorescent signals were observed to associate with the microtubule network in interphase (Fig. 6), whereas the wild type Epac was never observed to associate with the microtubules in interphase COS-7 cells. These data suggest that disrupting cAMP binding in Epac favors interaction with the microtubule cytoskeleton network. Taken together, our data suggest that high intracellular cAMP concentrations favor membrane association of Epac and low cAMP concentrations favor cytoskeleton association of Epac.

View larger version (59K): [in this window] [in a new window]   Fig. 6.   Association of Epac mutant defective in cAMP binding with microtubles. Cellular localization of the mutant EpacR279E-GFP fusion protein in a live COS-7 cell.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Intracellular signal transduction pathways consist of a complex network of interacting molecular switches, such as protein kinases and small GTPases. Many of these molecular switches belong to structurally and evolutionarily related protein superfamilies and share common upstream activators or downstream effectors. A fundamental question regarding intracellular signal transduction is how these seemingly similar signaling molecules maintain their specificity and exert their distinct physiological functions in vivo. Subcellular targeting has been proposed as a potential mechanism by which signaling molecules can maintain biological specificity, in part through assuming distinct cellular localization/co-localization inside the cells. There they can be accessed in an orderly fashion by upstream activators and, in turn, gain access to specific downstream targets. This specific targeting mechanism of intracellular signaling molecules allows optimal control of the flow of intracellular signal transduction in a temporal and spatial manner (24). Modulation of in vivo functions of PKA by a class of structurally diverse protein kinase A anchoring proteins exemplified the importance of this targeting mechanism in controlling the function and specificity of signaling molecules (25). Therefore, identifying the subcellular targeting mechanism of Epac may be critical for elucidating its biological functions.

Using C-terminal, GFP-tagged Epac fusion proteins, we have shown that Epac proteins are associated with the nuclear envelope and mitochondria in COS-7 cells in interphase. These observations were further confirmed by an independent approach, i.e. an immunofluorescent technique probing the localization of endogenous Epac using affinity-purified, Epac-specific antibodies. The results suggest that tagging of GFP at the C terminus did not affect the specific cellular targeting of Epac, which validated our strategy of using GFP fusion proteins to probe the subcellular localization of Epac. There are several advantages in using GFP-based fluorescent assays for monitoring subcellular localization of Epac. The exact subcellular localization of the Epac can be followed continuously in living cells. Potential translocalization of Epac in response to cAMP stimulation can also be examined. Furthermore, this technique does not require additional manipulations. This simplicity eliminates the chance of introducing possible artifacts associated with fixation and staining.

Deletion of the first 148 N-terminal amino acids completely abolishes Epac's nuclear membrane and mitochondrial localization whereas (72-148)Epac-GFP, which lacks the DEP domain, only affects the Epac nuclear membrane association. These results suggest the DEP domain is responsible for the Epac membrane association as implicated previously (20) while the mitochondrial targeting sequence is located at the N-terminal. The association of Epac with mitochondria is of particular interest, because PSORT analysis predicted Epac as a mitochondrion-associated protein with a mitochondrial presequence at its N-terminal (26). Although mitochondrial targeting signals share no apparent sequence homology, the precursors of nuclear-encoded mitochondrial proteins, in general, have distinct N-terminal presequences rich in positively charged residues, especially arginine and hydroxylated amino acids. In addition, these presequences also tend to form amphipathic -helices or -sheets (27). As shown in Fig. 3C, there are six Arg and six Ser/Thr residues within the first 37 amino acids at the N-terminal of Epac, while there is only one acidic residue (Glu) in this segment. A mitochondrial presequence cleavage site at position 32 is also predicted, based on Gavel analysis, because the N-terminal Epac amino acid sequence contains a well characterized mitochondrial targeting peptide cleavage site motif with conserved arginines at 2 and 10 positions (28).

Although the physiological significance of the association of Epac with mitochondria requires further investigation, the connection between cAMP and mitochondria has been established for decades. Adenylate cyclase has been reported to localize in mitochondria (29, 30), and it has been shown that cAMP can be transported into mitochondria and accumulated in the matrix (31). Both PKA regulatory and catalytic subunits and enzymatic activity have also been detected in mitochondria (32-35). More recently, several A-kinase anchoring proteins (AKAP) have been cloned and identified to target PKA specifically to mitochondrial subcompartments (36-39). These results suggest cAMP plays an intimate role in mitochondrial functions. Furthermore, GTP-binding proteins, including Rap1, have been reported in mitochondria (40, 41). The mitochondrial localization of Epac, a guanine nucleotide exchange factor directly regulated by cAMP, suggests Epac can potentially facilitate cAMP functions by directly connecting cAMP signaling with downstream GTPases in mitochondria.

Although Epac was originally cloned as an upstream activator for Rap1, experimental evidence is beginning to emerge that Epac may process cellular functions in addition to activating Rap1 (42). Our studies further suggest that Epac is localized to the mitotic spindle in metaphase and associated with the nuclear membrane in interphase. In addition, cAMP likely play a role in regulating the subcellular targeting of Epac, because high intracellular cAMP concentrations promote membrane association of Epac and low cAMP levels favor interaction with microtubules. These observations are consistent with the theory that cAMP concentration is lowest in metaphase where Epac is associated with the mitotic spindle and increases at the transition between mitosis and interphase where Epac is mainly located on the nuclear and cell membranes. Recent studies by Beaudouin et al. (43) and Salina et al. (44) suggest that microtubules in conjunction with dynein, a cytoplasmic motor protein, are responsible for the nuclear envelope disassembly/assembly during cell division. In light of these findings, our observation that Epac is associated with the mitotic spindle and nuclear membrane in a cell cycle-dependent manner suggests Epac may play an important role in coordinating the fundamental cellular processes of microtubule/motor-driven transport and organelle partition into daughter cells during mitosis.

	ACKNOWLEDGEMENTS

We thank Dr. Johannes L. Bos (University Medical Center Utrecht, The Netherlands) for providing the Epac cDNA.

	FOOTNOTES

^* This work was supported by American Cancer Society Research Scholar Grant RSG-01-035-01-TBE and National Institutes of Health Center Grant ES06676.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 409-772-9656; Fax: 409-772-9642; E-mail: xcheng@utmb.edu.

Published, JBC Papers in Press, May 8, 2002, DOI 10.1074/jbc.M203571200

	ABBREVIATIONS

The abbreviations used are: PKA, cAMP-dependent protein kinase; Epac, exchange protein directly activated by cAMP; CBD, cAMP-binding domain; GEF, guanine nucleotide exchange factors; Rap, Ras-proximate; GFP, green fluorescent protein; EGFP, enhanced GFP; PBS, phosphate-buffered saline; DEP domain, Disheveled, Egl-10, Pleckstrin domain; FITC, fluorescein isothiocyanate.

	REFERENCES

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				Y. Li, S. Asuri, J. F. Rebhun, A. F. Castro, N. C. Paranavitana, and L. A. Quilliam The RAP1 Guanine Nucleotide Exchange Factor Epac2 Couples Cyclic AMP and Ras Signals at the Plasma Membrane J. Biol. Chem., February 3, 2006; 281(5): 2506 - 2514. [Abstract] [Full Text] [PDF]