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Genes to Cells (2006) 11, 949-959. doi:10.1111/j.1365-2443.2006.00991.x
© 2006 Blackwell Publishing or its licensors
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AMY-1 (associate of Myc-1) localization to the trans-Golgi network through interacting with BIG2, a guanine-nucleotide exchange factor for ADP-ribosylation factors

Ray Ishizaki1,2, Hye-Won Shin1, Sanae M. M. Iguchi-Ariga3,4, Hiroyoshi Ariga2,4,* and Kazuhisa Nakayama1,*

1 Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan
2 Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan
3 Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
4 CREST, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
AMY-1 (associate of Myc-1) was originally identified as a c-Myc-binding protein that enhances the c-Myc transcription activity, and subsequently found to interact with A-kinase-anchoring proteins (AKAPs), including AKAP149, S-AKAP84 and AKAP95. We show here that, using anti-AMY-1 antibodies we raised, AMY-1 localizes to the trans-Golgi network (TGN) and the nucleus. To explore the possible function of AMY-1, we have undertaken a search for interacting partners by co-immunoprecipitation experiments using cells stably expressing FLAG-tagged AMY-1. Interestingly, we have found that AMY-1 interacts with BIG2 and BIG1, both of which are high molecular weight guanine-nucleotide exchange factors for ADP-ribosylation factors (ARFs) and mainly localize to the TGN. Furthermore, we have demonstrated that AMY-1 is associated with the TGN through interacting with BIG2 but not with BIG1 using an RNA interference approach, although AMY-1 can interact with both BIG1 and BIG2 in vitro. Taken together with the facts that BIG2 contains domains that bind to regulatory subunits of protein kinase A and that recruitment of ARF1 onto Golgi membranes is mediated, at least in part, by activation of protein kinase A, these results suggest that BIG2 alone or in concert with recruited AMY-1 coordinates ARF-mediated membrane trafficking and signaling pathways.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
AMY-1 (associate of Myc-1) was originally identified as an 11-kDa protein that interacts with c-Myc and regulates its E-box-dependent transcription activity (Taira et al. 1998). However, AMY-1 appears to function in a c-Myc-independent manner as well. For example, over-expression of AMY-1 but not that of c-Myc triggers differentiation of K562 cells to erythrocytes (Furusawa et al. 2000). Furthermore, we have previously shown that AMY-1 binds to some A-kinase anchoring proteins (AKAPs), including AKAP149, S-AKAP84 and AKAP95, through interacting with their binding domains for the regulatory subunit type II (RII) of protein kinase A (PKA) (Furusawa et al. 2001, 2002).

To date, more than ten AKAPs have been identified on the basis of their interactions with PKA regulatory subunits (Feliciello et al. 2001; Wong & Scott 2004). However, they also bind to other regulatory proteins, such as protein kinases and phosphatases. These AKAPs localize to various intracellular compartments where they recruit PKA and other regulatory proteins, and may serve as scaffolds that integrate signals from multiple pathways (Feliciello et al. 2001; Wong & Scott 2004). In the case of AMY-1, AKAP95 and S-AKAP84 recruit it into the nucleus and on to the mitochondrial outer membrane, respectively (Furusawa et al. 2001, 2002), suggesting organelle-specific roles of AMY-1. However, the molecular mechanisms underlying the AMY-1 functions remain to be elucidated.

Recently, Li et al. (2003) have reported that BIG2 (brefeldin A-inhibited guanine nucleotide exchange factor 2) has RII-binding domains (RIIBDs), which bind to RII subunits of PKA. BIG2 and its homolog, BIG1, are brefeldin A (BFA)-sensitive guanine nucleotide exchange factors (GEFs) for a family of small GTPases, ADP-ribosylation factors (ARFs) (reviewed in Donaldson & Jackson 2000; Jackson & Casanova 2000; Shin & Nakayama 2004), and are associated mainly with the trans-Golgi network (TGN), where they promote assembly of the clathrin/AP-1 coat through activating ARFs (Mansour et al. 1999; Yamaji et al. 2000; Shinotsuka et al. 2002a,b; Zhao et al. 2002; Shin et al. 2004). Human BIG1 and BIG2 consist of 1849- and 1785-amino acid residues, respectively, and the Sec7 catalytic domain covers only ~200 amino acids (Morinaga et al. 1997; Togawa et al. 1999). However, cellular functions of the regions outside of the Sec7 domain have been poorly understood, although some interacting partners have been identified (Li et al. 2003; Padilla et al. 2003; Xu et al. 2005).

We show here that endogenous AMY-1 localizes to the TGN and the nucleus, and interacts with BIG2 and BIG1 by binding to their RIIBDs. Furthermore, depletion of BIG2 but not that of BIG1 by RNA interference (RNAi) abrogates the TGN localization of AMY-1. These results suggest that AMY-1 is a modulator of AKAPs including BIG2 and that BIG2 may play a role as a scaffold in integrating membrane trafficking and signaling pathways.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Localization of AMY-1 to the trans-Golgi/TGN

To characterize endogenous AMY-1, we raised polyclonal rabbit and monoclonal rat antibodies to AMY-1. The affinity-purified rabbit and rat anti-AMY-1 antibodies recognized an ~11-kDa band on immunoblot analysis of HeLa cell extracts (Fig. 1A, lanes 2 and 4, respectively). When extracts from HeLa cells transfected with a FLAG-tagged AMY-1 vector were examined, both antibodies recognized an additional band (lanes 1 and 3) at a position corresponding to that of a band detected with anti-FLAG antibody (lane 5). These results indicate that both the rabbit and rat antibodies are able to specifically detect the AMY-1 protein. We used the rabbit polyclonal anti-AMY-1 antibody in the following experiments except for the cases noted, because the rabbit antibody appeared to be superior to the rat monoclonal antibody in the reactivity.


Figure 1
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  		Figure 1  Localization of endogenous AMY-1 to the trans-Golgi/TGN. (A) Extracts (containing 50 µg protein) of HeLa cells with (lanes 1, 3, and 5) or without (lanes 2, 4 and 6) AMY-1-FLAG transfection were separated on 15% SDS-polyacrylamide gel and subjected to immunoblot analysis with monoclonal rat anti-AMY-1 antibody (lanes 1 and 2), affinity-purified antibody to AMY-1 raised in a rabbit (lanes 3 and 4) or anti-FLAG M2 antibody (lanes 5 and 6). (B) HeLa cells that had been mock-treated (a, b) or treated with 5 µg/mL nocodazole for 2 h (c, d) were subjected to indirect immunofluorescence analysis with sheep anti-TGN46, rabbit anti-AMY-1 and mouse anti-GM130 antibodies, followed by Alexa488-conjugated anti-sheep, Alexa555-conjugated anti-rabbit and Cy5-conjugated anti-mouse IgGs.

 
When HeLa cells were stained with the anti-AMY-1 antibody, we observed a perinuclear staining reminiscent of the Golgi apparatus, along with faint staining for the nucleus and cytoplasm, under a confocal laser-scanning microscope (Fig. 1B). When cells were double-stained with anti-AMY-1 antibody and antibody to either TGN46 (a TGN marker) or GM130 (a cis-Golgi marker), the AMY-1 staining was significantly overlapped with those for both Golgi markers. Magnified images, however, showed that AMY-1 co-localizes almost completely with TGN46 (Ba), but not completely with GM130 (Bb).

To corroborate this observation that the AMY-1-positive structures may represent the trans-Golgi/TGN, HeLa cells were treated with a microtubule-depolymerizing drug, nocodazole, which causes fragmentation of the Golgi apparatus (Cole et al. 1996), and double-stained for AMY-1 and either TGN46 or GM130. Because the nocodazole-induced Golgi mini-stacks retain the cis-trans polarity, the treatment permits relative localization of Golgi proteins (Cole et al. 1996; Shima et al. 1997). Merging the image of the AMY-1 staining with that of the TGN46 or GM130 staining revealed that AMY-1 was co-localized well with TGN46 in the majority of the Golgi fragments (Bc), while juxtaposed to GM130 (Bd). These observations together indicate that AMY-1 is associated mainly with the trans-Golgi and/or the TGN.

Co-immunoprecipitation of BIG1 and BIG2 with AMY-1

We previously identified three AKAPs (AKAP149, S-AKAP84 and AKAP95) and AAT-1 as AMY-1-binding proteins by two-hybrid screening (Furusawa et al. 2001, 2002; Yukitake et al. 2002). In an attempt to identify other AMY-1-binding proteins, we here took another approach. We first established a CHO cell line that stably expresses C-terminally FLAG-tagged AMY-1 (named CHO-AMY). Extracts prepared from the CHO-AMY cells or control CHO cells were then applied to anti-FLAG M2 agarose beads, and the bound proteins were separated by SDS-PAGE (Fig. 2A). Five bands in the CHO-AMY lane, which appeared to be absent or present at extremely low levels in the control CHO lane, were excised from the gel and subjected to a MALDI-TOF MS/MS analysis. The five proteins thus identified were BIG1, BIG2, AKAP95, Grp78 and Hsc70 (Fig. 2A). Grp78 and Hsc70 are molecular chaperons and AKAP95 is a nuclear AKAP. On the other hand, BIG1 and BIG2 are ARF-GEFs, both of which are associated mainly with the TGN (for review see Shin & Nakayama 2004).


Figure 2
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  		Figure 2  Interaction of AMY-1 with BIG2 and BIG1. (A) Extracts from control CHO (lane 2) or CHO-AMY (lane 3) cells were precipitated with anti-FLAG M2-conjugated agarose beads, electrophoresed on 7.5% SDS-polyacrylamide gel, and stained with Coomassie Brilliant Blue. The five bands specifically detected in lane 3 were excised from the gel and subjected to MALDI-TOF MS/MS analysis. HC represents the immunoglobulin heavy chain. (B) Alignment of the sequences covering the RIIBDs in the BIG2-A, BIG2-B and BIG1-A regions. Conserved hydrophobic residues that are critical for formation of {alpha}-helical structures are shadowed. Sequences deleted in the BIG1-A {Delta}RII and BIG2-A {Delta}RII constructs are underlined. (C) Interaction between AMY-1 and either a BIG1 or BIG2 fragment was examined using a yeast two-hybrid system. Transformants grown on a filter were assayed for ß-galactosidase activity. The result shown was obtained by incubating the filter in the presence of X-gal for 30 min at 37 °C.

 
We thereafter focused on the interactions of AMY-1 with BIG1 and BIG2 for the following reasons: (i) AMY-1 localizes to the TGN (Fig. 1), (ii) AMY-1 binds to RIIBDs of AKAPs (Furusawa et al. 2001; 2002), (iii) BIG2 was reported to have RIIBDs and bind to the regulatory subunits of PKA (Li et al. 2003).

AMY-1 binds to an RIIBD of BIG2

The interactions of AMY-1 with BIG1 and BIG2 were confirmed by other approaches. First, we examined the interactions using a yeast two-hybrid system. BIG2 has two RIIBDs (Li et al. 2003). On the other hand, BIG1 has a sequence homologous to one of the RIIBDs of BIG2 (Fig. 2B). As shown in Fig. 2C, a BIG2 fragment containing the first RIIBD (BIG2-A) and a BIG1 fragment containing the RIIBD-like sequence (BIG1-A) interacted with AMY-1. By contrast, another BIG2 fragment covering the second RIIBD (BIG2-B) was unable to interact with AMY-1 in the two-hybrid assay. Deletion of the RIIBD (residues 283–301) from BIG2-A (BIG2-A {Delta}RII) or that (residues 320–341) from BIG1-A (BIG1-A {Delta}RII) abolished the interaction with AMY-1. Furthermore, mutation of a conserved Ile residue in the BIG2-A RIIBD to a Pro residue (BIG2-A I292P), which was predicted to disrupt the {alpha}-helical structure of the RIIBD, extremely reduced the interaction. These results indicate that the first RIIBD is responsible for interaction with AMY-1.

To confirm the interaction of AMY-1 with BIG2 in mammalian cells other than CHO-AMY, we transiently transfected HEK293T cells with a combination of expression vectors for AMY-1-FLAG and HA-BIG2, and subjected the extracts prepared from these transfected cells to immunoprecipitation with anti-FLAG or anti-HA agarose beads followed by immunoblotting with either of these antibodies. As shown in Fig. 3A, immunoprecipitation of AMY-1-FLAG brought down HA-BIG2 together from the transiently transfected HEK293T cell extracts (lanes 5–8). Conversely, AMY-1-FLAG was co-immunoprecipitated with HA-BIG2 as well (lanes 9–12). Furthermore, endogenous BIG2 was co-immunoprecipitated with AMY-1-FLAG (Fig. 3B, lane 7). Similarly, immunoprecipitation of AMY-1-FLAG brought down endogenous BIG1 (Fig. 3B, lane 3). These results indicate that AMY-1 is able to form a complex with BIG2 or BIG1 in the cell.


Figure 3
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  		Figure 3  Co-immunoprecipitation of AMY-1 with BIG2 and BIG1. (A) Extracts from HEK293T cells transfected with an expression vector for either AMY-1-FLAG or HA-BIG2 alone, or their combination, were immunoprecipitated with anti-FLAG (lanes 5–8) or anti-HA (lanes 9–12) antibody, and subjected to immunoblot analysis using anti-FLAG (upper panels) or anti-HA (lower panels) antibody. (B) Extracts from HEK293T cells transfected with an AMY-1-FLAG expression vector were immunoprecipitated with anti-FLAG antibody and subjected to immunoblot analysis using anti-FLAG (upper panels), anti-BIG1 (lower panels, lanes 1–4) or anti-BIG2 (lower panels, lanes 5–8) antibody. In the experiments shown in both A and B, proteins were separated on a 15% SDS-polyacrylamide gel to detect AMY-1 or on a 5% gel to detect BIG2 or BIG1.

 
Co-localization of AMY-1 with BIG1 and BIG2

As shown in Fig. 1, AMY-1 localizes mainly to the trans-Golgi/TGN. On the other hand, previous studies showed that both BIG1 and BIG2 localize mainly to the TGN (Mansour et al. 1999; Yamaji et al. 2000; Shinotsuka et al. 2002b; Zhao et al. 2002; Shin et al. 2004). To examine whether AMY-1 and BIG2 co-localize, HeLa cells were double-stained with rat monoclonal anti-AMY-1 antibody and affinity-purified rabbit anti-BIG2 antibody (Shin et al. 2004). As shown in Fig. 4B–B'', endogenous AMY-1 and BIG2 co-localized almost completely in the Golgi region. To examine whether AMY-1 and BIG1 co-localize, we raised an anti-BIG1 antibody in rabbit and used the affinity-purified antibody for double staining with rat anti-AMY-1 antibody. As shown in Fig. 4A–A'', endogenous AMY-1 and BIG1 also co-localized well in the Golgi region. However, as described below, the Golgi association of AMY-1 is not always coupled with that of BIG1.


Figure 4
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  		Figure 4  Co-localization of AMY-1 with BIG2 and BIG1. (A, B) HeLa cells were double-stained with rat anti-AMY-1 antibody (A, B) and rabbit anti-BIG1 (A') or anti-BIG2 (B') antibody followed by Cy3-conjugated anti-rat and Alexa488-conjugated anti-rabbit IgGs. (C, D) HeLa cells transiently transfected with an HA-BIG2 expression vector were mock-treated (C–C'') or treated with 5 µg/mL BFA for 15 min (D–D'') and immunostained with rabbit anti-AMY-1 (C and D) and mouse anti-HA (C' and D') antibodies followed by Alexa555-conjugated anti-rabbit and Alexa488-conjugated anti-mouse IgGs.

 
We previously showed that when BIG2-over-expressing cells were treated with BFA, the BIG2 protein was found on membrane tubules emanating from the TGN region (Shinotsuka et al. 2002b). We therefore examined the localization of AMY-1 when HA-BIG2-expressing cells were treated with BFA. As shown in Fig. 4D–D'' AMY-1 was found co-localized on the BFA-induced, HA-BIG2-positive tubular structures in cells expressing HA-BIG2, whereas disappeared from the Golgi region in surrounding non-transfected cells upon the BFA treatment. These observations indicate that the TGN association of AMY-1 is intimately coupled with that of BIG2.

Golgi association of AMY-1 depends on BIG2

To examine whether the association of AMY-1 with TGN membranes depends on BIG1 or BIG2, or both ARF-GEFs, we set out to deplete these ARF-GEFs by utilizing an RNAi approach. First, we confirmed the specific knockdown of BIG1 or BIG2, or both by immunoblot analysis of lysates from HeLa cells subjected to RNAi. In cells subjected to RNAi for the coding region of BIG1 (Fig. 5, lane 2; BIG1-coding), there was a significant decrease in the level of BIG1 but not in that of BIG2 or GM130 as a negative control. Reciprocally, RNAi for the coding region (lane 3; BIG2-coding) or 3'-untranslated region (lane 5; BIG2–3'UTR) of BIG2 suppressed specifically the expression of BIG2 but not that of BIG1. Cells subjected to RNAi for both ARF-GEFs showed depletion of both proteins (lane 4).


Figure 5
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  		Figure 5  Depletion of BIG1 or BIG2 by RNAi. Extracts from mock-treated HeLa cells (lane 1) or cells subjected to RNAi for either the BIG1-coding (lane 2) or BIG2-coding (lane 3) region, or both coding regions (lane 4), or the BIG2–3'-untranslated region (lane 5; BIG2–3'UTR) as described under Experimental procedures were analyzed by immunoblotting with antibody to BIG1 (top panel), BIG2 (middle panel) or GM130 (bottom panel). The band density of each lane was estimated using a LAS-3000 mini bioimaging analyzer (Fiji Photo Film, Co.) and represented as a percent density to the control.

 
We subsequently examined the effect of depletion of BIG1 or BIG2, or both, on the localization of AMY-1 (Fig. 6). Even though HeLa cells subjected to BIG1 RNAi no longer showed the typical Golgi-like staining for BIG1 (Fig. 6E), that for BIG2 (Fig. 6F), AMY-1 (Fig. 6G) or golgin-245 (Fig. 6H, a trans-Golgi/TGN marker) did not change. In striking contrast, depletion of BIG2 (Fig. 6J,R) resulted in disappearance of the Golgi staining for AMY-1 (Fig. 6K,S). However, the BIG2 depletion did not significantly affect the localization of BIG1 (Fig. 6I,Q) or that of golgin-245 (Fig. 6L,T), excluding a possibility that BIG1 was also responsible for recruitment of AMY-1 onto the Golgi and a formal possibility that the disappearance of the AMY-1 staining was due to disintegration of the Golgi structure. Note that, to confirm the Golgi integrity, the cells subjected to RNAi were double-stained for golgin-245 and either BIG1, BIG2 or AMY-1. Depletion of both BIG1 and BIG2 also abolished the Golgi localization of AMY-1 (Fig. 6O) but not that of golgin-245 (Fig. 6P). In addition, it is worth noting that the faint nuclear staining for AMY-1 remained unchanged in the BIG2-depleted cells (Fig. 6K,O,S; see bellow).


Figure 6
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  		Figure 6  Effect of BIG1 or BIG2 depletion of the AMY-1 localization. Mock-treated HeLa cells (A–D) or cells subjected to RNAi for either BIG1-coding (E–H) or BIG2-coding (I–L), or both BIG1-coding and BIG2-coding (M–P), or BIG2–3'UTR (Q–T) as described under Experimental procedures were stained for BIG1 (A, E, I, M, Q), BIG2 (B, F, J, N, R), AMY-1 (C, G, K, O, S) or golgin-245 (D, H, L, P, T).

 
In light of the above observations, we then examined the effect of BIG2 over-expression on the AMY-1 localization. As shown in Fig. 7, the perinuclear Golgi-like staining for endogenous AMY-1 was more prominent in cells with HA-BIG2 over-expression than those without exogenous HA-BIG2 expression (Fig. 7A,A'). This observation indicates that BIG2 recruits AMY-1 onto TGN membranes, although it is also possible that over-expressed BIG2 stabilized the AMY-1 protein on membranes.


Figure 7
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  		Figure 7  Effect of over-expression of BIG2 or AKAP95 on the AMY-1 localization. HeLa cells transiently transfected with an expression vector for either HA-BIG2 (A–A'') or FLAG-AKAP95 (B–B''), or both vectors (C–C'') were triple-stained with rat anti-HA (A–C), rabbit anti-AMY-1 (A'–C') and mouse anti-FLAG (A''–C'') antibodies, followed by Alexa488-conjugated anti-rat, Alexa555-conjugated anti-rabbit and Cy5-conjugated anti-mouse IgGs.

 
As described above, the BIG2 depletion appeared not to affect the faint nuclear staining for AMY-1 (Fig. 6,K,O and S). Taken together with our finding in the present study (Fig. 2A) as well as that in the previous study (Furusawa et al. 2002) that AKAP95, a nuclear AKAP, interacts with AMY-1, we speculated that AKAP95 determines, at least in part, the nuclear localization of AMY-1. To address this speculation, we examined the effect of AKAP95 over-expression on the localization of endogenous AMY-1. As shown in Fig. 7B',B'', over-expression of FLAG-AKPA95 extremely enhanced the nuclear staining for AMY-1, concomitant with the disappearance of its Golgi staining, indicating that AKAP95 is capable of recruiting AMY-1 into the nucleus.

In addition, we obtained an interesting result when BIG2 and AKAP95 were co-over-expressed (Fig. 7C–C''). Namely, cells with exogenous expression of both HA-BIG2 and FLAG-AKAP95 showed intense perinuclear Golgi-like staining for AMY-1. This observation suggests, albeit indirectly, that the affinity to AMY-1 of BIG2 is higher than that of AKAP95 in the cell, and makes it unlikely that AMY-1 can interact simultaneously with BIG2 and AKAP95.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
In the present study, we found that endogenous AMY-1 localizes to the TGN and the nucleus. Furthermore, we found that the TGN association of AMY-1 is mediated by BIG2, which is a high molecular weight ARF-GEF associated with the TGN and endosomes, and regulates membrane trafficking through activating ARFs (Yamaji et al. 2000; Shinotsuka et al. 2002a,b; Shin et al. 2004) (reviewed in Shin & Nakayama 2004). These results are in line with our previous data showing that AMY-1 interacts with some AKAPs through their RIIBDs (Furusawa et al. 2001, 2002) and with the data of Li et al. (2003) showing that BIG2 has RIIBDs. In addition, BIG1, a relative of BIG2, could also make some contribution to the TGN association of AMY-1, because in vitro and two-hybrid experiments showed an interaction between AMY-1 and BIG1.

AMY-1 was originally identified as a c-Myc-binding protein and found in the nucleus during the S phase, where it stimulates the transcriptional activity of c-Myc (Taira et al. 1998). It was later shown to be associated with mitochondria as well (Furusawa et al. 2001, 2002). The nuclear localization of AMY-1 might be mediated by its interaction with AKAP95 and the mitochondrial association might be mediated by S-AKAP84 (Furusawa et al. 2001, 2002). We demonstrate here the TGN association of AMY-1 through interaction with BIG2; an RIIBD outside of the Sec7 catalytic domain of BIG2 is responsible for the interaction with AMY-1. BIG2 is associated with the TGN and endosomes, where it promotes membrane recruitment of ARFs and in turn promotes formation of clathrin-coated vesicles by recrui-ting the adaptor protein complex AP-1 (Yamaji et al. 2000; Shinotsuka et al. 2002a,b; Shin et al. 2004) (reviewed in Shin & Nakayama 2004).

The broad range of intracellular distribution of AMY-1 suggests its diverse cellular functions. Some explanations are possible for the physiological relevance of the AMY-1-BIG2 interaction. The most intuitional one is that AMY-1 could modulate the catalytic activity of the ARF-GEF through interacting with the region outside of the Sec7 catalytic domain. Our preliminary experiments to date, however, failed to support this possibility; exogenous AMY-1 expression in cells does not change the production of GTP-bound ARF either in BIG2- or mock-transfected cells, or stimulate recruitment of ARF on to the TGN.

Secondly, AMY-1 might modulate a local function of PKA by binding to the RIIBD on BIG2. The RII subunits have been shown to be associated with the Golgi (Martin et al. 1999; Birkeli et al. 2003). Furthermore, PKA activation enhances association of ARF1 with Golgi membranes (Martin et al. 2000) and a PKA isozyme is required for the endosome-to-Golgi retrograde transport of the plant toxin ricin (Birkeli et al. 2003). These roles of PKA are overlapped with those of BIG2 (Yamaji et al. 2000; Shinotsuka et al. 2002a,b; Shin et al. 2004) (reviewed in Shin & Nakayama 2004). Therefore, local recruitment and/or activation of PKA at the Golgi, which might be regulated by BIG2 and AMY-1, are indispensable for membrane trafficking at the Golgi.

Another possible explanation is related to the large size of BIG2 (~190 kDa); namely, BIG2 might, like other AKAPs, serve as a scaffold for a variety of proteins including AMY-1 and coordinate multiple components of membrane trafficking and/or signal transduction pathways. In this context, it is noteworthy that BIG2 has recently been shown to interact with a component of the exocyst complex, which plays an important role in vesicle trafficking and tethering (Xu et al. 2005). In addition to BIG2, BIG1 and AKAP95, we identified two molecular chaperones, Hsc70 and Grp78, as AMY-1-binding partners, although we did not further investigate these proteins. Hsc70 is known as an uncoating ATPase that assembles with components of clathrin coats and stimulates release of adaptor protein complexes and clathrin from clathrin-coated vesicles (Jiang et al. 2000; Newmyer & Schmid 2001) (reviewed in Lemmon 2001). Because BIG2 promotes assembly of the AP-1 clathrin adaptor complex at the TGN through activating ARFs (Shinotsuka et al. 2002b), it is possible that BIG2 stimulates not only coat assembly but also Hsc70 entry mediated by AMY-1 into the coat complex. A very large AKAP, AKAP9 (its splicing variants are referred to as CG-NAP, AKAP350, AKAP450 and yotiao), is also associated with the Golgi apparatus and constitutes a scaffold for various kinases and phosphatases including PKA (Takahashi et al. 1999; Shanks et al. 2002) (reviewed in Feliciello et al. 2001; Wong & Scott 2004). Likewise, BIG2 and BIG1 may also constitute scaffolds to integrate various signaling pathways and membrane trafficking. Although BIG1 is able to bind to AMY-1 in vitro, it was dispensable for the AMY-1 association with Golgi membranes in vivo. The BIG1-AMY-1 interaction might be significant in the nucleus, because BIG1 has recently been shown to translocate to the nucleus under certain conditions (Padilla et al. 2003, 2004).

In summary, we described the molecular interactions that may underlie the coordination of vesicular trafficking and the signaling pathway at the Golgi. Further experiments will be necessary to define the molecular mechanism that couples the PKA-mediated signaling and the ARF-mediated membrane trafficking by BIG2 and BIG1. In addition, the identification of novel binding proteins of these high molecular weight ARF-GEFs will help to address their potential functions.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Antibodies

A GST-AMY-1 fusion protein was expressed and purified from Escherichia coli BL21(DE3) cells as previously described (Taira et al. 1998). The AMY-1 portion was excised from the fusion protein with a PreScission protease (Amersham Biosciences) and used to raise polyclonal antibody in a rabbit and to prepare monoclonal rat anti-AMY-1 antibody. These anti-AMY-1 antibodies were affinity-purified using the recombinant AMY-1 protein immobilized on NHS-activated Sepharose 4B beads (Amersham Biosciences). Anti-BIG1 antiserum was raised in a rabbit against a synthetic peptide (C)SQPPEQELGINKQ (the C-terminal 13-amino acids of human BIG1; Morinaga et al. 1997), conjugated with keyhole limpet hemocyanin, and affinity-purified using the antigen peptide immobilized on Sulfolink beads (Pierce Chemical). Preparation and affinity purification of anti-BIG2 antibody was previously described (Shin et al. 2004). Monoclonal mouse antibodies against GM130 and golgin-245 were purchased from BD Biosciences; that against the FLAG epitope (M2) was from Sigma. Polyclonal sheep anti-TGN46 antibody was from Serotec. Monoclonal rat (3F10) and monoclonal mouse (12CA5) antibodies to the HA epitope were from Roche Diagnostics. Alexa488- and Alexa555-conjugated secondary antibodies were from Molecular Probes, and Cy3- and Cy5-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories. Anti-FLAG M2 agarose beads and anti-HA agarose beads (HA-probe) were purchased from Sigma and Santa Cruz Biotechnology, respectively.

Plasmids

AMY-1 vectors for two-hybrid analysis and for expression in mammalian cells and an AKAP95 expression vector, pcDNA3-FLAG-AKAP95, were constructed as previously described (Taira et al. 1998; Furusawa et al. 2001). An expression vector for HA-tagged BIG2, pcDNA4-HA-BIG2, was constructed as previously described (Shinotsuka et al. 2002b). Two-hybrid prey vectors for fragments of BIG1 and BIG2 (BIG1-A, residues 248–375; BIG2-A, residues 209–327; BIG2-B, residues 450–648) were constructed by subcloning of the corresponding human cDNA fragments into the pGADGH vector (Clontech).

Establishment of a CHO-AMY cell line, immunoprecipitation, and MALDI-TOF MS/MS analysis

CHO cells grown on a 10-cm dish were transfected with 10 µg of pEF-AMY-1-FLAG together with 0.5 µg of pSV-bsr, a blasticidin S-resistant vector, by a calcium phosphate method and cultured in the presence of 4 µg/mL blasticidin S. Two weeks after transfection, blasticidin S-resistant colonies were selected. One (designated CHO-AMY) of the cell lines thus isolated was used for the following experiments.

An extract from CHO-AMY cells or control CHO cells that carry pSV-bsr alone was allowed to immunoprecipitate with anti-FLAG M2 agarose beads, followed by 7.5% SDS-polyacrylamide gel electrophoresis and staining with Coomassie Brilliant Blue. The protein bands specific for CHO-AMY cells were cut out from the gel and subjected to a MALDI-TOF MS/MS analysis.

Yeast two-hybrid analysis

A yeast two-hybrid analysis using combinations of pGLex-AMY-1 and a pGAD vector for a fragment of BIG1 or BIG2 were performed as previously described (Furusawa et al. 2001).

Immunoprecipitation experiments

HEK293T cells were transfected with either pEF-AMY-1-FLAG or pcDNA4-HA-BIG2, or with both vectors by a calcium phosphate method. Two days after transfection, the cells were extracted with G buffer (1 mM Tris-HCl, pH 7.8, 0.1 mM CaCl2, 0.01% Tween 20). The extracts were then immunoprecipitated with anti-FLAG M2 or anti-HA agarose beads, followed by immunoblot analysis with either of these antibodies. For analysis of interaction between AMY-1 and endogenous BIG1 or BIG2, an extract prepared from cells transfected with pEF-AMY-1-FLAG were immunoprecipitated with anti-FLAG antibody and subjected to immunoblot analysis using anti-BIG1 or anti-BIG2 antibody. The blot was detected using peroxidase-conjugated anti-mouse or anti-rabbit IgG and ECL reagents (Amersham Biosciences).

Indirect immunofluorescence analysis

Indirect immunofluorescence analysis was performed as previously described (Shin et al. 1997, 2004; Shinotsuka et al. 2002b).

RNAi suppression

To generate siRNAs against BIG1 and BIG2, we used a BLOCK-iT RNAi TOPO transcription kit and a BLOCK-iT Dicer RNAi kit (Invitrogen). Briefly, a human BIG1 cDNA fragment was PCR-amplified using a set of primers [5'-GCATTACAGGAAGCAAAACAAATG-3' and 5'-GCATTAATAAACATCTCATTTGTCC-3' (BIG1-coding)], and a human BIG2 cDNA fragment was amplified using a set of primers [either 5'-AGTGTTGCAGGAGGCCAGAG-3' and 5'-GAGAGAGCTCAAAGACATCAGG-3' (BIG2-coding) or 5'-CCCTGGCTGCCCAGGCCAGTG-3' and 5'-TAGGTGGCAGTTAGCCGTTAC-3' (BIG2–3'UTR)]. The PCR fragment was ligated to T7 linkers and amplified again by PCR. Sense and anti-sense single-stranded RNAs were transcribed from the fragment using T7 RNA polymerase and annealed with each other to generate double-stranded RNA, which was then cleaved with the Dicer enzyme to create 20–23 base pair fragments. HeLa cells grown to ~50% confluency in one well of a 6-well plate were transfected with the double-stranded RNA fragments (0.5 µg/well) using Lipofectamine 2000 (Invitrogen) and incubated overnight. The transfected cells were then transferred to a 10-cm plate containing coverslips, further incubated for 48 h, and processed for immunofluorescence and immunoblot analyses.


   Acknowledgements
 
This work was supported in part from the Ministry of Education, Culture, Sports, Science and Technology of Japan; the Japan Society for Promotion of Science; the Protein 3000 Project; the Naito Foundation; and the Takeda Science Foundation. Ray Ishizaki was supported as a Research Assistant by the 21st Century COE Program, "Knowledge Information Infrastructure for Genome Science."


   Footnotes
 
Communicated by: Akihiko Nakano

* Correspondence: E-mail: kazunaka{at}pharm.kyoto-u.ac.jp; hiro{at}pharm.hokudai.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
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Received: 6 March 2006
Accepted: 17 May 2006




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