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J. Biol. Chem., Vol. 280, Issue 30, 28007-28014, July 29, 2005
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From the Center for Cardiovascular Research in the Aab Institute of Biomedical Sciences, University of Rochester School of Medicine, Rochester, New York 14642
Received for publication, December 14, 2004 , and in revised form, May 10, 2005.
| ABSTRACT |
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| INTRODUCTION |
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The intrinsic targeting and protein-protein interaction domains of each AKAP direct correct spatial and temporal assembly of protein kinase A-containing signaling complexes. Such compartmentalization simultaneously shields neighboring complexes from activated signal cascades and confers specificity to signaling complexes utilizing ubiquitous components (4). Accordingly, these targeting and interaction domains comprise the primary determinants of the role of each AKAP. For example, the ability of AKAP5 (also known as AKAP75, AKAP79, or AKAP150) to amplify cAMP signals to the nucleus is disrupted either by deletion of its membrane-targeting sequence or by mutation of the protein kinase A binding site (5). Moreover, localization of AKAPs can be dynamically regulated through the phosphorylation of critical residues within their targeting motifs, such as the membrane-targeting sequence of AKAP5 (6), to allow for mobilization of AKAP-anchored signaling complexes. Characterization of the domains that direct the localization of each AKAP is thus an essential foundational step toward elucidating AKAP physiological functions.
AKAP12, also known as SSeCKS and Gravin, is an anchoring protein that coordinates the assembly of a multiprotein complex that may include protein kinase A, protein kinase C, protein phosphatase 2B, the
-adrenergic receptor, and calmodulin (7-12). We recently demonstrated that the AKAP12 gene encodes three transcriptionally separate AKAP12 isoforms,
,
, and
, that have distinct spatio-temporal expression patterns (13). Although all three isoforms share >95% of their coding sequence, an N-terminal myristoylation motif directs AKAP12
to the endoplasmic reticulum. The remaining isoforms are localized in the cytosol and at discrete locations at the cell periphery, indicating that their targeting information is contained in domains common to all three isoforms. Characterization of the domains controlling AKAP12 targeting will provide a foundation to address how localization of this protein controls its ability to dually regulate nuclear/perinuclear compartmentalization of cyclin D1 (14) and
-adrenergic receptor functions at the cell membrane (10).
In the present study we have characterized the localization of the non-myristoylated AKAP12 isoforms. AKAP12 is a highly charged protein composed of an N terminus of alternating acidic and basic residue-rich regions and a large acidic C-terminal tail. Here we used cross-species sequence analysis and deletion mapping to facilitate the identification of targeting motifs. Seven conserved basic regions in the N terminus were found to be important in determining the localization of AKAP12. The first three basic regions display similarity to the membrane-targeting domain of the MARCKS protein and are important determinants of AKAP12 targeting to ganglioside-rich regions at the cell periphery. Interestingly, the remaining basic regions each contain an SV40-like nuclear localization signal (NLS). Constructs spanning this region are localized to the nucleus. A fifth NLS, revealed through deletion mapping and mutagenesis, represents a novel class of NLS. Nuclear localization, however, is suppressed by the acidic C terminus. The ability of the C terminus to suppress nuclear localization of green fluorescent protein (GFP) chimeras could not be localized to any specific region of the C terminus but instead appears to be related to net charge. Together, our data indicate that the charged residues in the AKAP12 protein are the main targeting determinants and reveal new insight into the interplay between charged residue-rich targeting domains in regulating subcellular localization. Additionally, our data suggest that localization of AKAP12-anchored signaling complexes may be dynamically regulated by factors or modifications (e.g. phosphorylation) that alter the local charge of the AKAP12-targeting domains.
| EXPERIMENTAL PROCEDURES |
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Plasmid ConstructsAKAP12
-enhanced GFP deletion constructs were constructed using either internal restriction enzymes or by amplifying the indicated region by PCR using restriction site-clamped primers. We used our recently described AKAP12
-GFP reporter construct, which displays similar localization as endogenous AKAP12, as the template for the deletions reported in this study (13). Mutagenesis was performed using the QuikChange site-directed mutagenesis kit (Stratagene) with primers incorporating the indicated amino acid changes. All amplified constructs were sequenced to confirm fidelity and in-frame fusion to GFP. Sequences of the primers used in this report are available upon request.
MicroscopyLocalization of GFP fusion proteins was assessed 48 h post-transfection using a BX51 microscope and a 60x water immersion lens (Olympus) as described previously (13). Images were digitized with a charge-coupled device camera (Spot) and processed in Photoshop (Adobe). Ganglioside-rich (GR) membrane regions were labeled by incubating cells (48 h post-transfection) with 1 µg/ml Alexa Fluor 594-conjugated cholera toxin subunit B (Molecular Probes) for 5 min at 37 °C (15). Cells were then rinsed twice with PBS and immediately visualized. In some experiments the number of cells with peripheral AKAP12
-GFP localization was quantitated by counting 100 cells per condition. Data presented are the means of three separate experiments.
Localization of AKAP12 in hCASMC cells was examined by seeding cells on glass chamber slides (Nalgene) and culturing as indicated. Cells were fixed with 4% freshly prepared paraformaldehyde followed by permeabilization with 0.1% Triton X-100. AKAP12 was detected using rabbit anti-SSeCKS antibody (kindly provided by Dr. Irwin Gelman, Roswell Park Cancer Institute) and goat anti-rabbit fluorescein (Pierce). Smooth muscle actin, a marker of differentiated smooth muscle cells, was detected with mouse anti-smooth muscle actin (Sigma) and goat anti-mouse rhodamine (Pierce). Nuclei were stained by labeling nucleic acids with 4,6'-diamidino-2-phenylindole (Molecular Probes). Cells were visualized as described previously (13).
Computer AnalysisThe charge plot profile for AKAP12 was generated using the charge program of EMBOSS via web interface (bioweb.pasteur.fr/seqanal/interfaces/charge.html) (16). The rat AKAP12
sequence (AY695057
[GenBank]
) was used. Because of window size constraints, the charge over the first and last 10 amino acids is not included in analysis. The evolutionary conservation profile plot was created using eSHADOW (17). Briefly, sequences for rat, mouse (fusion of open reading frames of BY002721
[GenBank]
and NM_031185
[GenBank]
to create mouse AKAP12
), and human (NM_144497
[GenBank]
) AKAP12
were submitted via Internet interface (eshadow.dcode.org/). The returned ClustalW alignment was then checked and manually edited as necessary to ensure proper alignment prior to final submission for visualization of conserved regions.
| RESULTS |
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,
, and
(13). These transcripts encode distinct but largely similar proteins. Although an N-terminal myristoylation motif targets the AKAP12
protein to the endoplasmic reticulum, mutants lacking this motif localize in a similar manner as the
and
isoforms. This finding indicates that the common AKAP12 coding sequence contains targeting information that directs localization of the AKAP12 isoforms in the absence of myristoylation. Because the subcellular distributions of endogenous AKAP12
and AKAP12
as well as those of the AKAP12
myristoylation mutants are highly similar, we have used AKAP12
(hereafter referred to as AKAP12) here to identify the targeting domains regulating non-myristoylated AKAP12 localization. AKAP12 is a large, highly charged protein that is enriched in acidic and basic amino acid residues (nearly 1 of every 3) and has a predicted net charge of -196 at physiological pH. These charged residues are not equally distributed across the protein but rather are organized into alternating acidic and basic residue-rich (AR and BR, respectively) regions (Fig. 1A). The N-terminal half of AKAP12 is composed of seven alternating AR and BR regions. The C-terminal half, in contrast, is more uniformly negatively charged because of the wide distribution of acidic residues across this region. Comparison of the distribution of these regions with the profile of evolutionary conserved regions reveals elevated conservation of the BR regions, as compared with neutral and AR regions (Fig. 1B). Given that targeting motifs regulate a key property of AKAP function and are therefore likely to be well conserved across species, we hypothesized that these basic regions may be important determinants of AKAP12 localization.
Three Regions Regulate AKAP12 LocalizationTo determine whether the targeting regions of AKAP12 are localized to the BR regions, we performed a first pass deletion study to identify general regions important in AKAP12 targeting (Fig. 1C). Whereas deletion of the C-terminal most 404 amino acids does not affect localization, further truncation of the C terminus (
791-1607) results in redistribution of AKAP12-GFP chimeras to the nucleus (Fig. 1D). Nuclear targeting is abolished and normal targeting is restored when we further deleted the region from amino acids 486 to 790 (data not shown). Finally, specific targeting is eliminated by deleting all but the first 78 amino acids.
Our deletion analysis suggests the AKAP12 targeting domains are subdivided into three regions. Interestingly, these three regions span the first two BR regions, the fourth through seventh BR region, and the AR region C terminus, respectively. The first region spans amino acids 79-286 and directs the cytosolic distribution of AKAP12 as well as targeting at the cell periphery. The second region, spanning amino acids 486-790, directs localization to the nucleus. A third region at the C terminus appears to be involved in suppressing nuclear localization, because a construct containing only the first 78 and last 404 (1-78, 1203-1607) amino acids is excluded from the nucleus (Fig. 1D). However, nuclear localization of AKAP12 appears to be suppressed by an additional C-terminal sequence, as constructs containing the region spanning amino acids 791-1202 were similarly excluded from the nucleus. Thus, based on our deletion analysis, the role of the three AKAP12 targeting regions appears to be subdivided into two functions: 1) nuclear/cytoplasmic partitioning; and 2) cytosolic and peripheral targeting.
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expression, we noted nuclear AKAP12 in a subpopulation of mesenchymal cells of the bladder and lung (data not shown).
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Identification of a Novel Class of NLSUsing mutagenesis, we assessed whether the second SV40 NLS is required for nuclear targeting (Fig. 4A). Mutagenesis of NLS2 in a construct spanning amino acids 559-612 does not affect nuclear localization (Fig. 4B). As this region lacks the other three NLSs, this finding indicates that the targeting of this region is not conferred by NLS2 but instead by another motif contained in this region. To identify the nuclear targeting sequence within amino acids 559-612 we constructed a series of GFP chimeras tiled across this region. Consistent with our mutagenesis data, a sub-region containing NLS2 was not sufficient to target to the nucleus; however, two C-terminal sub-regions displayed specific nuclear localization (Fig. 4C). This region contains several basic residues but does not represent either an SV40-type or a bipartite NLS, the two main NLS classes. Interestingly, this region is similar to the nuclear localization signal of the transcription factor, serum response factor (SRF) (19, 20) (Fig. 4D), and thus represents a novel third class of NLS. This class tentatively has a consensus of GXX(K/R)(K/R)XX(K/R)(K/R)XX- SXX(D/E), although identification of further NLSs of this class will be necessary to define a precise consensus. Based on the spacing of the consensus amino acids, we propose that this new class of NLS be termed X2-NLS to reflect the di-amino acid spacing between the pairs of basic residues.
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The Five NLSs Contribute to Nuclear Localization PotentialTo clarify which class of NLS confers targeting to the AKAP12 nuclear localization region, we mutated either the four SV40-type NLSs or the X2-NLS and then examined localization. Mutation of all four SV40-type NLSs abolishes specific nuclear localization, indicating that these NLSs are important for localization and that the X2-NLS is not sufficient on its own for nuclear targeting in the context of the AKAP12 nuclear targeting region (Fig. 5). Mutation of the X2-NLS alone reduces the extent of nuclear localization, indicating it also contributes to nuclear targeting. However, in support of the X2-NLS being stronger than the SV40-type NLS, mutation of any single SV40-type NLS had no affect on localization (data not shown). Taken together, these data demonstrate that all five NLSs contribute to the overall nuclear localizing potential of the region between amino acids 486 and 790 of AKAP12. In support of the combinatorial nature of the NLS motifs of AKAP12, inspection of the conservation of these sites revealed that the X2-NLS and at least three of the four SV40-type NLSs are highly conserved in all species examined (human, mouse, rat, dog, chicken, Xenopus, zebrafish, and Tetraodon; data not shown).
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To explore other potential targeting mechanisms, we searched the C terminus for conserved regions with similarity to other targeting or protein interaction domains. The only candidate domain we identified was a potential 14-3-3 binding site (data not shown). Because 14-3-3 proteins have been shown to regulate the nuclear localization of other proteins (21), we used deletion mapping to assess if this site was important for targeting. Deletion of the region from amino acid 787 to amino acid 883, which contains the putative 14-3-3 site, does not affect targeting, therefore excluding this site as a mediator (Fig. 6).
Because we were unable to identify any known nuclear exclusion motifs by similarity or pattern searches, we instead used deletion mapping to identify sub-regions in the C terminus that mediate nuclear exclusion. In support of our initial deletion study (Fig. 1), division of the C terminus into two approximate halves does not affect nuclear exclusion, suggesting that at least two domains are capable of this function (Fig. 6). Further division of these sub-regions, however, failed to identify a specific nuclear localization domain, as none of these deletion chimeras is capable of preventing nuclear localization. Thus, the nuclear exclusion property of this region may be conferred by a pair of large complex domains or by the physical composition of the C terminus. For instance, the ability of the C-terminal sub-regions to prevent nuclear localization appears to be correlated with their net charge (Fig. 6), suggesting that the predominance of acidic residues across the C terminus is related to the targeting role of this region.
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The First Three BR Regions Regulate Peripheral TargetingOur data suggest that the interplay between the C terminus and the central region of AKAP12 determines the partitioning of AKAP12 between the cytosol and nucleus. Although further studies will be needed to identify the conditions that regulate the nuclear exclusion function of the C terminus, these studies suggest that the C terminus suppresses the nuclear targeting region of AKAP12. When nuclear targeting is suppressed, the targeting of AKAP12 is regulated by the N terminus, as GFP chimeras containing only the first 286 amino acids recapitulate the localization pattern of the full-length AKAP12 (Fig. 1B). For instance, these chimeras display both the typical perinuclear enrichment and targeting to discrete regions at the cell periphery.
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In contrast to cytosolic targeting, peripheral targeting could be specifically assigned to the BR regions, as each BR region can direct peripheral targeting on its own (Fig. 8C). However, the inclusion of flanking AR regions results in nonspecific distribution throughout the cell, indicating that these regions antagonize the targeting ability of the BR regions. To achieve peripheral localization in the context of these acidic regions, multiple BR regions are required. For instance, GFP chimeras containing the first BR region and first two AR regions are nonspecifically targeted. Extension of this region by the addition of the next 45 amino acids containing the second BR region, however, redirects targeting to the cell periphery.
In the context of the full-length AKAP12 protein, the additive effect of the three BR regions on peripheral targeting is apparent. Internal deletion of these BR regions alone, in pairs, or in total reduces the ability of the full-length AKAP12 to target to the cell periphery in a correlative manner (Fig. 9A). Specifically, deletion of a single BR region only modestly affects peripheral targeting. Compound deletion of any pair of BR regions has a more pronounced effect. The most pronounced reduction in peripheral targeting is observed when all three BR regions are deleted, as would be expected. These data confirm that the BR regions regulate peripheral targeting and demonstrate that maximal targeting requires the contribution of all three BR regions.
Local Charge Controls the Targeting Ability of the N-terminal BR RegionsEach of the N-terminal BR regions contains two sets of sequences similar to that identified as being important for the MARCKS electrostatic switch mechanism of membrane interaction. This motif, which we identify here as a SFKK motif, is also present in a number of other membrane-associated proteins such as Src (data not shown). In this motif, the basic residues interact with the negatively charged phospholipids, the aromatic residue integrates into the membrane to stabilize the interaction, and phosphorylation of the serine residues destabilizes association with the membrane by introducing a negative charge within the local region (23). To address whether charge plays a role in determining the localization of AKAP12 to the cell periphery, we replaced the serines and threonines flanking the MARCKS-like domains with aspartic acid, a substitution that introduces a negative charge (Fig. 9B). This substitution may also mimic phosphorylation of AKAP12 at these residues. Whereas replacing the serines and threonines with a non-charged amino acid, alanine, did not affect targeting (data not shown), substitution with aspartic acid greatly attenuated the localization of AKAP12 to peripheral regions of the cell (Fig. 9C). These data indicate that the charged residues contained in the N terminus, specifically the three BR regions, are the determinants of AKAP12 localization to the cell periphery.
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| DISCUSSION |
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to the endoplasmic reticulum, our present study indicates that, by virtue of their high degree of similarity, all AKAP12 isoforms, including AKAP12
, retain the targeting information to localize to these subcellular sites. Control of AKAP12 targeting to these two distinct locales appears to be regulated by the same domain, which is found at the AKAP12 N terminus. However, localization to these compartments is dependent on different properties of this targeting domain. For instance, whereas sub-fragments of the N terminus retained their ability to target to the cell periphery, the entire N-terminal targeting domain was required for correct cytosolic distribution. One potential explanation for this discrepancy is that correct cytosolic targeting is controlled by interplay between the N-terminal AR and BR regions that is disrupted by any amount of truncation. In contrast, the BR regions alone are sufficient for targeting to GR regions at the membrane.
The three BR regions of the N-terminal targeting domain are similar to the membrane-targeting sequence found in MARCKS. Like the MARCKS-membrane interaction, we found that the charge across these regions underlies their targeting ability. For instance, introduction of acidic residues into the N-terminal BR regions disrupts peripheral targeting. Furthermore, AR regions that flank the N-terminal BR regions antagonize the latter's function. Interestingly, several serines within these BR regions have been shown to be phosphorylated (26). Taken with previous reports that show translocation of AKAP12 from the membrane to the perinuclear region upon phosphorylation (11), our data suggest that AKAP12 targeting to GR regions is dynamically regulated by introduction of negative charges into the BR regions through phosphorylation. Specifically, because our mutagenesis overlaps known AKAP12 phosphorylation sites, we propose that phosphorylation of these residues modulates the ability of AKAP12 to target to the membrane. Future studies are warranted to fully characterize the nature of AKAP12 localization to GR regions and agonist-stimulated AKAP12 redistribution.
AKAP12 is most often observed to localize to the cell periphery and the perinuclear region, although recently expression has been documented in nuclear protein fractions (14). Our data support a heretofore unrealized nuclear localization potential for AKAP12 and suggest that under certain conditions AKAP12 can be targeted to the nucleus. Although its function in the nucleus is not clear at this time, AKAP12 may continue to act as a multivalent scaffold inside the nucleus. Nuclear localization of AKAP12 may also be related to its control of cell-cycle progression. For example, AKAP12 has been shown to interact with and redistribute cyclin D1 to the cytosol (14). Although AKAP12 could be detected in nuclear extracts, how and where AKAP12 initially associates with the nuclear localized cyclin D1 is unclear. Taken with our findings, it is tempting to speculate that at some time during the cell cycle AKAP12 localization is altered and redistributed to the nucleus. In support of this notion, in actively growing primary derived hCASMCs a substantial pool of AKAP12 is localized in the nucleus. Upon differentiation of these cells, however, AKAP12 appears to be redirected from the nucleus to the cytosol. Investigation of the mechanism regulating AKAP12 nuclear/cytoplasmic partitioning will not only reveal insight into how the relationship between the central NLSs and the C-terminal exclusion domain regulates AKAP12 localization, it will help elucidate the function of nuclear localized AKAP12.
As a whole, our data suggest that a hierarchy of targeting domains determines AKAP12 localization. In this hierarchy, the C-terminal nuclear exclusion domain is the most dominant. However, because this domain lacks the ability to direct specific targeting, subordinate domains must regulate the specific distribution of AKAP12. The myristoylation domain follows in this hierarchy, superseding the central nuclear targeting domain.2 Interestingly, at the bottom of the AKAP12 targeting hierarchy is the N-terminal targeting domain, which is responsible for the majority of AKAP12 distribution.
The presence of such a targeting hierarchy suggests a means to dynamically regulate AKAP12 localization. For instance, protein modifications such as phosphorylation may functionally inactivate one or more dominant domains, resulting in targeting being directed by alternate domains. Indeed, this notion is supported by our demonstration that alterations in charge, such as those caused by phosphorylation, can affect membrane-targeting ability. A thorough understanding of how the AKAP12 targeting domain hierarchy is maintained and modified will be necessary to elucidate the role of this mobile signaling scaffold at its multiple subcellular locales.
| FOOTNOTES |
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Supported by National Institutes of Health Institutional Training Grant T32HL07949. ![]()
To whom correspondence should be addressed: Center for Cardiovascular Research, Aab Inst. of Biomedical Sciences, University of Rochester School of Medicine, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 585-273-1664; Fax: 585-273-1497; E-mail: j.m.miano{at}rochester.edu.
1 The abbreviations used are: AKAP, A kinase anchoring protein; AR, acidic residue-rich; BR, basic residue-rich; GFP, enhanced green fluorescent protein; GR, ganglioside-rich; hCASMC, human coronary artery smooth muscle cell; MARCKS, myristoylated alanine-rich C kinase substrate; NLS, nuclear localization signal. ![]()
2 J. W. Streb and J. M. Miano, unpublished data. ![]()
| ACKNOWLEDGMENTS |
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| REFERENCES |
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