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• W2008355265 abstract "A kinase anchoring proteins (AKAPs) assemble and compartmentalize multiprotein signaling complexes at discrete subcellular locales and thus confer specificity to transduction cascades using ubiquitous signaling enzymes, such as protein kinase A. Intrinsic targeting domains in each AKAP determine the subcellular localization of these complexes and, along with protein-protein interaction domains, form the core of AKAP function. As a foundational step toward elucidating the relationship between location and function, we have used cross-species sequence analysis and deletion mapping to facilitate the identification of the targeting determinants of AKAP12 (also known as SSeCKS or Gravin). Three charged residue-rich regions were identified that regulate two aspects of AKAP12 localization, nuclear/cytoplasmic partitioning and perinuclear/cell periphery targeting. Using deletion mapping and green fluorescent protein chimeras, we uncovered a heretofore unrecognized nuclear localization potential. Five nuclear localization signals, including a novel class of this type of signal termed X2-NLS, are found in the central region of AKAP12 and are important for nuclear targeting. However, this nuclear localization is suppressed by the negatively charged C terminus that mediates nuclear exclusion. In this condition, the distribution of AKAP12 is regulated by an N-terminal targeting domain that simultaneously directs perinuclear and peripheral AKAP12 localization. Three basic residue-rich regions in the N-terminal targeting region have similarity to the MARCKS proteins and were found to control AKAP12 localization to ganglioside-rich regions at the cell periphery. Our data suggest that AKAP12 localization is regulated by a hierarchy of targeting domains and that the localization of AKAP12-assembled signaling complexes may be dynamically regulated. A kinase anchoring proteins (AKAPs) assemble and compartmentalize multiprotein signaling complexes at discrete subcellular locales and thus confer specificity to transduction cascades using ubiquitous signaling enzymes, such as protein kinase A. Intrinsic targeting domains in each AKAP determine the subcellular localization of these complexes and, along with protein-protein interaction domains, form the core of AKAP function. As a foundational step toward elucidating the relationship between location and function, we have used cross-species sequence analysis and deletion mapping to facilitate the identification of the targeting determinants of AKAP12 (also known as SSeCKS or Gravin). Three charged residue-rich regions were identified that regulate two aspects of AKAP12 localization, nuclear/cytoplasmic partitioning and perinuclear/cell periphery targeting. Using deletion mapping and green fluorescent protein chimeras, we uncovered a heretofore unrecognized nuclear localization potential. Five nuclear localization signals, including a novel class of this type of signal termed X2-NLS, are found in the central region of AKAP12 and are important for nuclear targeting. However, this nuclear localization is suppressed by the negatively charged C terminus that mediates nuclear exclusion. In this condition, the distribution of AKAP12 is regulated by an N-terminal targeting domain that simultaneously directs perinuclear and peripheral AKAP12 localization. Three basic residue-rich regions in the N-terminal targeting region have similarity to the MARCKS proteins and were found to control AKAP12 localization to ganglioside-rich regions at the cell periphery. Our data suggest that AKAP12 localization is regulated by a hierarchy of targeting domains and that the localization of AKAP12-assembled signaling complexes may be dynamically regulated. Multicellular organisms require extensive signaling networks to coordinate physiological responses to a diverse array of stimuli. As organismal complexity increases, so theoretically does the requirement for an increased complement of signal transduction machinery. Whereas expansion of some kinase families appears to parallel increasing organismal complexity (1Gu J. Gu X. Gene. 2003; 317: 49-57Crossref PubMed Scopus (40) Google Scholar), the protein kinase A family has undergone little expansion (2Canaves J.M. Taylor S.S. J. Mol. Evol. 2002; 54: 17-29Crossref PubMed Scopus (93) Google Scholar) despite the large number of its physiological substrates distributed throughout the cell. Thus, intrinsic differences between protein kinase A family members cannot fully account for such diversity because, on their own, they do not provide sufficient specificity. Instead, specificity appears to be conferred by an ever expanding pseudo-family of anchoring proteins, the A kinase anchoring proteins (AKAPs), 1The 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. that compartmentalize protein kinase A-containing signaling complexes to discrete subcellular locales (3Michel J.J. Scott J.D. Annu. Rev. Pharmacol. 2002; 42: 235-257Crossref Scopus (289) Google Scholar). 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 (4Colledge M. Scott J.D. Trends Cell Biol. 1999; 9: 216-221Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar). 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 (5Indolfi C. Stabile E. Coppola C. Gallo A. Perrino C. Allevato G. Cavuto L. Torella D. Di Lorenzo E. Troncone G. Feliciello A. Avvedimento E.V. Chiariello M. Circ. Res. 2001; 88: 319-324Crossref PubMed Scopus (45) Google Scholar). 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 (6Dell'Acqua M.L. Faux M.C. Thorburn J. Thorburn A. Scott J.D. EMBO J. 1998; 17: 2246-2260Crossref PubMed Scopus (203) Google Scholar), 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 β-adrenergic receptor, and calmodulin (7Nauert J.B. Klauck T.M. Langeber L.K. Scott J.D. Curr. Biol. 1997; 7: 52-62Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 8Chapline C. Mousseau B. Ramsay K. Duddy S. Li Y. Kiley S.C. Jaken S. J. Biol. Chem. 1996; 271: 6417-6422Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 9Shih M. Lin F. Scott J.D. Wang H. Malbon C.C. J. Biol. Chem. 1999; 274: 1588-1595Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 10Tao J. Wang H.Y. Malbon C.C. EMBO J. 2003; 22: 6419-6429Crossref PubMed Scopus (99) Google Scholar, 11Lin X. Tombler E. Nelson P.J. Gelman I.H. J. Biol. Chem. 1996; 271: 28430-28438Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 12Lin X. Gelman I.H. Biochem. Biophys. Res. Commun. 2002; 290: 1368-1375Crossref PubMed Scopus (42) Google Scholar). We recently demonstrated that the AKAP12 gene encodes three transcriptionally separate AKAP12 isoforms, α, β, and γ, that have distinct spatio-temporal expression patterns (13Streb J.W. Kitchen C.M. Gelman I.H. Miano J.M. J. Biol. Chem. 2004; 279: 56014-56023Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Although all three isoforms share >95% of their coding sequence, an N-terminal myristoylation motif directs AKAP12α 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 (14Lin X. Nelson P.J. Gelman I.H. Mol. Cell. Biol. 2000; 20: 7259-7272Crossref PubMed Scopus (106) Google Scholar) and β-adrenergic receptor functions at the cell membrane (10Tao J. Wang H.Y. Malbon C.C. EMBO J. 2003; 22: 6419-6429Crossref PubMed Scopus (99) Google Scholar). 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. Cell Culture—COS-7 cells were maintained at 37 °C in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and transfected with the indicated plasmids using FuGENE 6 (Roche Applied Science) per the manufacturer's directions. Primary human coronary artery smooth muscle cells (hCASMCs) were obtained from Cascade Biologics and cultured per the manufacturers' directions. For differentiation experiments, growth media were replaced with SMC differentiation media (Cascade Biologics) for 7 days. hCASMCs were used prior to passage 10. Experiments using hCASMC cells were repeated with independent isolates. Plasmid Constructs—AKAP12β-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 AKAP12β-GFP reporter construct, which displays similar localization as endogenous AKAP12, as the template for the deletions reported in this study (13Streb J.W. Kitchen C.M. Gelman I.H. Miano J.M. J. Biol. Chem. 2004; 279: 56014-56023Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). 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. Microscopy—Localization of GFP fusion proteins was assessed 48 h post-transfection using a BX51 microscope and a 60× water immersion lens (Olympus) as described previously (13Streb J.W. Kitchen C.M. Gelman I.H. Miano J.M. J. Biol. Chem. 2004; 279: 56014-56023Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). 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 (15Kenworthy A.K. Petranova N. Edidin M. Mol. Biol. Cell. 2000; 11: 1645-1655Crossref PubMed Scopus (392) Google Scholar). Cells were then rinsed twice with PBS and immediately visualized. In some experiments the number of cells with peripheral AKAP12β-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 (13Streb J.W. Kitchen C.M. Gelman I.H. Miano J.M. J. Biol. Chem. 2004; 279: 56014-56023Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Computer Analysis—The charge plot profile for AKAP12 was generated using the charge program of EMBOSS via web interface (bioweb.pasteur.fr/seqanal/interfaces/charge.html) (16Rice P. Longden I. Bleasby A. Trends Genet. 2000; 16: 276-277Abstract Full Text Full Text PDF PubMed Scopus (6537) Google Scholar). The rat AKAP12 β sequence (AY695057) 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 (17Ovcharenko I. Boffelli D. Loots G.G. Genome Res. 2004; 14: 1191-1198Crossref PubMed Scopus (46) Google Scholar). Briefly, sequences for rat, mouse (fusion of open reading frames of BY002721 and NM_031185 to create mouse AKAP12β), and human (NM_144497) AKAP12β 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. Charge and Conservation of AKAP12—We recently characterized the AKAP12 gene locus and identified three separate AKAP12 transcripts, α, β, and γ (13Streb J.W. Kitchen C.M. Gelman I.H. Miano J.M. J. Biol. Chem. 2004; 279: 56014-56023Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). These transcripts encode distinct but largely similar proteins. Although an N-terminal myristoylation motif targets the AKAP12α protein to the endoplasmic reticulum, mutants lacking this motif localize in a similar manner as the β and γ 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 AKAP12β and AKAP12γ as well as those of the AKAP12α myristoylation mutants are highly similar, we have used AKAP12β (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 Localization—To 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 (Δ791-1607) 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. Nuclear Localization Potential of AKAP12—The nuclear localization potential of AKAP12 is intriguing because a recent study has demonstrated the presence of AKAP12 in nuclear fractions, a finding that likely relates to its ability to control cell cycle progression through regulation of cyclin D1 compartmentalization (14Lin X. Nelson P.J. Gelman I.H. Mol. Cell. Biol. 2000; 20: 7259-7272Crossref PubMed Scopus (106) Google Scholar). In addition, we have observed differential compartmentalization of AKAP12 in some primary cell lines. In growing hCASMCs, for example, AKAP12 is predominantly localized in the nucleus with some expression in the cytosol (Fig. 2). Upon differentiation, however, AKAP12 expression diminishes greatly in the nucleus and appears to be redirected to the perinuclear region of the cytosol (Fig. 2) The reduction of AKAP12 nuclear expression is not due to reduced protein expression as detected by Western blotting (data not shown). AKAP12 also appears at times to be localized to the nucleus in vivo. When we examined tissues with high levels of AKAP12β expression, we noted nuclear AKAP12 in a subpopulation of mesenchymal cells of the bladder and lung (data not shown). Because our deletion study suggested the determinants for nuclear targeting are contained in the region spanning amino acids 486-790, we assessed whether this region was sufficient for nuclear targeting. A fusion construct comprising GFP and a sub-fragment of this region (amino acids 501-767) localizes predominantly to the nucleus, confirming that the nuclear targeting information is retained in this region (Fig. 3, B and C). As this region was localized in the same fashion as larger constructs, we focused our investigation of nuclear targeting on this subregion of AKAP12. The nuclear targeting region spans the fourth to seventh BR regions. Using PSORT (psort.org/), we scanned this region and the rest of AKAP12 to identify potential NLSs (18Nakai K. Horton P. Trends Biochem. Sci. 1999; 24: 34-36Abstract Full Text Full Text PDF PubMed Scopus (1838) Google Scholar). Several bipartite and SV40-type NLSs were found. The bipartite NLSs were found outside of the nuclear targeting region and do not appear to contribute to nuclear localization (data not shown). However, four SV40-type NLSs were identified in the nuclear targeting region, with one identified in each BR region (Fig. 3A). Because the region encompassing only these four NLSs is sufficient for nuclear localization (Fig. 3, B and C), we constructed further deletions to determine whether the NLSs are necessary for nuclear targeting. Deletion of a single NLS does not affect targeting, nor is the targeting of constructs containing only the two N-terminal NLSs or the two central NLSs altered (Fig. 3D). In contrast, a construct containing only the two C-terminal most NLSs is not specifically targeted to the nucleus, suggesting that the NLSs are not equal in their ability to target to the nucleus. This notion is supported by further deletions, as only the region spanning the second NLS is sufficient on its own for nuclear targeting. Taken with the other deletions, this last finding indicates that nuclear localization is only observed when the second NLS is present. Although NLS2 appears to be the dominant NLS, the result was somewhat surprising because NLS2 differs little in sequence with the other three NLSs (Fig. 3A). Identification of a Novel Class of NLS—Using 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) (19Rech J. Barlat I. Veyrune J.L. Vie A. Blanchard J.M. J. Cell Sci. 1994; 107: 3029-3036Crossref PubMed Google Scholar, 20Gauthier-Rouviere C. Vandromme M. Lautredou N. Cai Q.Q. Girard F. Fernandez A. Lamb N. Mol. Cell. Biol. 1995; 15: 433-444Crossref PubMed Scopus (43) Google Scholar) (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. Both pairs of basic residues are necessary for the X2-NLS to target to the nucleus. Substitution of either pair with alanines abolishes specific nuclear localization (Fig. 4E). In contrast, substitution of the serines with alanines does not affect localization, indicating that the pairs of basic residues are the primary targeting determinant of this class of NLS (data not shown). It should be noted that the other two classes of NLS, the SV40-type and the bipartite, are also composed of two pairs of basic residues, though some substitution is allowed (e.g. proline at position 1 of the SV40-type NLS). In these cases, the spacing between pairs is 0 and 10 amino acids, respectively. In addition to differing in spacing, at least two of the NLS types are substantially different in their ability to target to the nucleus. Whereas no specific nuclear localization is observed with any of the single SV40-type NLS chimaeras, the single X2-NLS is sufficient for targeting to the nucleus (Figs. 3D and 4C). Together, these data indicate that the nuclear targeting region of AKAP12 (amino acids 501-767) contains five NLSs split between two NLS classes that differ in their ability to confer nuclear targeting. The Five NLSs Contribute to Nuclear Localization Potential—To 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). The C Terminus of AKAP12 Prevents Nuclear Localization—The presence of five functional NLSs suggests that AKAP12 should be localized to the nucleus. However, nuclear localization of AKAP12 is not always observed, indicating that AKAP12 is being excluded from the nucleus. Our initial deletion mapping experiments indicate that this exclusion is mediated by the AKAP12 C-terminal region spanning amino acids 787-1607 (Fig. 1). Moreover, fusion of this region to GFP is sufficient to exclude GFP from the nucleus (Fig. 6). To determine whether nuclear export mediates AKAP12 exclusion, we scanned this region for nuclear export sequences and examined the effect of the nuclear export inhibitor, leptomycin B, on targeting. Presently known nuclear export mechanisms are unlikely to be the mechanisms by which the C terminus of AKAP12 mediates nuclear exclusion, as this region lacks any clear nuclear export signals, and its targeting is not affected by leptomycin B (data not shown). 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 (21Grozinger C.M. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7835-7840Crossref PubMed Scopus (504) Google Scholar), 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. The C Terminus of AKAP12 Antagonizes NLSs—As the GFP reporter we used to assess nuclear exclusion is not specifically targeted and lacks a NLS, we sought to determine whether the C terminus can specifically suppress nuclear targeting conferred by a NLS. To generate a specifically targeted GFP reporter, we tandemly fused three copies of the SV40 NLS to the C terminus of GFP (GFP-3×NLS) (Fig. 7). An AKAP12 C terminus-3×NLS chimera was then created to test the ability of this region to antagonize specific nuclear targeting. Whe" @default.
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• W2008355265 title "Cross-species Sequence Analysis Reveals Multiple Charged Residue-rich Domains That Regulate Nuclear/Cytoplasmic Partitioning and Membrane Localization of A Kinase Anchoring Protein 12 (SSeCKS/Gravin)" @default.
• W2008355265 cites W1604547377 @default.
• W2008355265 cites W1986449829 @default.
• W2008355265 cites W1996423252 @default.
• W2008355265 cites W1998510219 @default.
• W2008355265 cites W2000990440 @default.
• W2008355265 cites W2006211612 @default.
• W2008355265 cites W2011092125 @default.
• W2008355265 cites W2020645222 @default.
• W2008355265 cites W2041305500 @default.
• W2008355265 cites W2043804549 @default.
• W2008355265 cites W2062574001 @default.
• W2008355265 cites W2090576076 @default.
• W2008355265 cites W2104471705 @default.
• W2008355265 cites W2105375733 @default.
• W2008355265 cites W2112534728 @default.
• W2008355265 cites W2116450965 @default.
• W2008355265 cites W2133446638 @default.
• W2008355265 cites W2136259733 @default.
• W2008355265 cites W2137211481 @default.
• W2008355265 cites W2137500801 @default.
• W2008355265 cites W2142682942 @default.
• W2008355265 cites W2144796569 @default.
• W2008355265 cites W2171058117 @default.
• W2008355265 cites W2172247457 @default.
• W2008355265 cites W2182357005 @default.
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