Regulation of the Subcellular Localization of the G-protein Subunit Regulator GPSM3 through Direct Association with 14-3-3 Protein*

Background: GPSM3, a guanine nucleotide dissociation inhibitor acting on Gαi subunit family members, is both cytoplasm- and nucleus-localized. Results: GPSM3 interacts directly with 14-3-3 through a phosphorylation-dependent mechanism. Conclusion: Interaction with 14-3-3 stabilizes a cytoplasmic pool of GPSM3. Significance: Our findings represent the first demonstration of post-translational modulation of GPSM3, a hematopoietic-restricted G-protein subunit regulator. G-protein signaling modulator-3 (GPSM3), also known as G18 or AGS4, is a member of the Gαi/o-Loco (GoLoco) motif containing proteins. GPSM3 acts through its two GoLoco motifs to exert GDP dissociation inhibitor activity over Gαi subunits; recently revealed is the existence of an additional regulatory site within GPSM3 directed toward monomeric Gβ subunits during their biosynthesis. Here, using in silico and proteomic approaches, we have found that GPSM3 also interacts directly with numerous members of the 14-3-3 protein family. This interaction is dependent on GPSM3 phosphorylation, creating a mode II consensus 14-3-3 binding site. 14-3-3 binding to the N-terminal disordered region of GPSM3 confers stabilization from protein degradation. The complex of GPSM3 and 14-3-3 is exclusively cytoplasmic, and both moieties mutually control their exclusion from the nucleus. Phosphorylation of GPSM3 by a proline-directed serine/threonine kinase and the resultant association of 14-3-3 is the first description of post-translational regulation of GPSM3 subcellular localization, a process that likely regulates important spatio-temporal aspects of G-protein-coupled receptor signaling modulation by GPSM3.

exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP) by the heterotrimeric G-protein ␣ subunit. This active, GTP-bound G␣ subunit will thus dissociate from the G␤␥ dimer and modulate intracellular effectors such as adenylyl cyclases and phospholipases (5,6). Signal termination is regulated by the action of "regulator of G-protein signaling" (RGS) proteins that enhance the intrinsic GTPase activity of the G␣ subunit (7,8). The inactive, GDP-bound G␣ subunit is generally thought to reassociate with the G␤␥ dimer to form the inactive heterotrimer. However, a more recently discovered family of G-protein modulators, containing the conserved GoLoco 2 motif, also interacts with inactive, GDP-bound G␣ subunits of the G i subfamily known to inhibit adenylyl cyclases (8 -10). The GoLoco motif interaction inhibits spontaneous GDP release by the bound G␣ i subunit (11,12) and also occludes its G␤␥ binding site (13), thereby inhibiting reassociation with G␤␥ (14).

EXPERIMENTAL PROCEDURES
Commercial Antibodies, Constructs, and Other Reagents-Horseradish peroxidase (HRP)-conjugated anti-hemagglutinin (HA) monoclonal antibody (clone 3F10) was obtained from Roche Applied Science. Anti-␤-actin, anti-FLAG M2 antibody, and agarose-conjugated anti-FLAG M2 antibody were purchased from Sigma. HRP-conjugated goat anti-mouse and goat anti-rabbit antibodies were from GE Healthcare. Polyclonal pan anti-14-3-3 antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-GPSM3 antibody was produced in-house and has been previously described (20). All cDNAs used in this study were cloned in the pcDNA3.1 backbone vector (Invitrogen), with FLAG or HA epitope tag sequences included in the forward PCR primer to produce N-terminal tagged open reading frames as described previously (20) or in the reverse PCR primer to produce C-terminal tagged GSK3␣. All mutagenesis was performed using the QuikChange site-directed mutagenesis kit following the manufacturer's recommendations (Agilent Technologies, Santa Clara, CA).
Cell Culture and Transfection-Human embryonic kidney 293 (HEK293) and THP-1 cell lines were each obtained from the American Type Culture Collection (ATCC) and maintained in DME medium or RPMI 1640 medium (Invitrogen), respectively, supplemented with 10% fetal bovine serum (Cellgro, Manassas, VA) at 37°C in a humidified atmosphere containing 5% CO 2 . Transient transfections of cell monolayers grown to 75-90% confluence were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.
Immunoprecipitation and Immunoblotting-Cells were lysed with ice-cold lysis buffer (20 mM HEPES, pH 7.5, 1 mM EDTA, 150 mM NaCl, 1% Nonidet P-40, and Complete protease inhibitor mixture tablets (Roche Applied Science)) at 4°C on a rocker platform for 30 min. Lysates were clarified by centrifugation at 16,000 ϫ g for 15 min at 4°C and quantified by the bicinchoninic acid (BCA) protein content assay (Pierce). For immunoprecipitation, lysates were incubated with specific antibody for 2 h at 4°C followed by overnight incubation with protein-A/G agarose (Santa Cruz Biotechnology) or directly incubated with agarose-conjugated anti-FLAG M2 antibody overnight. Pelleted antibody/bead complexes were then washed three times with lysis buffer, and proteins were eluted in Laemmli buffer. Eluted proteins or lysate samples were resolved on 4 -12% precast SDS-polyacrylamide gels (Novex/ Invitrogen), transferred to nitrocellulose, immunoblotted using primary and HRP-conjugated secondary antibodies, and visualized by chemiluminescence (ECL, GE Healthcare).
Mass Spectrometry Analysis-Immunoprecipitation of FLAGtagged GPSM3 and subsequent mass spectrometric identification of co-immunoprecipitating proteins were performed as described previously (20). For post-translational modification analysis, FLAG-tagged GPSM3 immunocomplex beads were submitted to the Systems Proteomics Center of University of North Carolina School (UNC)-Chapel Hill (directed by Dr. Oscar Alzate) and analyzed for post-translational modification using two-dimensional-differential in gel electrophoresis (23). Phosphorylation detected within GPSM3 was further analyzed using tandem mass spectrometry.
Bioluminescence Resonance Energy Transfer (BRET)-HEK293 cells were seeded in 12-well plates (3.5 ϫ 10 5 cells/well) and transfected with a fixed amount of 14-3-3-RLuc vector DNA (50 ng), increasing amounts of GFP10-GPSM3 vector DNA (0 -1500 ng), and corresponding (decreasing) amounts of pcDNA3 empty vector (1500 -0 ng) to obtain a saturation curve. The other BRET assays were performed by transfecting cells with 50 ng of the Renilla luciferase (RLuc) fusion proteinexpressing vector with 750 ng of GFP10 fusion protein expressing vector with 750 ng of additional "challenge" constructs or pcDNA3 vector. 24 h after transfection, cells were washed once, harvested, resuspended in BRET buffer (phosphate-buffered saline with 1 mM CaCl 2 , 0.5 mM MgCl 2 , 0.1% glucose), and distributed in white 96-well microplates. BRET was initiated by adding coelenterazine-400a at a final concentration of 5 M. Measurements of emitted light were collected on a Mithras LB-940 plate reader (Berthold Technologies) using a BRET 2 filter set.
Bimolecular Fluorescence Complementation (BiFC)-Fusion constructs were made similar to those previously described for G␤/GPSM3 tracking (20): namely, a fusion of the N-terminal fragment (amino acids 1-158) of yellow fluorescent protein (YN) to the N terminus of full-length GPSM3 (YN-GPSM3) and the C-terminal fragment (amino acids 159 -238) of YFP (YC) to the C terminus of 14-3-3 (14-3-3-YC). HEK293 cells were transfected with an equal amount of plasmids encoding the fusion proteins YN-GPSM3 and 14-3-3-YC, and cells were incubated at 37°C for 24 h. Total DNA quantity was normalized using empty pcDNA3.1 vector DNA. To measure fluorescence from formed complexes, transfected cells were washed, harvested, and resuspended in PBS. BiFC signal was acquired using a Mithras LB-940 plate reader using an excitation/emission filter set of 485 and 510 nm. The level of expression of each fusion protein was quantified by Western blotting using a polyclonal antibody directed against the GFP.
Immunofluorescence Microscopy-HEK293 cells were seeded in a 12-well plate and transfected with 0.4 g of each DNA: YN-GPSM3 and 14-3-3-YC. The following day, cells were transferred to a poly-D-lysine-coated coverslip in a 6-well plate and grown overnight. Cells were then fixed with 4% paraformaldehyde plus PBS for 10 min at room temperature. Cells were then washed with PBS and permeabilized with 0.1% Triton X-100 plus PBS for 30 min at room temperature. Nonspecific binding was blocked with 0.1% Triton X-100 plus PBS containing 5% nonfat dry milk for 30 min at room temperature.
Cells were then incubated with an anti-HA FITC conjugate antibody (Roche Applied Science) for 1 h at room temperature in PBS supplemented with 5% nonfat dry milk. Cells were then washed three times with permeabilization buffer. Finally, coverslips were mounted using VECTASHIELD mounting medium containing DAPI (Vector Laboratories, Burlingame, CA) and examined by inverted fluorescence microscopy (Olympus IX70) using a 40ϫ objective.

RESULTS AND DISCUSSION
Interaction between GPSM3 and 14-3-3 Family Members Stabilizes GPSM3-The N-terminal 50 amino acids of GPSM3 are of low complexity and predicted (44) to be disordered (e.g. supplemental Fig. S1). Truncation of the first 30 amino acids has little or no effect on ectopic GPSM3 expression (Fig. 1A). However, N-terminal deletion beyond 30 amino acids was observed to dramatically reduce levels of expressed GPSM3. Analysis of the primary sequence revealed the presence of a potential binding site for 14-3-3 between amino acids 31 and 40 (supplemental Fig. S2A). 14-3-3 binding sites are often localized within disordered regions of a protein; it is thought that interaction with disordered regions facilitates ligand capture and transition from a disordered to ordered state that stabilizes complex formation (25,26).
As described previously (20), a yeast two-hybrid screen of a human leukocyte cDNA library was performed to identify novel interacting partners of GPSM3; as shown in Fig. 1B, numerous clones and isoforms of 14-3-3 were identified in this screen. Fig.  1C demonstrates the specific interaction between full-length GPSM3 used as bait and one of the retrieved 14-3-3 clones, identified as the full-length 14-3-3 isoform. An additional approach taken to identify GPSM3 binding partners, also described previously (20), employed tandem mass spectrometry identification of proteins present in an immunoprecipitated complex of ectopically expressed, FLAG-tagged GPSM3, confirming the presence of multiple isoforms of 14-3-3 (Fig. 1D).
Mapping the Interaction between GPSM3 and 14-3-3 and Its Consequence on GPSM3 Stability-To confirm whether GPSM3 interacts with 14-3-3 proteins and identify the motif involved, we introduced point mutations into full-length, FLAG-tagged GPSM3 and its interaction with HA-tagged 14-3-3 was assessed by co-immunoprecipitation. We first observed that point mutation of arginines 31 and 34 (R31,34A), 3 serine 35 (S35A), or serine 39 (S39A) within GPSM3 each reduced the level of ectopically expressed GPSM3 protein observed ( Fig. 2A); doubling the quantity of cDNA of each mutant resulted in a similar level of expression to that of wild-type GPSM3. We then used these altered transient transfection conditions to test their relative capability to co-immunoprecipitate with HA-tagged 14-3-3. The R31,34A and S35A point mutants of GPSM3 failed to interact with 14-3-3, and a considerable reduction of the interaction was observed with the S39A mutant (Fig. 2, B and C); these observations confirm site 1 of GPSM3 as important to the binding of 14-3-3. Mutations to GPSM3 residues R31,34 and Ser-35 were also seen to affect its interactions with the ␣/␤ and isoforms of 14-3-3, whereas the S39A mutation reduced interaction with 14-3-3 ␣/␤ and isoforms but had little effect with the 14-3-3 isoform (Fig. 2, C, D, and E). As already described for other 14-3-3 substrates (27), mutation of GPSM3 residue serine 35 or 39 to aspartic acid was found to be ineffective in providing a phosphomimetic residue that enhances 14-3-3 binding (Fig. 2, C, D, and E). No effect on 14-3-3 binding was observed with S56A, S153A, or T157A mutations to GPSM3 (Fig. 2B), excluding the other two sites identified in silico.
Two additional point mutants of 14-3-3 isoforms have been well characterized; mutation of serine 58, known to be phosphorylated by PKA or PKB/AKT1, to alanine (S58A) induces constitutive dimerization of 14-3-3, whereas mutation of this serine to aspartic acid (S58E) abrogates dimerization (31)(32)(33). However, as shown in Fig. 2G, neither of these mutations to serine 58 affected interaction with GPSM3, suggesting that the GPSM3 interaction with 14-3-3 is independent of the dimeriza-tion status of 14-3-3. Homo-and heterodimerization of 14-3-3 proteins is thought to be important for their cellular functions (33). The 14-3-3 dimer adopts a cup-like shape that can interact simultaneously with two binding sites within the same protein, thereby inducing conformational change and regulating protein activity or regulating other interactions via competition or occlusion. The presence of only one 14-3-3 binding site in GPSM3 and the binding of either dimeric or monomeric 14-3-3 to GPSM3 suggest that the 14-3-3 interaction could represent an important scaffolding function and/or stabilize formation of a multiprotein complex (21,25).
The decreased stability of the 14-3-3 binding loss-of-function GPSM3 mutant R31,34A was further explored. FLAGtagged GPSM3 was expressed in HEK293 cells in the presence or absence of 200 g/ml cycloheximide to block de novo protein synthesis, and GPSM3 protein levels were subsequently tracked over time by Western blotting. Only prolonged incubation (Ͼ12 h) with cycloheximide was seen to dramatically reduce the protein level of wild-type GPSM3 (Fig. 3). In contrast, the R31,34A mutant of GPSM3 was found to have reduced expression following 6 h of cycloheximide treatment and was almost undetectable at 12 h after treatment (Fig. 3), indicating that the loss-of-function mutant has reduced stability as compared with wild-type GPSM3.
Endogenous GPSM3 Associates with Endogenous 14-3-3 in a Monocytic Cell Line-GPSM3 is proposed to be a hematopoietic restricted protein, and we have previously shown that the human monocytic cell line THP-1 expresses detectable levels of endogenous GPSM3 (20). The interaction between endogenous GPSM3 and 14-3-3 proteins was confirmed in THP-1 cells using co-immunoprecipitation with an in-house anti-GPSM3 monoclonal antibody as well as a polyclonal pan-14-3-3 antibody (Fig. 2H).
Characterization of the Interactions between GPSM3 and Its Binding Partners Using BRET-We also examined the interactions between GPSM3, 14-3-3, and G-protein subunits in a cellular context using BRET between RLuc-and green fluorescent protein (GFP10)-tagged fusions. Saturation BRET has been established as a robust measure of the specificity of an interaction (34). HEK293 cells transfected with a constant amount of 14-3-3-expressing donor construct (14-3-3-RLuc) and increasing amounts of the GPSM3 acceptor fusion expression vector (GFP10-GPSM3) were observed to produce a net BRET signal resulting in a saturable curve (Fig. 4A); mutation of the two arginines within the 14-3-3 binding site of GPSM3 (mutant R31,34A; Fig. 2C) eliminated BRET under the same experimental conditions (Fig. 4A). In addition, S35A and S39A point mutations within this site 1 in GPSM3 also greatly reduced the observed BRET signal as compared with the use of wild-type GPSM3 (Fig. 4B), confirming the results obtained by co-immunoprecipitation (Fig. 2B).
We previously reported that GPSM3 associates with heterotrimeric G-protein ␤ subunits (G␤) during their biosynthetic pathway toward formation of the G␤␥ heterodimer (20). This GPSM3/G␤ interaction was found to be independent of the well established G␣ i /GDP interactions mediated by two of three GoLoco motifs within GPSM3 (19). We employed the BRET assay to evaluate the effect of 14-3-3 on both of these 3 R31,34A ϭ R31A and R34A.
Conversely, overexpression of G␤ 1 was seen to greatly reduce the interaction between GPSM3 and 14-3-3, as shown both by BRET (Fig. 4E) and by co-immunoprecipitation studies (Fig.  4F). The mechanism underlying this unidirectional modulation of the GPSM3/14-3-3 interaction by G␤ subunit expression, while 14-3-3 expression did not cause a reciprocal decrease in GPSM3/G␤ association, remains to be established, but it is unlikely to be direct steric competition between G␤ and 14-3-3 as this effect would have been seen to be bidirectional.

FIGURE 2. Identification of the 14-3-3 binding motif within GPSM3 and the endogenous GPSM3/14-3-3 interaction within THP-1 cells. A, point mutations within a region comprising amino acids 31 and 40 of GPSM3 perturb its stable expression. Mutagenized, FLAG-tagged GPSM3 expression constructs were transfected into HEK293 cells, and GPSM3 protein was immunoprecipitated (IP) using anti-FLAG M2 antibody. Immunoprecipitates were resolved by 4 -12% SDS-PAGE and immunoblotted (IB) with anti-FLAG antibody. Lysates were resolved on 4 -12% SDS-PAGE and immunoblotted with anti-␤-actin as loading control. B-E, serines 35 and 39, as well as arginines 31 and 34, were identified as essential determinants for the interaction with 14-3-3 isoforms. Point-mutated, FLAG-tagged GPSM3 constructs were co-transfected with HA-tagged, wild type 14-3-3 (B and C), ␣/␤ (D), (E), 14-3-3 R56A mutant (F), and 14-3-3 S58A or S58E mutants (G); immunoprecipitation was performed as in panel A and immunoblotted with anti-HA-and anti-FLAG-HRP conjugates. H, detection of an endogenous GPSM3/14-3-3 interaction. Whole cell lysate from THP-1 cells was immunoprecipitated with the anti-
Direct Interaction between Recombinant GPSM3 and 14-3-3-To confirm the direct interaction between GPSM3 and 14-3-3, both proteins were first purified from recombinant expression systems. His-tagged 14-3-3 was expressed in Escherichia coli, purified by Ni-NTA affinity and size-exclusion chromatographies, and immobilized on a surface plasmon resonance (SPR) biosensor chip as described previously (24); the E. coli-derived 14-3-3 was dimeric, as assessed by migration during calibrated size-exclusion chromatography. Although GPSM3 was expressed and purified from E. coli, we also purified recombinant GPSM3 protein using baculovirus-mediated expression in Sf9 insect cells in which endogenous protein serine/threonine kinases are present. Recombinant GPSM3 purified from E. coli was observed to interact with immobilized 14-3-3 (Fig. 5A), but apparently with lower affinity than GPSM3 purified from insect cells; i.e. the Sf9-sourced protein bound immobilized 14-3-3 with higher apparent affinity (Fig. 5, A  and B), suggesting its phosphorylation during insect cell expression. Given difficulties in attaining a true equilibrium state or single-phase association/dissociation kinetics using this SPR biosensor approach, we were unable to assign a numerical dissociation constant (K D ) value to the interaction using either equilibrium or kinetic approaches; however, based on the dose-response data obtained (e.g. Fig. 5B), we estimate the K D for the GPSM3/14-3-3 interaction to be in the submicromolar range.
Analysis of the GPSM3/14-3-3 Interaction and Subcellular Localization by BiFC-BiFC is based on the reconstitution of two unfolded counterparts, such as the N-and C-terminal halves (YN and YC) of yellow fluorescent protein (YFP), to form a stable fluorescent protein. In addition to confirming the interaction between two proteins, the fluorescent signal generated can be used to locate the site of interaction within the cell by simple fluorescence microscopy visualization (35). For exam-ple, we previously used BiFC to show that the interaction between GPSM3 and G␤ subunits occurs in the cytoplasm and can also form juxtanuclear aggregates (20). Here, the N-terminal portion of YFP was fused to the N terminus of GPSM3 (YN-GPSM3), whereas the C-terminal fragment of YFP was fused to the C terminus of 14-3-3 (14-3-3-YC). Expression of each construct alone (YN-GPSM3 or 14-3-3-YC) or expression of each fusion paired with the cognate fluorescent protein fragment lacking the fusion bait gave no appreciable fluorescence, as expected. In contrast, combining expression of YN-GPSM3 with 14-3-3-YC generated a strong BiFC signal (Fig. 6A).
Co-expression of nontagged 14-3-3 significantly inhibited the BiFC signal (Fig. 6A), a measure of the specificity of the GPSM3/14-3-3 interaction. Unlike the interaction between GPSM3 and G␤ subunits that was found to lead to juxtanuclear aggregates over time (20), the interaction between GPSM3 and 14-3-3 observed by BiFC was diffusely located throughout the cytoplasm and completely excluded from the nucleus. This pattern of subcellular localization did not change over time after transfection and was not affected by the level of expression (e.g. Fig. 6B).
GPSM3 and 14-3-3 Mutually Affect Their Subcellular Localizations-GPSM3 expression has previously been observed throughout the cell including the nucleus, either when overexpressed or at the endogenous level (20); therefore, we next evaluated whether the interaction with 14-3-3 could regulate the subcellular localization of GPSM3. mCherry-tagged, wild-type GPSM3 was seen to be distributed throughout transfected cells, including the nucleus, but with a more pronounced cytoplasmic localization than that seen in the nucleus (Fig. 7A). In contrast, the 14-3-3 binding mutant of GPSM3 (R31,34A; as an mCherry fusion) was observed to be evenly distributed throughout transfected cells, without any distinction between nuclear and cytosolic compartments (Fig. 7A). The presence of ectopically expressed 14-3-3 increased the observed exclusion of wild-type GPSM3 from the nucleus (Fig. 7B, panel ii), but had no effect on the R31,34A mutant (Fig. 7B, panel iii). In these studies, we found HA-tagged 14-3-3 to be distributed throughout the cell when expressed alone (Fig. 7B, panel i) or with the loss-of-function R31,34A GPSM3 mutant (Fig. 7B,  panel iii). On the other hand, HA-tagged 14-3-3 was excluded from the nucleus in the presence of wild type mCherry-GPSM3 (Fig. 7B, panel ii).
In silico analyses of potential serine/threonine kinases that could phosphorylate serine 35 (e.g. Scansite) reveal the mode II consensus 14-3-3 binding sequence within GPSM3 (R 31   phorylation target, X is any amino acid, and pS/T is the priming phosphorylation site. GSK3 has an unusual preference for substrates that are prephosphorylated at a priming, C-terminal Ser/Thr residue; this priming process, although not strictly required, increases phosphorylation efficiency of the N-terminal Ser/Thr by 100 -1000-fold (37). Moreover, GSK3 favors the presence of proline in the recognition site; for example, one of the GSK3 phosphorylation sites in glycogen synthase is SVPPpS (38). GPSM3 serine 35, required for the 14-3-3 interaction, fits perfectly into this GSK3 recognition sequence. Furthermore, serine 39 was found to be phosphorylated in the mass spectrometry analysis of GPSM3 post-translational modifications (supplemental Fig. S3), and mutation of the serine 39 to alanine (S39A) reduces the GPSM3/14-3-3 interaction. As GPSM3 serine 39 is followed by a proline, we focused our studies on GSK3, a potential proline-directed kinase (39 -41), and other well characterized, proline-directed serine/threonine kinases (e.g. cyclin-dependent kinases (CDKs), c-Jun N-terminal kinases (JNKs), p38, and extracellular-signal-regulated kinases 1/2 (ERK1/2).

FIGURE 4. Characterization of the GPSM3/14-3-3 interaction using BRET. A, the interaction between GPSM3 and 14-3-3 was confirmed by a BRET saturation experiment. A constant amount of 14-3-3-RLuc fusion expression vector was co-transfected into HEK293 cells with increasing amounts of GFP10-GPSM3 fusion expression vector
; the resultant net BRET ratio was plotted as a function of the acceptor/donor ratio. The specificity of the interaction is indicated by the saturable nature of the BRET signal. B, point mutations, previously found by co-immunoprecipitation to disrupt ((S35A) or (R31,34A)) or strongly reduce (S39A) the GPSM3 interaction with 14-3-3, were introduced in the GFP10-GPSM3 construct and assayed by BRET for 14-3-3 interaction. C and D, the effect of 14-3-3 on the previously described interactions between GPSM3 and G␣ i1 or G␤ 1 (20) was assayed by BRET. G␣ i1 -RLuc or G␤ 1 -Rluc expression constructs were cotransfected in HEK293 cells with GFP10-GPSM3 (wild type or R31,34A mutant) in the presence or absence of 14-3-3 expression vector. E and F, G␤ 1 overexpression reduces interaction between GPSM3 and 14-3-3. E, nontagged G␤ 1 was co-expressed with the 14-3-3-RLuc/GFP10-GPSM3 BRET pair, and the effect on BRET signal was measured. Error bars in panels A-E indicate S.E. F, FLAG-tagged GPSM3 (WT or R31,34A) constructs were co-transfected with HA-tagged, wild-type 14-3-3 expression vector in the presence or absence of HA-tagged G␤ 1 , and immunoprecipitation was performed using agarose-conjugated anti-FLAG M2 antibody. Immunoprecipitates (IP) were resolved on a 4 -12% SDS-PAGE and immunoblotted (IB) with anti-HA-and anti-FLAG-HRP conjugates.
A similar effect was observed on endogenous GPSM3 expression in THP-1 cells, with expression reduced by more than 75% (Fig. 8B). None of the other, above mentioned inhibitors were seen to have any effect on endogenous GPSM3 levels in THP-1 cells, although H89 treatment was found to drastically reduce THP-1 cell viability.
Mammalian cells express two isoforms of GSK3, GSK3␣ and GSK3␤, which share 98% sequence similarity in their kinase domain. However, the isoforms are structurally and functionally distinct (42). There is an N-terminal glycine-rich extension in   GSK3␣ as well as distinct C-terminal regions (only 38% similarity between isoforms). When co-expressed in HEK293 cells, GSK3␣, but not GSK3␤, was found in complex with FLAG-tagged GPSM3 (Fig. 8C). The exclusion of GSK3␤, even when overexpressed, highlights the unique nature and specificity of this GSK3␣/ GPSM3 interaction. To date, only the protein adaptor RACK1 has been shown to selectively interact with GSK3␣, an interaction that involves the unique N-terminal extension of GSK3␣ (43). The mechanism by which GSK3␣ is recruited to GPSM3 as well as the regulation of this interaction is unknown.
Conclusions-A single 14-3-3 binding site exists in GPSM3 and corresponds to a consensus mode II binding motif, RX(/ S)(ϩ)(pS)XP, in which position P 0 is an obligatory pS known to interact with positively charged arginine 56 and 127 within the 14-3-3 binding groove ( isoform numbering). The arginine at position P Ϫ4 forms an intramolecular salt bridge with the P 0 phosphoserine, and the proline at position P ϩ2 orients the main chain direction of the binding peptide (25). The binding site within GPSM3 (RPWRS 35 AP) contains all requirements for an optimal, high affinity interaction; mutagenesis confirmed the critical role of these elements as the R31,34A and S35A mutants of GPSM3 completely abolish interaction with 14-3-3. Additionally, the R56A 14-3-3 mutant exhibited reduced interaction with GPSM3. Co-immunoprecipitation of endogenous GPSM3 and 14-3-3 proteins from the human monocytic cell line THP-1 suggests basal phosphorylation of GPSM3 at serine 35 as potentially mediated by GSK3␣. The GPSM3/14-3-3 interaction is seen to stabilize GPSM3 from degradation and also support the nuclear exclusion of both proteins. Future studies will be directed at establishing the specific roles of these newly described 14-3-3 and GSK3␣ interactions in the G-protein modulatory functions of GPSM3 within immune system cells.