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J. Biol. Chem., Vol. 278, Issue 38, 36323-36327, September 19, 2003
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Is Controlled by Phosphorylation of Ser58 at the Dimer Interface*
From the
Cytokine Receptor Laboratory, Division of Human Immunology, Hanson Institute, Institute of Medical and Veterinary Science, G. P. O. Box 14 Rundle Mall, Adelaide, SA 5000, and the Department of Biochemistry and Molecular Biology, Monash University, Clayton VIC 3168, Australia
Received for publication, May 6, 2003 , and in revised form, July 1, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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phosphorylated on Ser58 and employing native-PAGE and cross-linking techniques, we found that 14-3-3 phosphorylated on Ser58 is monomeric both in vitro and in vivo. Phosphorylated 14-3-3 was detected solely as a monomer, indicating that phosphorylation of a single monomer within a dimer is sufficient to disrupt the dimeric structure. Significantly, phosphorylation-induced monomerization did not prevent 14-3-3 binding to a phosphopeptide target. We propose that this regulated monomerization of 14-3-3 controls its ability to modulate the activity of target proteins and thus may have significant implications for 14-3-3 function and the regulation of many cellular processes. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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, 
, 
, 
, 
, 
, and , which are expressed from separate genes and are relatively abundant in all tissues (with the exception of and that are predominantly expressed in epithelium and T-cells, respectively). In most cases, the regulated phosphorylation of the client protein controls their binding to 14-3-3 proteins. However, the regulation of the 14-3-3 proteins themselves by phosphorylation and the consequences are virtually unknown.
14-3-3 proteins are dimeric, being composed of two 30-kDa monomers that are each capable of binding a phosphoserine motif. Each monomer is composed of nine 
helices arranged in antiparallel fashion to form an amphipathic groove that mediates phosphoserine target binding. The resolved crystal structures of 14-3-3 and 14-3-3 demonstrate the structural similarity between isoforms and explain the high degree of conservation across the family, with conserved residues lining the phosphoserine binding groove (6, 7). The N-terminal helices of 14-3-3 mediate dimer formation with helix 1 of monomer A interacting with helices 3 and 4 of monomer B. Heteroas well as homodimerization of isoforms occurs (8, 9). However, it is not known whether 14-3-3 monomers exist in vivo and if so what control mechanisms may exist to regulate their formation.
It has remained a mystery exactly how 14-3-3 can regulate so many client proteins with such diverse functions. A recently proposed model provides a possible explanation (5), describing 14-3-3 as a molecular anvil that deforms a bound phosphoserine client protein, thus affecting its conformation, altering enzymatic activity, or masking/revealing phosphorylation sites or nuclear transport sequences. This deforming activity of 14-3-3 is wholly dependent on 14-3-3 protein being dimeric and binding to multiple sites in the client protein, although initially a single 14-3-3 binding motif in the client protein may act as a dominant or "gatekeeper" site for 14-3-3 interaction. In the context of this model, monomeric 14-3-3 would retain binding to a gatekeeper site but would not exhibit any "molecular anvil" activity and would therefore be unable to perform deforming functions.
Previous studies demonstrated that a sphingosine-dependent kinase (SDK)1 isolated from Balb/c 3T3 fibroblasts phosphorylated 14-3-3 proteins in vitro on Ser58 in helix 3; however, the effect of this phosphorylation was not known (10). From the crystal structure of 14-3-3 this residue is located in the dimer interface just 5.8 Å from a conserved arginine (Arg18) in helix 1 of the opposing monomer. We have now determined the functional consequence of Ser58 phosphorylation on the structure of 14-3-3 and show that phosphorylation of this residue disrupts dimer formation. Moreover, 14-3-3 phosphorylated on Ser58 is detected in vivo in response to sphingolipid and is also found to be a monomer. These results demonstrate for the first time that the dimeric status of 14-3-3 can be regulated in vivo by site-specific phosphorylation at the dimer interface, a finding that may have profound implications for 14-3-3 function and the regulation of many cellular processes.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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(GST-14-3-3) as the immunogen. Polyclonal anti-14-3-3 antibodies were subsequently purified from rabbit serum using a GST-14-3-3 affinity column and used for immunoblotting at a dilution of 1:2000. Anti-phospho-Ser58 14-3-3 antibody (anti-pS58) was raised by immunizing New Zealand White rabbits with the phosphorylated CGARRSpSWRVVS peptide conjugated to keyhole limpet hemocyanin. The phospho-specific Ser58 antibodies were subsequently affinity purified from rabbit serum using the phospho- and an unphosphorylated form of the immunizing peptide, essentially as described previously (11). The purified anti-phospho-Ser58 was used for immunoblotting at a concentration of 1 µg/ml. Horseradish peroxidase-conjugated anti-rabbit antibody was purchased from Pierce.
Cell Culture—Balb/c 3T3 fibroblasts were maintained in Dulbecco's modified Eagle's medium supplemented with 10% v/v fetal calf serum. CTLL-2 cells were maintained in RPMI 1640 supplemented with 10% v/v fetal calf serum, 50 µM -mercaptoethanol, and 100 units/ml bacterially synthesized mouse interleukin-2.
Expression Constructs and Recombinant Protein Production—Recombinant 14-3-3 was produced in Escherichia coli from pGEX2T-14-3-3 (12). The recombinant 14-3-3 was cleaved from the immobilized GST-14-3-3 by resuspending 1 ml of GST-14-3-3 on glutathione resin (Sigma) in 2 ml of thrombin cleavage buffer (50 mM Tris-Cl, pH 8, 150 mM NaCl, 2.5 mM CaCl2, 2 mM dithiothreitol) and incubating overnight with 200 units of Thrombostat (Pfizer). The cleaved protein was then purified on a Mono Q HR5/5 (Amersham Biosciences) column after dialysis into Mono Q buffer (20 mM Tris-Cl, pH 7.5, 1 mM dithiothreitol). Cleaved 14-3-3 was eluted with a 0–0.5 M NaCl gradient in Mono Q buffer, and the purity of the 14-3-3 was determined by SDS-PAGE and Coomassie staining. Mutations were generated in the 14-3-3 cDNA by QuikChange site-directed mutagenesis (Stratagene), and mutant protein was produced and purified as described above.
In Vitro SDK Reactions—An S100 cytosolic fraction was prepared from serum-starved, PMA-stimulated Balb/c 3T3 fibroblasts as described previously (10). Aliquots of extract were frozen away at –70 °C and were subsequently used in SDK reactions to phosphorylate 14-3-3. SDK assays were carried out essentially as described previously (10) with the following changes: assays were carried out in 20-µl volume with 100 ng of recombinant 14-3-3. S100 extract made up half the assay volume, and SDK assay buffer was 20 mM Tris-Cl, pH 7.4, 15 mM MgCl2, 25 µM ATP, 3 mM dithiothreitol with or without 2 µCi of [-32P]ATP. Sphingolipid was added to a final concentration of 100 µM in 0.5% w/v n-octyl glucoside. Reactions were incubated at 37 °C for 15 min.
Cross-linking Studies—14-3-3 phosphorylated by SDK was subjected to cross-linking with bis(sulfosuccinimidyl) suberate (BS3) (Pierce). BS3 (50 µg/ml) was added to an SDK reaction after incubation and the mixture incubated for a further 5 min at 37 °C. Cross-linking was terminated by addition of ethanolamine to a final concentration on 100 µM. Cross-linked samples were subjected to SDS-PAGE, Western transferred, and immunoblotted as indicated.
Peptide Pull-downs—14-3-3 phosphorylated by SDK was precipitated after incubation for 1 h at 4 °C with 20 µl of streptavidin-agarose (Pierce) and 2 µg of biotinylated peptide (Chiron) from 0.5 ml volume of Nonidet P-40 lysis buffer (10 mM Tris-HCl, pH 7.4, 137 mM NaCl, 10% v/v glycerol, 1% v/v Nonidet P-40). The precipitated material was washed four times in Nonidet P-40 lysis buffer prior to separation by SDS-PAGE, Western transfer, and immunoblotting as indicated.
Cell Stimulation and Lysate Preparation—Balb/c 3T3 cells were stimulated at 80% confluence in 10-cm dishes. After stimulation as indicated, cells were put on ice and washed twice with phosphate-buffered saline. Cells were released from the dishes by scraping and pelletted by centrifuging at 1000 x g for 5 min at 4 °C. The cell pellets were lysed on ice in 100 µl of Nonidet P-40 lysis buffer containing protease inhibitors (10 µg/ml leupeptin, 2 mM phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin) and phosphatase inhibitors (2 mM sodium vanadate, 1 mM sodium fluoride, 1 mM sodium molybdate, 1 mM sodium pyrophosphate, and 1 mM -glycerophosphate). Cell debris was removed by centrifugation at 10,000 x g for 15 min at 4 °C and protein concentration determined by BCA assay (Pierce). 50 µg of lysate was subjected to native-PAGE and immunoblotted as indicated. CTLL-2 cells (2 x 106 per treatment) were stimulated as indicated and after pelletting by centrifugation at 400 x g for 5 min were washed with phosphate-buffered saline containing 1 mM -glycerophosphate and lysed on ice in 50 µl of Nonidet P-40 lysis buffer containing protease and phosphatase inhibitors. Cell debris was removed by centrifugation at 10,000 x g for 15 min at 4 °C, and 25 µl of lysate was subjected to native-PAGE and immunoblotted as indicated.
PAGE and Immunoblotting—Native-PAGE was carried out on 12.5% PAGE gels with 5% stacking gels. Gels were prepared using the standard Laemmli recipe but excluding SDS. SDS was also excluded from the sample load buffer. Benchmark prestained protein markers (Invitrogen) were used as an indicator of protein migration. All gels were transferred to nitrocellulose by electroblotting using standard buffers, and blots were blocked in TNT (10 mM Tris-Cl, pH 8, 150 mM NaCl, and 0.05% (v/v) Tween 20) containing 1% (w/v) blocking reagent 1096 176 (Roche Applied Science). Immunoblotting was carried overnight at 4 °C with anti-phospho-Ser58 antibody and for 1 h at room temperature with the anti-14-3-3 antibody. All washes were performed in TNT with 0.5% (w/v) blocking reagent added to antibody incubations. Immunoblots were developed by enhanced chemiluminescence using proprietary reagents (Amersham Biosciences).
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RESULTS |
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on Ser58 by an SDK Activity Disrupts 14-3-3 Dimers—A SDK activity that phosphorylates Ser58 of 14-3-3 was previously identified in cytosolic extracts of PMA-stimulated Balb/c 3T3 fibroblasts (10). We have reproduced the in vitro SDK assay system described in the original report. As shown previously (10), phosphorylation of recombinant wild type 14-3-3 was induced by sphingosine (data not shown) and the sphingosine analogue, dimethyl sphingosine (DMS) (Fig. 1A) but not by sphingosine 1-phosphate (data not shown). Furthermore, recombinant 14-3-3 mutated at Ser58 is not phosphorylated in response to DMS confirming that Ser58 is the sole site of phosphorylation by SDK (Fig. 1A). We have now raised a polyclonal antibody to a phosphopeptide corresponding to the amino acid sequence encompassing phospho-Ser58 14-3-3: CGARRSpSWRVVS, where pS represents phosphoserine. The antibody specifically recognized the phosphorylated, but not the unphosphorylated, form of the Ser58 peptide nor a phosphopeptide corresponding to another known phosphorylation site in 14-3-3, Ser185 (Fig. 1B). Using the in vitro SDK assay, we demonstrated that this anti-phosphopeptide antibody (anti-pS58) strongly recognized 14-3-3 after phosphorylation by SDK (Fig. 1C), verifying the specificity of the antibody for Ser58-phosphorylated 14-3-3. Using this unique phospho-specific antibody, we have examined the effect of Ser58 phosphorylation on 14-3-3 dimer formation.
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14-3-3 proteins are characteristically acidic proteins with pI values of between 4 and 5. This being so, they resolve well on native-PAGE when run under neutral conditions. Native-PAGE has been employed previously in 14-3-3 studies to distinguish between monomeric and dimeric 14-3-3 forms (13). Using native-PAGE in conjunction with immunoblotting with the Ser58 phosphopeptide-specific polyclonal antibody, we have examined the effect of Ser58 phosphorylation on 14-3-3 dimerization. In vitro SDK reactions were carried out either in the presence or absence of added recombinant 14-3-3, subjected to native-PAGE, and immunoblotted after Western transfer with either the phospho-specific Ser58 antibody (Fig. 2A) or with a polyclonal anti-14-3-3 antibody (Fig. 2B). Phosphorylation of both endogenous and recombinant 14-3-3 was detected with the Ser58 phospho-specific antibody only when DMS was present in the kinase reaction, corresponding to stimulation of the SDK activity (Fig. 2A). Prestained markers were used as an aid to compare the migration of immunoreactive species between blots. The mobility of the phosphorylated 14-3-3 coincides with the mobility of the seventh protein marker, whereas immunoblotting of the same reactions with anti-14-3-3 indicates that both the endogenous and recombinant 14-3-3 migrate with the sixth protein marker (Fig. 2B). Furthermore, this mobility coincides with the mobility of purified recombinant 14-3-3 as demonstrated by Coomassie Blue staining of native gels (Fig. 2C). In addition, the anti-14-3-3 antibody also detected a small amount of protein running with the seventh marker that corresponds to the protein detected by the anti-phospho-specific Ser58 antibody (Fig. 2B), identifying the faster migrating species as 14-3-3. This increase in mobility detected by the phospho-specific antibody is greater than can be accounted for by the addition of a phosphate group alone which adds two extra negative charges and 80 Da molecular mass to the protein. This implies that the difference between the two differentially migrating 14-3-3 species is due to conformational effects and strongly suggests that 14-3-3 phosphorylated on Ser58 by SDK is monomeric, whereas the unphosphorylated 14-3-3 is dimeric. Furthermore, no phosphorylated 14-3-3 is detected migrating at the sixth marker (Fig. 2A), suggesting that phosphorylation of a single monomer within a 14-3-3 dimer is sufficient to disrupt the dimeric structure of 14-3-3.
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Demonstration That 14-3-3 Phosphorylated on Ser58 Is Monomeric by Cross-linking—We employed cross-linking to examine the dimeric status of Ser58-phosphorylated 14-3-3 compared with unphosphorylated 14-3-3. 14-3-3 was phosphorylated in vitro in an SDK reaction and then subjected to cross-linking with BS3. Cross-linking was terminated using ethanolamine and the cross-linked complexes separated by SDS-PAGE. Gels were Western transferred and immunoblotted either with Ser58 phospho-specific or anti-14-3-3 antibody (Fig. 3). As can be seen, the anti-14-3-3 antibody detected protein of molecular mass 28 kDa, corresponding to the size of denatured monomeric 14-3-3 and also a protein of 56 kDa when the SDK reaction was treated with BS3, corresponding in size to covalently cross-linked 14-3-3 dimers (Fig. 3). In contrast, the phospho-specific antibody only detected protein of the size of denatured monomeric 14-3-3 and not covalently cross-linked dimeric 14-3-3 (Fig. 3), indicating that 14-3-3 phosphorylated on Ser58 is monomeric. The absence of cross-linked phosphorylated 14-3-3 dimers re-affirms the previous result (Fig. 2A), showing that phosphorylation of a single monomer within a dimer is sufficient to disrupt the dimeric structure of 14-3-3. Therefore, Ser58 phosphorylation has a dominant effect with respect to dimer disruption, increasing the potency of the event.
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14-3-3 Phosphorylated on Ser58 Retains the Ability to Bind Phosphoserine Peptide—Serine 58 of 14-3-3 is in the middle of helix 3 and from the crystal structure is buried at the dimer interface (7). Other residues in helix 3 are involved in client protein binding in the amphipathic groove; specifically Arg56 and Arg60 that flank Ser58 have been demonstrated to be critical for binding to phosphoserine peptide (14). Therefore, it is important to determine whether phosphorylation of Ser58 disrupts the amphipathic groove and consequently the ability of 14-3-3 to bind to phosphoserine client proteins. Thus, the ability of 14-3-3 phosphorylated on Ser58 to bind a peptide corresponding to a 14-3-3 binding motif in the cytoplasmic domain of the granulocyte-macrophage colony-stimulating factor receptor chain (12) was determined. Phosphorylated and unphosphorylated peptides (16) were used to pull-down 14-3-3 from SDK reactions carried out either in the presence or absence of DMS (Fig. 4). Immunoblotting of the pull-down material with anti-14-3-3 antibody showed that 14-3-3 bound to the phosphorylated but not the unphosphorylated peptide, demonstrating that interaction of 14-3-3 with peptide is dependent on phosphorylation of the peptide. Similarly, Ser58-phosphorylated 14-3-3 also bound to phosphorylated peptide and, as expected, was detected only in SDK reactions carried out in the presence of DMS (Fig. 4). This indicates that Ser58-phosphorylated monomeric 14-3-3 retains the ability to bind to a 14-3-3 binding site and that binding is dependent on phosphorylation of the site, thus demonstrating that the amphipathic binding groove in the phosphorylated 14-3-3 monomer is not structurally perturbed by phosphorylation.
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Phosphorylation and Monomerization of 14-3-3 Can Be Induced in Vivo by Sphingolipids—Having demonstrated that phosphorylation of Ser58 by SDK renders 14-3-3 monomeric in vitro, it is important to show that this process also occurs in vivo. We have examined the phosphorylation and dimerization of 14-3-3 in vivo in Balb/c 3T3 cells after sphingolipid treatment using the phospho-specific Ser58 antibody (Fig. 5A). DMS was employed for these studies being a naturally occurring nonmetabolizable analogue of sphingosine. Whole cell lysates from Balb/c 3T3 cells treated with 25 µM DMS for varying lengths of time were run on native-PAGE and immunoblotted with phospho-specific Ser58 and anti-14-3-3 antibodies (Fig. 5A). The phospho-specific Ser58 antibody detected a protein migrating with the seventh protein marker after 10 min of treatment with DMS corresponding to monomeric 14-3-3. This demonstrates that DMS treatment of Balb/c 3T3 fibroblasts does indeed disrupt dimeric 14-3-3 in vivo presumably by inducing phosphorylation of Ser58.
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We extended these studies to other cell types and investigated the effect of DMS treatment on 14-3-3 phosphorylation and dimeric status in the murine lymphoid cell line, CTLL-2. Cells were stimulated with increasing doses of DMS for 30 min and whole cell lysates prepared and run on native-PAGE. After Western blotting the lysates were immunoblotted with either the phospho-specific Ser58 antibody or anti-14-3-3 antibody (Fig. 5B). As can be seen, when cells were treated with 25 µM DMS, phosphorylated 14-3-3 was detected with the Ser58 phospho-specific antibody migrating exclusively with the seventh protein marker (Fig. 5B). This indicates that sphingolipid-induced phosphorylation of 14-3-3 on Ser58 disrupts the dimeric structure of the endogenous protein in CTLL-2 cells. Furthermore, as seen in vitro, no phosphorylated 14-3-3 is detected as a dimer, indicating that phosphorylation of a single monomer within a 14-3-3 dimer is sufficient to disrupt the dimer.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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phosphorylated on Ser58, and this unique tool has allowed us to probe the effect of SDK-mediated phosphorylation of this site on 14-3-3 dimeric structure. We have employed native-PAGE and cross-linking studies to qualitatively discriminate between monomeric and dimeric forms of 14-3-3 and have found that sphingolipid induced phosphorylation of Ser58 disrupts the dimeric structure of 14-3-3 both in vitro and in vivo (Figs. 2, 3, and 5). Furthermore, phosphorylation of a single monomer unit within a dimer of 14-3-3 is sufficient to disrupt the dimer, increasing the potency of the event. We show that phosphorylated monomeric 14-3-3 is competent at phosphoserine peptide binding, thereby demonstrating that phosphorylation does not have gross conformational effects on the amphipathic phosphoserine binding groove. We propose that this regulated phosphorylation and monomerization of 14-3-3 controls it's deforming activity and may have profound implications for 14-3-3 function and the regulation of many cellular processes.
Phosphorylation of Ser58 by SDK is not restricted to the 14-3-3 isoform. The original SDK report also demonstrated phosphorylation of the equivalent residues in and (10), and equivalent sites are found in all isoforms with the exception of and . Moreover, the amino acid sequence surrounding the serine in all phosphorylatable isoforms is highly conserved, suggesting that the ability to regulate monomerization by phosphorylation has been functionally conserved. Interestingly, not only is the sequence in the corresponding region of 14-3-3 and 14-3-3 more divergent but the homologous residue to Ser58 is an alanine and therefore not susceptible to phosphorylation. It is intriguing that these more tissue-restricted isoforms are not phosphorylatable at this site and suggests that they may perform specific roles for which regulation of monomerization is not desirable.
Our findings are at odds with a recent report in which phosphorylation of Ser58 in 14-3-3 by PKB/Akt was said to have no effect on dimer stability (13). However, as noted by others (8), the recombinant 14-3-3 used as kinase substrate in those studies already comprised a significant amount of monomeric 14-3-3, as determined by native-PAGE analysis. The extent of phosphorylation obtained coincided with the amount of monomer present, suggesting that 14-3-3 monomer rather than dimer may have been the substrate for Akt phosphorylation (13). In contrast, the recombinant 14-3-3 used in our in vitro studies is solely dimeric as determined by native-PAGE analysis, with no monomeric component detectable by immunoblotting (Fig. 2B) or Coomassie staining (Fig. 2C). Additionally, we detect monomeric 14-3-3 in vivo using the phospho-specific Ser58 antibody (Fig. 5), indicating that the monomerization observed is physiological.
It is not possible from our studies to gauge the exact stoichiometry of 14-3-3 phosphorylation in vivo as the native-PAGE system used to discriminate monomers from dimers resolves only proteins and complexes with relatively acidic isoelectric characteristics. As shown (Fig. 4), phosphorylated 14-3-3 is still able to bind to phosphoserine peptide and so would therefore be expected to interact with phosphoserine client proteins and the resulting complexes may not resolve on native-PAGE. Therefore, the amount of phosphorylated 14-3-3 detected under these conditions may underestimate the level of phosphorylation actually occurring.
The proposed functional role for Ser58 in regulating monomerization is consistent with its location in the dimer interface (7). However, as previously noted (8), how a kinase gains access to this buried residue remains unknown. The dimeric form of the protein is inherently stable and not prone to dissociation (9), and we have found no evidence for free exchange of monomers in mixing experiments using differentially tagged forms of 14-3-3 (data not shown). An alternative explanation could be that 14-3-3 protein is flexible, allowing access of SDK activity to the dimer interface, but evidence suggests that this is not the case as the crystal structures of 14-3-3 unbound or bound to a protein are remarkably similar (17). It is conceivable that sphingolipid may have some direct effect on 14-3-3 that allows access of the kinase to Ser58. Such modulation of Src kinase substrates by sphingolipids has been observed previously (18). Alternatively, in the original description of SDK activity, it was shown that the enzyme activity co-purified with 14-3-3 and could be co-immunoprecipitated with endogenous 14-3-3 (10), indicating that the kinase responsible for SDK activity is in intimate contact with 14-3-3, even in the absence of sphingolipid. Therefore, SDK may gain access to Ser58 by conformationally altering 14-3-3 directly.
Previous studies with 14-3-3 mutants defective in dimerization indicate that although monomeric forms of 14-3-3 are capable of binding to phosphoserine target proteins, in many cases the monomer is unable to regulate client protein functions (19–22). Furthermore, 14-3-3 mutants that are defective in client protein binding owing to substitutions in the phosphoserine binding amphipathic groove exhibit dominant negative effects on several pathways due to their ability to dimerize with endogenous 14-3-3, resulting in functionally monomeric forms (23, 24). In the light of these studies we believe that the regulated monomerization of 14-3-3 demonstrated here has significant implications for the regulation of many 14-3-3 functions in cells.
To date, the most clearly defined role of 14-3-3 is to protect cells against apoptosis. A peptide with high affinity for 14-3-3, difopein, induces apoptosis when expressed in cells, due to its ability to compete with cellular proteins for 14-3-3 binding (25). Strikingly, expression of a functionally monomeric dominant negative form of 14-3-3 in mouse fibroblasts enhances the cells apoptotic response to UVC irradiation, serum withdrawal, and tumor necrosis factor-, highlighting the role of dimeric 14-3-3 in protecting cells against apoptosis (23). Therefore, regulated monomerization may play a role in inducing apoptosis by disrupting the anti-apoptotic function of dimeric 14-3-3. Consistent with this hypothesis, the sphingolipids sphingosine and DMS have both been shown to induce apoptosis in many cell types, including CTLL-2 cells (15, 26–31). Indeed, under the conditions used here to induce 14-3-3 phosphorylation in CTLL-2 cells (Fig. 5B), significant cell death was observed after 5 h of treatment with DMS (data not shown). Therefore, it is conceivable that phosphorylation and consequent monomerization of 14-3-3 contributes to apoptosis induced by these sphingolipids and may have a more general role in functions where 14-3-3 is involved.
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FOOTNOTES |
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To whom correspondence should be addressed. Tel.: 61-8-8222-3713; Fax: 61-8-8222-3538; E-mail: joanna.woodcock{at}imvs.sa.gov.au.
1 The abbreviations used are: SDK, sphingosine-dependent kinase; PMA, phorbol 12-myristate 13-acetate; BS3, bis(sulfosuccinimidyl) suberate; DMS, dimethyl sphingosine.
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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D. Bridges and G. B. G. Moorhead 14-3-3 Proteins: A Number of Functions for a Numbered Protein Sci. Signal., July 20, 2004; 2004(242): re10 - re10. [Abstract] [Full Text] [PDF]
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