Involvement of 14-3-3 protein post-translational modifications in Giardiaduodenalis encystation

14-3-3s are a family of phosphoserine/phosphothreonine binding proteins directly affecting many protein functions by regulating enzyme activity, intracellular localisation or mediating protein-protein interaction. The single 14-3-3 (g14-3-3) of the flagellated parasite Giardia duodenalis is phosphorylated at residue threonine 214 (T214) and polyglycylated at the extreme C-terminus in a stage-specific manner. To define the role of each post-translational modification, Giardia transgenic lines expressing a N-terminally FLAG-tagged g14-3-3, or the single point mutant T214A, or the E246A and the E247A mutants of the putative polyglycylation sites, were generated in this study. By affinity chromatography and MALDI-MS analysis, Glu246 was identified as the only site of polyglycylation. The absence of a polyglycine chain results in the nuclear localisation of the protein at any parasite life-stage, suggesting a role for polyglycylation in 14-3-3 nucleo/cytoplasm shuttling. Moreover, cyst formation was strongly induced in parasites expressing the E246A mutant and delayed in those harbouring the T214A mutant. Finally, in vitro overlay assays with a GST_T214E mutant indicated that phosphorylation can alter in vitro the binding properties of 14-3-3. The present data suggest that g14-3-3 post-translational modifications act in combination to affect encystation efficiency in Giardia.     

Interaction network of the 14-3-3 protein in the ancient protozoan parasite Giardia duodenalis

14-3-3s are phosphoserine/phosphotreonine binding proteins that play pivotal roles as regulators of multiple cellular processes in eukaryotes. The flagellated protozoan parasite Giardia duodenalis, the causing agent of giardiasis, is a valuable simplified eukaryotic model. A single 14-3-3 isoform (g14-3-3) is expressed in Giardia, and it is directly involved in the differentiation of the parasite into cyst. To define the overall functions of g14-3-3, the protein interactome has been investigated. A transgenic G. duodenalis strain was engineered to express a FLAG-tagged g14-3-3 under its own promoter. Affinity chromatography coupled with tandem mass spectrometry analysis have been used to purify and identify FLAG-g14-3-3-associated proteins from trophozoites and encysting parasites. A total of 314 putative g14-3-3 interaction partners were identified, including proteins involved in several pathways. Some interactions seemed to be peculiar of one specific stage, while others were shared among the different stages. Furthermore, the interaction of g14-3-3 with the giardial homologue of the CDC7 protein kinase (gCDC7) was characterized, leading to the identification of a multiprotein complex containing not only g14-3-3 and gCDC7 but also a newly identified and highly divergent homologue of DBF4, the putative regulatory subunit of gCDC7. The relevance of g14-3-3 interactions in G. duodenalis biology was discussed.     

Interkingdom Complementation Reveals Structural Conservation and Functional Divergence of 14-3-3 Proteins

The 14-3-3s are small acidic cytosolic proteins that interact with multiple clients and participate in essential cellular functions in all eukaryotes. Available structural and functional information about 14-3-3s is largely derived from higher eukaryotes, which contain multiple members of this protein family suggesting functional specialization. The exceptional sequence conservation among 14-3-3 family members from diverse species suggests a common ancestor for 14-3-3s, proposed to have been similar to modern 14-3-3ε isoforms. Structural features of the sole family member from the protozoan Giardia duodenalis (g14-3-3), are consistent with this hypothesis, but whether g14-3-3 is functionally homologous to the epsilon isoforms is unknown. We use inter-kingdom reciprocal functional complementation and biochemical methods to determine whether g14-3-3 is structurally and functionally homologous with members of the two 14-3-3 conservation groups of the metazoan Drosophila melanogaster. Our results indicate that although g14-3-3 is structurally homologous to D14-3-3ε, functionally it diverges presenting characteristics of other 14-3-3s. Given the basal position of Giardia in eukaryotic evolution, this finding is consistent with the hypothesis that 14-3-3ε isoforms are ancestral to other family members.     

The uptake and conversion of L-[U14C-] aspartate and L-[U14C-] alanine to 14CO2 by intact trophozoites of Giardia duodenalis.

1. Intact trophozoites of Giardia duodenalis (clone P1C10) took up and metabolised L-[U14C-] aspartate to 14CO2 at rates of 10.27 +/- 0.76 and 27.6 +/- 2.07 ng hr-1 10(-6) cells in a simple maintenance medium (MM) and in a complex bile supplemented (BIS-33) medium respectively. 2. Intact trophozoite of G. duodenalis (clone P1C10) also took up and metabolised L-[U14C-] alanine to 14CO2 at rates of 20.6 +/- 1.1 and 91.4 +/- 17.5 ng hr-1 10(-6) cells in the simple (MM) and complex (BIS-33) medium respectively. 3. trophozoite sonicates contained significant levels of aspartate-2-oxoglutarate transaminase (AST; EC 2.6.1.1) and alanine-2-oxoglutarate transaminase (ALT; EC 2.6.2.2.). Specific activities (at 23 degrees C) were 95.1 +/- 11.3 and 87.3 +/- 9.8 nmol (min)-1 (mg protein)-1 respectively. 4. These observations suggest that Giardia has the capacity to utilise aspartate and alanine and possibly other amino acids as alternative sources of energy. 5. The extrusion or uptake of alanine by Giardia trophozoites may be dictated by the intracellular redox-status of the protozoan parasite or components in the external mileu.     

Regulation of yeast Yak1 kinase by PKA and autophosphorylation-dependent 14-3-3 binding

Yak1 is a member of an evolutionarily conserved family of Ser/Thr protein kinases known as dual-specificity Tyr phosphorylation-regulated kinases (DYRKs). Yak1 was originally identified as a growth antagonist, which functions downstream of Ras/PKA signalling pathway. It has been known that Yak1 is phosphorylated by PKA in vitro and is translocated to the nucleus upon nutrient deprivation. However, the regulatory mechanisms for Yak1 activity and localization are largely unknown. In the present study, we investigated the role of PKA and Bmh1, a yeast 14-3-3 protein, in regulation of Yak1. We demonstrate that PKA-dependent phosphorylation of Yak1 on Ser295 and two minor sites inhibits nuclear localization of Yak1. We also show that intramolecular autophosphorylation on at least four Ser/Thr residues in the non-catalytic N-terminal domain is required for full kinase activity of Yak1. The most potent autophosphorylation site, Thr335, plays an essential role for Bmh1 binding in collaboration with a yet unidentified second binding site in the N-terminal domain. Bmh1 binding decreases the catalytic activity of Yak1 without affecting its subcellular localization. Since the binding of 14-3-3 proteins to Yak1 coincides with PKA activity, such regulatory mechanisms might allow cytoplasmic retention of an inactive form of Yak1 under high glucose conditions.     

14-3-3 proteins: regulation of subcellular localization by molecular interference

14-3-3 family of proteins plays a key regulatory role in signal transduction, checkpoint control, apoptotic, and nutrient-sensing pathways. 14-3-3 proteins act by binding to partner proteins, and this binding often leads to the altered subcellular localization of the partner. 14-3-3 proteins promote the cytoplasmic localization of many binding partners, including the pro-apoptotic protein BAD and the cell cycle regulatory phosphatase Cdc25C, but they can also promote the nuclear localization of other partners, such as the catalytic subunit of telomerase (TERT). In some cases, 14-3-3 binding has no effect on the subcellular localization of a partner. 14-3-3 may affect the localization of a protein by interfering with the function of a nearby targeting sequence, such as a nuclear localization sequence (NLS) or a nuclear export sequence (NES), on the binding partner.     

The 14-3-3s

Multiple members of the 14-3-3 protein family have been found in all eukaryotes so far investigated, yet they are apparently absent from prokaryotes. The major native forms of 14-3-3s are homo- and hetero-dimers, the biological functions of which are to interact physically with specific client proteins and thereby effect a change in the client. As a result, 14-3-3s are involved in a vast array of processes such as the response to stress, cell-cycle control, and apoptosis, serving as adapters, activators, and repressors. There are currently 133 full-length sequences available in GenBank for this highly conserved protein family. A phylogenetic tree based on the conserved middle core region of the protein sequences shows that, in plants, the 14-3-3 family can be divided into two clearly defined groups. The core region encodes an amphipathic groove that binds the multitude of client proteins that have conserved 14-3-3-recognition sequences. The amino and carboxyl termini of 14-3-3 proteins are much more divergent than the core region and may interact with isoform-specific client proteins and/or confer specialized subcellular and tissue localization.     

14-3-3 dimers probe the assembly status of multimeric membrane proteins

BACKGROUND: Arginine-based endoplasmic reticulum (ER) localization signals are involved in the heteromultimeric assembly of membrane protein complexes like ATP-sensitive potassium channels (K(ATP)) or GABA(B) G protein-coupled receptors. They constitute a trafficking checkpoint that prevents ER exit of unassembled subunits or partially assembled complexes. For K(ATP) channels, the mechanism that leads to masking of the ER localization signals in the fully assembled octameric complex is unknown. RESULTS: By employing a tetrameric affinity construct of the C terminus of the K(ATP) channel alpha subunit, Kir6.2, we found that 14-3-3 isoforms epsilon and zeta specifically recognize the arginine-based ER localization signal present in this cytosolic tail. The interaction was reconstituted by using purified 14-3-3 proteins. Competition with a nonphosphorylated 14-3-3 high-affinity binding peptide implies that the canonical substrate binding groove of 14-3-3 is involved. Comparison of monomeric CD4, dimeric CD8, and artificially tetramerized CD4 fusions correlates the copy number of the tail containing the arginine-based signal with 14-3-3 binding, resulting in the surface expression of the membrane protein. Binding experiments revealed that the COPI vesicle coat can specifically recognize the arginine-based ER localization signal and competes with 14-3-3 for the binding site. CONCLUSIONS: The COPI vesicle coat and proteins of the 14-3-3 family recognize arginine-based ER localization signals on multimeric membrane proteins. The equilibrium between these two competing reactions depends on the valency and spatial arrangement of the signal-containing tails. We propose a mechanism in which 14-3-3 bound to the correctly assembled multimer mediates release of the complex from the ER.     

Plant receptor kinases bind and phosphorylate 14-3-3 proteins

14-3-3 proteins are pSer/pThr-binding proteins that interact with a wide array of cellular ‘client’ proteins. The plant brassinosteroids (BRs) receptor, BRASSINOSTEROID INSENSITIVE 1 (BRI1), is a member of the large family of leucine-rich repeat receptor-like kinases (LRR-RLKs) that contain cytoplasmic protein kinase domains. At least two LRR-RLKs are involved in BR perception and signal transduction: BRI1 and BRI1-associated receptor kinase 1 (BAK1). We determined that several 14-3-3 proteins bind to BRI1-CD and are phosphorylated by BRI1, BAK1 and At3g21430 receptor kinases in vitro. Moreover, we observed14-3-3 s are phosphorylated on threonine residue(s) with BR-dependent manner. To reveal the function of 14-3-3 proteins interacting with LRR-RLKs, we treated tyrosine phosphatase (PTP1B) to the BRI1-CD recombinant protein, which is autophosphorylated on tyrosine residue(s). Tyrosine autophosphorylation signal was disappeared, suggesting that 14-3-3 proteins cannot protect BRI1 tyrosine phosphorylation from PTP1B phosphatase. Our study suggests that 14-3-3 proteins may be important for plant growth and development through BR signaling.     

Interaction of apoptosis signal-regulating kinase 1 with isoforms of 14-3-3 proteins

Apoptosis signal-regulating kinase 1 (ASK1) is a critical mediator of apoptotic signaling pathways initiated by a variety of death stimuli. Its activity is tightly controlled by various mechanisms such as covalent modification and protein-protein interaction. One of the proteins that control ASK1 function is 14-3-3zeta, a member of the 14-3-3 protein family. Here, we report that ASK1 is capable of binding to other isoforms of 14-3-3, suggesting that binding ASK1 is a general property of the 14-3-3 family. In support of this notion, mutational analysis revealed that the ASK1/14-3-3 interaction was mediated by the conserved amphipathic groove of 14-3-3 with some residue selectivity. Functionally, expression of various isoforms of 14-3-3 suppressed ASK1-induced apoptosis. To understand how 14-3-3 controls the ASK1 activity, we examined intracellular localization of ASK1 upon 14-3-3 co-expression. We found that 14-3-3 co-expression is correlated with the translocation of ASK1 from the cytoplasm to a perinuclear localization, likely the ER compartment. Consistent with this notion, ASK1(S967A), a 14-3-3 binding defective mutant of ASK, showed no change in intracellular distribution upon 14-3-3 co-expression. These data support a model that 14-3-3 proteins regulate the proapoptotic function of ASK1 in part by controlling its subcellular distribution.     

Phosphorylation of transglutaminase 2 by PKA at Ser216 creates 14-3-3 binding sites

Transglutaminase 2 (TG2) is a multifunctional ubiquitous enzyme which is present in various cellular compartments and is subject to phosphorylation by PKA. To better understand the relevance of PKA induced phosphorylation of TG2, we performed pull-down assays using phosphorylated biotinylated-TG2(209-223) peptides spanning PKA induced phosphorylation sites as a bait. Subsequent analysis of pull-down protein by SDS-PAGE and LC/MS identified 14-3-3epsilon as the binding partner for TG2 which was further confirmed by immunoblotting with 14-3-3 specific antiserum. In contrast, non-phosphorylated and/or phosphorylation site substituted peptides fail to pull-down 14-3-3. Furthermore, we demonstrate that 14-3-3 co-immunoprecipitated with TG2 antiserum after activation of PKA from mouse embryonic fibroblasts (MEF)(TG2+/+) cells but not from MEF(TG2-/-) cells. In summary, we provide convincing evidence that phosphorylation of TG2 by PKA creates binding site(s) for 14-3-3 both in vitro and in vivo.     

Phosphorylation of S776 and 14-3-3 Binding Modulate Ataxin-1 Interaction with Splicing Factors

Ataxin-1 (Atx1), a member of the polyglutamine (polyQ) expanded protein family, is responsible for spinocerebellar ataxia type 1. Requirements for developing the disease are polyQ expansion, nuclear localization and phosphorylation of S776. Using a combination of bioinformatics, cell and structural biology approaches, we have identified a UHM ligand motif (ULM), present in proteins associated with splicing, in the C-terminus of Atx1 and shown that Atx1 interacts with and influences the function of the splicing factor U2AF65 via this motif. ULM comprises S776 of Atx1 and overlaps with a nuclear localization signal and a 14-3-3 binding motif. We demonstrate that phosphorylation of S776 provides the molecular switch which discriminates between 14-3-3 and components of the spliceosome. We also show that an S776D Atx1 mutant previously designed to mimic phosphorylation is unsuitable for this aim because of the different chemical properties of the two groups. Our results indicate that Atx1 is part of a complex network of interactions with splicing factors and suggest that development of the pathology is the consequence of a competition of aggregation with native interactions. Studies of the interactions formed by non-expanded Atx1 thus provide valuable hints for understanding both the function of the non-pathologic protein and the causes of the disease.     

14-3-3 Protein mediates phosphorylation of microtubule-associated protein tau by serum- and glucocorticoid-induced protein kinase 1

The microtubule-associated protein, tau, is involved in numerous neuronal processes such as vesicle transport, microtubule-plasma membrane interaction and the intracellular localization of proteins. Tau is known to be phosphorylated by several kinases such as mitogen activated protein kinase, microtubule affinity regulating kinase, and protein kinase A. We found a putative serum- and glucocorticoid-induced protein kinase 1 (SGK1) phosphorylation site within the 207GSRSRTPSLP216 tau amino acid sequence. We report here that SGK1 phosphorylates Ser214 of Tau. Using a pull-down assay, we found that 14-3-3q interacts with SGK1 and tau to form a ternary protein complex that leads to phosphorylation of tau. 14-3-3 and phosphorylated tau were mainly co-localized in the nucleus of COS-1 cells. These results demonstrate that 14-3-3 scaffolds tau with SGK1 to facilitate the phosphorylation of tau at Ser214 and to regulate its subcellular localization.     

A combined proteome and ultrastructural localization analysis of 14-3-3 proteins in transformed human amnion (AMA) cells: definition of a framework to study isoform-specific differences

The 14-3-3 proteins constitute a family of highly conserved and broadly expressed multifunctional polypeptides that are involved in a variety of important cellular processes that include cell cycle progression, growth, differentiation, and apoptosis. Although the exact cellular function(s) of 14-3-3 proteins is not fully elucidated, as a rule these proteins act by binding to protein ligands, thus regulating their activity; so far more than 300 cellular proteins have been reported to interact with 14-3-3 proteins. Binding to cognate interacting partners is isoform-specific, but redundancy also exists as several binding peptides can be recognized by all isoforms, and some functions can be carried out by any isoform indistinctly. Moreover by interacting with different ligands in a spatially and temporally regulated fashion the same isoform can play multiple possibly even opposing roles where the resultant cellular outcome will be determined by the integration of the various effects. Although there is a large body of literature on specific aspects of 14-3-3 biology, not much is known on the coordinated aspects of 14-3-3 isoform expression, post-translational modifications, and subcellular localization. To address the question of isoform-specific differences, we carried out a comparative analysis of the patterns of expression, phosphorylation, and subcellular localization of the 14-3-3 beta, epsilon, sigma, tau, and zeta protein isoforms in transformed human amnion (AMA) cells. To validate as well as broaden our observations we analyzed the occurrence of the various isoforms in a large number of established cell lines and mammary and urothelial tissue specimens. Given the systematic approach we undertook and our application of isoform-discriminating technologies to the analysis of various cellular systems, we expect the data presented in this study to serve as an enabling resource for researchers working with 14-3-3 proteins.