A structural basis for 14-3-3sigma functional specificity

The 14-3-3 family of proteins includes seven isotypes in mammalian cells that play numerous diverse roles in intracellular signaling. Most 14-3-3 proteins form homodimers and mixed heterodimers between different isotypes, with overlapping roles in ligand binding. In contrast, one mammalian isoform, 14-3-3sigma, expressed primarily in epithelial cells, appears to play a unique role in the cellular response to DNA damage and in human oncogenesis. The biological and structural basis for these 14-3-3sigma-specific functions is unknown. We demonstrate that endogenous 14-3-3sigma preferentially forms homodimers in cells. We have solved the x-ray crystal structure of 14-3-3sigma bound to an optimal phosphopeptide ligand at 2.4 angstroms resolution. The structure reveals the presence of stabilizing ring-ring and salt bridge interactions unique to the 14-3-3sigma homodimer structure and potentially destabilizing electrostatic interactions between subunits in 14-3-3sigma-containing heterodimers, rationalizing preferential homodimerization of 14-3-3sigma in vivo. The interaction of the phosphopeptide with 14-3-3 reveals a conserved mechanism for phospho-dependent ligand binding, implying that the phosphopeptide binding cleft is not the critical determinant of the unique biological properties of 14-3-3sigma. Instead, the structure suggests a second ligand binding site involved in 14-3-3sigma-specific ligand discrimination. We have confirmed this by site-directed mutagenesis of three sigma-specific residues that uniquely define this site. Mutation of these residues to the alternative sequence that is absolutely conserved in all other 14-3-3 isotypes confers upon 14-3-3sigma the ability to bind to Cdc25C, a ligand that is known to bind to other 14-3-3 proteins but not to sigma.     

CaMKK2 is inactivated by cAMP-PKA signaling and 14-3-3 adaptor proteins

The calcium-calmodulin–dependent protein kinase kinase-2 (CaMKK2) is a key regulator of cellular and whole-body energy metabolism. It is known to be activated by increases in intracellular Ca²⁺, but the mechanisms by which it is inactivated are less clear. CaMKK2 inhibition protects against prostate cancer, hepatocellular carcinoma, and metabolic derangements induced by a high-fat diet; therefore, elucidating the intracellular mechanisms that inactivate CaMKK2 has important therapeutic implications. Here we show that stimulation of cAMP-dependent protein kinase A (PKA) signaling in cells inactivates CaMKK2 by phosphorylation of three conserved serine residues. PKA-dependent phosphorylation of Ser⁴⁹⁵ directly impairs calcium-calmodulin activation, whereas phosphorylation of Ser¹⁰⁰ and Ser⁵¹¹ mediate recruitment of 14-3-3 adaptor proteins that hold CaMKK2 in the inactivated state by preventing dephosphorylation of phospho-Ser⁴⁹⁵. We also report the crystal structure of 14-3-3ζ bound to a synthetic diphosphorylated peptide that reveals how the canonical (Ser⁵¹¹) and noncanonical (Ser¹⁰⁰) 14-3-3 consensus sites on CaMKK2 cooperate to bind 14-3-3 proteins. Our findings provide detailed molecular insights into how cAMP-PKA signaling inactivates CaMKK2 and reveals a pathway to inhibit CaMKK2 with potential for treating human diseases.     

Phosphorylation of Arabidopsis Ubiquitin Ligase ATL31 Is Critical for Plant Carbon/Nitrogen Nutrient Balance Response and Controls the Stability of 14-3-3 Proteins

Ubiquitin ligase plays a fundamental role in regulating multiple cellular events in eukaryotes by fine-tuning the stability and activity of specific target proteins. We have previously shown that ubiquitin ligase ATL31 regulates plant growth in response to nutrient balance between carbon and nitrogen (C/N) in Arabidopsis. Subsequent study demonstrated that ATL31 targets 14-3-3 proteins for ubiquitination and modulates the protein abundance in response to C/N-nutrient status. However, the underlying mechanism for the targeting of ATL31 to 14-3-3 proteins remains unclear. Here, we show that ATL31 interacts with 14-3-3 proteins in a phosphorylation-dependent manner. We identified Thr²⁰⁹, Ser²⁴⁷, Ser²⁷⁰, and Ser³⁰³ as putative 14-3-3 binding sites on ATL31 by motif analysis. Mutation of these Ser/Thr residues to Ala in ATL31 inhibited the interaction with 14-3-3 proteins, as demonstrated by yeast two-hybrid and co-immunoprecipitation analyses. Additionally, we identified in vivo phosphorylation of Thr²⁰⁹ and Ser²⁴⁷ on ATL31 by MS analysis. A peptide competition assay showed that the application of synthetic phospho-Thr²⁰⁹ peptide, but not the corresponding unphosphorylated peptide, suppresses the interaction between ATL31 and 14-3-3 proteins. Moreover, Arabidopsis plants overexpressing mutated ATL31, which could not bind to 14-3-3 proteins, showed accumulation of 14-3-3 proteins and growth arrest in disrupted C/N-nutrient conditions similar to wild-type plants, although overexpression of intact ATL31 resulted in repression of 14-3-3 accumulation and tolerance to the conditions. Together, these results demonstrate that the physiological role of phosphorylation at 14-3-3 binding sites on ATL31 is to modulate the binding ability and stability of 14-3-3 proteins to control plant C/N-nutrient response.     

Interactions Crucial for Three-Dimensional Domain Swapping in the HP-RNase Variant PM8

The structural determinants that are responsible for the formation of higher order associations of folded proteins remain unknown. We have investigated the role on the dimerization process of different residues of a domain-swapped dimer human pancreatic ribonuclease variant. This variant is a good model to study the dimerization and swapping processes because dimer and monomer forms interconvert, are easily isolated, and only one dimeric species is produced. Thus, simple models for the swapping process can be proposed. The dimerization (dissociation constant) and swapping propensity have been studied using different variants with changes in residues that belong to different putative molecular determinants of dimerization. Using NMR spectroscopy, we show that these mutations do not substantially alter the overall conformation and flexibility, but affect the residue level stability. Overall, the most critical residues for the swapping process are those of one subunit that interact with the hinge loop of another one-subunit residue, stabilizing it in a conformation that favors the interchange. Tyr²⁵, Gln¹⁰¹, and Pro¹⁹, with Asn¹⁷, Ser²¹, and Ser²³, are found to be the most significant; notably, Glu¹⁰³ and Arg¹⁰⁴, which were postulated to form salt bridges that would stabilize the dimer, are not critical for dimerization.     

Determinants of 14-3-3σ Protein Dimerization and Function in Drug and Radiation Resistance

Many proteins exist and function as homodimers. Understanding the detailed mechanism driving the homodimerization is important and will impact future studies targeting the “undruggable” oncogenic protein dimers. In this study, we used 14-3-3σ as a model homodimeric protein and performed a systematic investigation of the potential roles of amino acid residues in the interface for homodimerization. Unlike other members of the conserved 14-3-3 protein family, 14-3-3σ prefers to form a homodimer with two subareas in the dimeric interface that has 180° symmetry. We found that both subareas of the dimeric interface are required to maintain full dimerization activity. Although the interfacial hydrophobic core residues Leu¹² and Tyr⁸⁴ play important roles in 14-3-3σ dimerization, the non-core residue Phe²⁵ appears to be more important in controlling 14-3-3σ dimerization activity. Interestingly, a similar non-core residue (Val⁸¹) is less important than Phe²⁵ in contributing to 14-3-3σ dimerization. Furthermore, dissociating dimeric 14-3-3σ into monomers by mutating the Leu¹², Phe²⁵, or Tyr⁸⁴ dimerization residue individually diminished the function of 14-3-3σ in resisting drug-induced apoptosis and in arresting cells at G₂/M phase in response to DNA-damaging treatment. Thus, dimerization appears to be required for the function of 14-3-3σ.     

Residues of 14-3-3 zeta required for activation of exoenzyme S of Pseudomonas aeruginosa.

Exoenzyme S (ExoS) is a mono-ADP-ribosyltransferase secreted by the opportunistic pathogen Pseudomonas aeruginosa. ExoS requires a eukaryotic factor, the 14-3-3 protein, for enzymatic activity. Here, two aspects of the activation of the ADP-ribosyltransferase activity of ExoS by 14-3-3 proteins are examined. Initial studies showed that several isoforms of 14-3-3, including beta, zeta, eta, sigma, and tau, activated ExoS with similar efficiency. This implicates a conserved structure in 14-3-3 that contributes to the interaction between 14-3-3 and ExoS. One candidate structure is the conserved amphipathic groove that mediates the 14-3-3/Raf-1 interaction. The next series of experiments examined the role of individual amino acids of the amphipathic groove of 14-3-3 zeta in ExoS activation and showed that ExoS activation required the basic residues lining the amphipathic groove of 14-3-3 zeta without extensive involvement of the hydrophobic residues. Strikingly, mutations of Val-176 of 14-3-3 zeta that disrupted its interaction with Raf-1 did not affect the binding and activation of ExoS by 14-3-3. Thus, ExoS selectively employs residues in the Raf-binding groove for its association with 14-3-3 proteins.     

The Heterologous Interactions among Plant 14-3-3 Proteins and Identification of Regions That Are Important for Dimerization

The 14-3-3 proteins constitute a family of dimeric proteins that are involved in many cellular functions. At least two mammalian 14-3-3 proteins can form heterodimers and the approximate regions important for dimerization have been identified. In this study, we demonstrate that eightArabidopsisand one maize 14-3-3 protein can dimerize with each other and with themselves. Native gel Western analysis ofArabidopsiscell extract also suggests the presence of 14-3-3 heterodimersin vivo.Finally, we identified the domains of one 14-3-3 protein that are sufficient for homodimerization and heterodimerization. These data support the hypothesis that evolutionarily divergent 14-3-3 proteins can interact with each other to form diverse molecular modulators or adapters in signaling pathways.     

Mitotic Phosphorylation of Cdc25B Ser321 Disrupts 14-3-3 Binding to the High Affinity Ser323 Site

Cdc25B is a key regulator of entry into mitosis, and its activity and localization are regulated by binding of the 14-3-3 dimer. There are three 14-3-3 binding sites on Cdc25B, with Ser³²³ being the highest affinity binding and is highly homologous to the Ser²¹⁶ 14-3-3 binding site on Cdc25C. Loss of 14-3-3 binding to Ser³²³ increases cyclin/Cdk substrate access to the catalytic site, thereby increasing its activity. It also affects the localization of Cdc25B. Thus, phosphorylation and 14-3-3 binding to this site is essential for down-regulating Cdc25B activity, blocking its mitosis promoting function. The question of how this inhibitory signal is relieved to allow Cdc25B activation and entry into mitosis is yet to be resolved. Here, we show that Ser³²³ phosphorylation is maintained into mitosis, but phosphorylation of Ser³²¹ disrupts 14-3-3 binding to Ser³²³, mimicking the effect of inhibiting Ser³²³ phosphorylation on both Cdc25B activity and localization. The unphosphorylated Ser³²¹ appears to have a role in stabilizing 14-3-3 binding to Ser³²³, and loss of the Ser hydroxyl group appears to be sufficient to significantly reduce 14-3-3 binding. A consequence of loss of 14-3-3 binding is dephosphorylation of Ser³²³. Ser³²¹ is phosphorylated in mitosis by Cdk1. The mitotic phosphorylation of Ser³²¹ acts to maintain full activation of Cdc25B by disrupting 14-3-3 binding to Ser³²³ and enhancing the dephosphorylation of Ser³²³ to block 14-3-3 binding to this site.     

Molecular evolution of the 14-3-3 protein family

Members of the highly conserved and ubiquitous 14-3-3 protein family modulate a wide variety of cellular processes. To determine the evolutionary relationships among specific 14-3-3 proteins in different plant, animal, and fungal species and to initiate a predictive analysis of isoform-specific differences in light of the latest functional and structural studies of 14-3-3, multiple alignments were constructed from forty-six 14-3-3 sequences retrieved from the GenBank and SwissProt databases and a newly identified second 14-3-3 gene fromCaenorhabditis elegans. The alignment revealed five highly conserved sequence blocks. Blocks 2–5 correlate well with the alpha helices 3, 5, 7, and 9 which form the proposed internal binding domain in the three-dimensional structure model of the functioning dimer. Amino acid differences within the functional and structural domains of plant and animal 14-3-3 proteins were identified which may account for functional diversity amongst isoforms. Protein phylogenic trees were constructed using both the maximum parsimony and neighbor joining methods of the PHYLIP(3.5c) package; 14-3-3 proteins fromEntamoeba histolytica, an amitochondrial protozoa, were employed as an outgroup in our analysis. Epsilon isoforms from the animal lineage form a distinct grouping in both trees, which suggests an early divergence from the other animal isoforms. Epsilons were found to be more similar to yeast and plant isoforms than other animal isoforms at numerous amino acid positions, and thus epsilon may have retained functional characteristics of the ancestral protein. The known invertebrate proteins group with the nonepsilon mammalian isoforms. Most of the current 14-3-3 isoform diversity probably arose through independent duplication events after the divergence of the major eukaryotic kingdoms. Divergence of the seven mammalian isoforms beta, zeta, gamma, eta, epsilon, tau, and sigma (stratifin/ HME1) occurred before the divergence of mammalian and perhaps before the divergence of vertebrate species. A possible ancestral 14-3-3 sequence is proposed. Correspondence to: D.C. Shakes     

Concomitant Action of Structural Elements and Receptor Phosphorylation Determines Arrestin-3 Interaction with the Free Fatty Acid Receptor FFA4

In addition to being nutrients, free fatty acids act as signaling molecules by activating a family of G protein-coupled receptors. Among these is FFA4, previously called GPR120, which responds to medium and long chain fatty acids, including health-promoting ω-3 fatty acids, which have been implicated in the regulation of metabolic and inflammatory responses. Here we show, using mass spectrometry, mutagenesis, and phosphospecific antibodies, that agonist-regulated phosphorylation of the human FFA4 receptor occurred primarily at five residues (Thr³⁴⁷, Thr³⁴⁹, Ser³⁵⁰, Ser³⁵⁷, and Ser³⁶⁰) in the C-terminal tail. Mutation of these residues reduced both the efficacy and potency of ligand-mediated arrestin-3 recruitment as well as affecting recruitment kinetics. Combined mutagenesis of all five of these residues was insufficient to fully abrogate interaction with arrestin-3, but further mutagenesis of negatively charged residues revealed additional structural components for the interaction with arrestin-3 within the C-terminal tail of the receptor. These elements consist of the acidic residues Glu³⁴¹, Asp³⁴⁸, and Asp³⁵⁵ located close to the phosphorylation sites. Receptor phosphorylation thus operates in concert with structural elements within the C-terminal tail of FFA4 to allow for the recruitment of arrestin-3. Importantly, these mechanisms of arrestin-3 recruitment operate independently from G_q/11 coupling, thereby offering the possibility that ligands showing stimulus bias could be developed that exploit these differential coupling mechanisms. Furthermore, this provides a strategy for the design of biased receptors to probe physiologically relevant signaling.     

Determinants of Versican-V1 Proteoglycan Processing by the Metalloproteinase ADAMTS5

Proteolysis of the Glu⁴⁴¹-Ala⁴⁴² bond in the glycosaminoglycan (GAG) β domain of the versican-V1 variant by a disintegrin-like and metalloproteinase domain with thrombospondin type 1 motif (ADAMTS) proteases is required for proper embryo morphogenesis. However, the processing mechanism and the possibility of additional ADAMTS-cleaved processing sites are unknown. We demonstrate here that if Glu⁴⁴¹ is mutated, ADAMTS5 cleaves inefficiently at a proximate upstream site but normally does not cleave elsewhere within the GAGβ domain. Chondroitin sulfate (CS) modification of versican is a prerequisite for cleavage at the Glu⁴⁴¹-Ala⁴⁴² site, as demonstrated by reduced processing of CS-deficient or chondroitinase ABC-treated versican-V1. Site-directed mutagenesis identified the N-terminal CS attachment sites Ser⁵⁰⁷ and Ser⁵²⁵ as essential for processing of the Glu⁴⁴¹-Ala⁴⁴² bond by ADAMTS5. A construct including only these two GAG chains, but not downstream GAG attachment sites, was cleaved efficiently. Therefore, CS chain attachment to Ser⁵⁰⁷ and Ser⁵²⁵ is necessary and sufficient for versican proteolysis by ADAMTS5. Mutagenesis of Glu⁴⁴¹ and an antibody to a peptide spanning Thr⁴³²-Gly⁴⁴⁵ (i.e. containing the scissile bond) reduced versican-V1 processing. ADAMTS5 lacking the C-terminal ancillary domain did not cleave versican, and an ADAMTS5 ancillary domain construct bound versican-V1 via the CS chains. We conclude that docking of ADAMTS5 with two N-terminal GAG chains of versican-V1 via its ancillary domain is required for versican processing at Glu⁴⁴¹-Ala⁴⁴². V1 proteolysis by ADAMTS1 demonstrated a similar requirement for the N-terminal GAG chains and Glu⁴⁴¹. Therefore, versican cleavage can be inhibited substantially by mutation of Glu⁴⁴¹, Ser⁵⁰⁷, and Ser⁵²⁵ or by an antibody to the region of the scissile bond.     

Arsenite Stress Down-regulates Phosphorylation and 14-3-3 Binding of Leucine-rich Repeat Kinase 2 (LRRK2), Promoting Self-association and Cellular Redistribution

Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are a common genetic cause of Parkinson disease, but the mechanisms whereby LRRK2 is regulated are unknown. Phosphorylation of LRRK2 at Ser⁹¹⁰/Ser⁹³⁵ mediates interaction with 14-3-3. Pharmacological inhibition of its kinase activity abolishes Ser⁹¹⁰/Ser⁹³⁵ phosphorylation and 14-3-3 binding, and this effect is also mimicked by pathogenic mutations. However, physiological situations where dephosphorylation occurs have not been defined. Here, we show that arsenite or H₂O₂-induced stresses promote loss of Ser⁹¹⁰/Ser⁹³⁵ phosphorylation, which is reversed by phosphatase inhibition. Arsenite-induced dephosphorylation is accompanied by loss of 14-3-3 binding and is observed in wild type, G2019S, and kinase-dead D2017A LRRK2. Arsenite stress stimulates LRRK2 self-association and association with protein phosphatase 1α, decreases kinase activity and GTP binding in vitro, and induces translocation of LRRK2 to centrosomes. Our data indicate that signaling events induced by arsenite and oxidative stress may regulate LRRK2 function.     

Functional conservation of 14-3-3 isoforms in inhibiting bad-induced apoptosis.

14-3-3 proteins are a family of homologous eukaryotic molecules with seven distinct isoforms in mammalian cells. Isoforms of 14-3-3 proteins interact with diverse ligands and are involved in the regulation of mitogenesis, cell cycle progression, and apoptosis. However, whether different 14-3-3 isoforms are responsible for distinct functions remains elusive. Here we report that multiple isoforms of 14-3-3 proteins were capable of binding to several ligands, Bad, Raf-1, and Cbl. In a functional assay of 14-3-3 isoforms, all mammalian 14-3-3 isoforms could inhibit Bad-induced apoptosis. Thus, 14-3-3 function in regulating one of its ligands, Bad, is conserved among mammalian isoforms. We addressed whether 14-3-3 isoforms are differentially expressed in tissues, which may in part determine isoform-specific interactions. In situ hybridization revealed that 14-3-3zeta was present in most tissues tested, but sigma was preferentially expressed in epithelial cells. Thus, isoforms of 14-3-3 can interact and control the function of selected protein ligands, and differential tissue distribution of 14-3-3 isoforms may contribute to their specific interactions and subsequent downstream signaling events.     

Mutational Analysis Identifies Residues Crucial for Homodimerization of Myeloid Differentiation Factor 88 (MyD88) and for Its Function in Immune Cells

Myeloid differentiation factor 88 (MyD88) is an adaptor protein that transduces intracellular signaling pathways evoked by the Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs). MyD88 is composed of an N-terminal death domain (DD) and a C-terminal Toll/IL-1 receptor (TIR) domain, separated by a short region. Upon ligand binding, TLR/IL-1Rs hetero- or homodimerize and recruit MyD88 through their respective TIR domains. Then, MyD88 oligomerizes via its DD and TIR domain and interacts with the interleukin-1 receptor-associated kinases (IRAKs) to form the Myddosome complex. We performed site-directed mutagenesis of conserved residues that are located in exposed regions of the MyD88-TIR domain and analyzed the effect of the mutations on MyD88 signaling. Our studies revealed that mutation of Glu¹⁸³, Ser²⁴⁴, and Arg²⁸⁸ impaired homodimerization of the MyD88-TIR domain, recruitment of IRAKs, and activation of NF-κB. Moreover, overexpression of two green fluorescent protein (GFP)-tagged MyD88 mini-proteins (GFP-MyD88_151–189 and GFP-MyD88_168–189), comprising the Glu¹⁸³ residue, recapitulated these effects. Importantly, expression of these dominant negative MyD88 mini-proteins competed with the function of endogenous MyD88 and interfered with TLR2/4-mediated responses in a human monocytic cell line (THP-1) and in human primary monocyte-derived dendritic cells. Thus, our studies identify novel residues of the TIR domain that are crucially involved in MyD88 homodimerization and TLR signaling in immune cells.     

14‐3‐3 Ligand Prevents Nuclear Import of c‐ABL Protein in Chronic Myeloid Leukemia

Here we demonstrated that the ‘loss of function’ of not‐rearranged c‐ABL in chronic myeloid leukemia (CML) is promoted by its cytoplasmic compartmentalization bound to 14‐3‐3 sigma scaffolding protein. In particular, constitutive tyrosine kinase (TK) activity of p210 BCR‐ABL blocks c‐Jun N‐terminal kinase (JNK) phosphorylation leading to 14‐3‐3 sigma phosphorylation at a critical residue (Ser¹⁸⁶) for c‐ABL binding in response to DNA damage. Moreover, it is associated with 14‐3‐3 sigma over‐expression arising from epigenetic mechanisms (promoter hyper‐acetylation). Accordingly, p210 BCR‐ABL TK inhibition by the TK inhibitor Imatinib mesylate (IM) evokes multiple events, including JNK phosphorylation at Thr¹⁸³, p38 mitogen‐activated protein kinase (MAPK) phosphorylation at Thr¹⁸⁰, c‐ABL de‐phosphorylation at Ser residues involved in 14‐3‐3 binding and reduction of 14‐3‐3 sigma expression, that let c‐ABL release from 14‐3‐3 sigma and nuclear import, and address BCR‐ABL‐expressing cells towards apoptotic death. Informational spectrum method (ISM), a virtual spectroscopy method for analysis of protein interactions based on their structure, and mathematical filtering in cross spectrum (CS) analysis identified 14‐3‐3 sigma/c‐ABL binding sites. Further investigation on CS profiles of c‐ABL‐ and p210 BCR‐ABL‐containing complexes revealed the mechanism likely involved 14‐3‐3 precluded phosphorylation in CML cells.