MADM,a novel adaptor protein that mediates phosphorylation of the 14-3-3 binding site of myeloid leukemia factor 1

A yeast two-hybrid screen was conducted to identify binding partners of Mlf1, an oncoprotein recently identified in a translocation with nucleophosmin that causes acute myeloid leukemia. Two proteins isolated in this screen were 14-3-3zeta and a novel adaptor, Madm. Mlf1 contains a classic RSXSXP sequence for 14-3-3 binding and is associated with 14-3-3zeta via this phosphorylated motif. Madm co-immunoprecipitated with Mlf1 and co-localized in the cytoplasm. In addition, Madm recruited a serine kinase, which phosphorylated both Madm and Mlf1 including the RSXSXP motif. In contrast to wild-type Mlf1, the oncogenic fusion protein nucleophosmin (NPM)-MLF1 did not bind 14-3-3zeta, had altered Madm binding, and localized exclusively in the nucleus. Ectopic expression of Madm in M1 myeloid cells suppressed cytokine-induced differentiation unlike Mlf1, which promotes maturation. Because the Mlf1 binding region of Madm and its own dimerization domain overlapped, the levels of Madm and Mlf1 may affect complex formation and regulate differentiation. In summary, this study has identified two partner proteins of Mlf1 that may influence its subcellular localization and biological function.     

Significance of 14-3-3 self-dimerization for phosphorylation-dependent target binding

14-3-3 proteins via binding serine/threonine-phosphorylated proteins regulate diverse intracellular processes in all eukaryotic organisms. Here, we examine the role of 14-3-3 self-dimerization in target binding, and in the susceptibility of 14-3-3 to undergo phosphorylation. Using a phospho-specific antibody developed against a degenerated mode-1 14-3-3 binding motif (RSxpSxP), we demonstrate that most of the 14-3-3-associated proteins in COS-7 cells are phosphorylated on sites that react with this antibody. The binding of these phosphoproteins depends on 14-3-3 dimerization, inasmuch as proteins associated in vivo with a monomeric 14-3-3 form are not recognized by the phospho-specific antibody. The role of 14-3-3 dimerization in the phosphorylation-dependent target binding is further exemplified with two well-defined 14-3-3 targets, Raf and DAF-16. Raf and DAF-16 can bind both monomeric and dimeric 14-3-3; however, whereas phosphorylation of specific Raf and DAF-16 sites is required for binding to dimeric 14-3-3, binding to monomeric 14-3-3 forms is entirely independent of Raf and DAF-16 phosphorylation. We also find that dimerization diminishes 14-3-3 susceptibility to phosphorylation. These findings establish a significant role of 14-3-3 dimerization in its ability to bind targets in a phosphorylation-dependent manner and point to a mechanism in which 14-3-3 phosphorylation and dimerization counterregulate each other.     

Proteomic, functional, and domain-based analysis of in vivo 14-3-3 binding proteins involved in cytoskeletal regulation and cellular organization

BACKGROUND: 14-3-3 proteins are abundant and conserved polypeptides that mediate the cellular effects of basophilic protein kinases through their ability to bind specific peptide motifs phosphorylated on serine or threonine. RESULTS: We have used mass spectrometry to analyze proteins that associate with 14-3-3 isoforms in HEK293 cells. This identified 170 unique 14-3-3-associated proteins, which show only modest overlap with previous 14-3-3 binding partners isolated by affinity chromatography. To explore this large set of proteins, we developed a domain-based hierarchical clustering technique that distinguishes structurally and functionally related subsets of 14-3-3 target proteins. This analysis revealed a large group of 14-3-3 binding partners that regulate cytoskeletal architecture. Inhibition of 14-3-3 phosphoprotein recognition in vivo indicates the general importance of such interactions in cellular morphology and membrane dynamics. Using tandem proteomic and biochemical approaches, we identify a phospho-dependent 14-3-3 binding site on the A kinase anchoring protein (AKAP)-Lbc, a guanine nucleotide exchange factor (GEF) for the Rho GTPase. 14-3-3 binding to AKAP-Lbc, induced by PKA, suppresses Rho activation in vivo. CONCLUSION: 14-3-3 proteins can potentially engage around 0.6% of the human proteome. Domain-based clustering has identified specific subsets of 14-3-3 targets, including numerous proteins involved in the dynamic control of cell architecture. This notion has been validated by the broad inhibition of 14-3-3 phosphorylation-dependent binding in vivo and by the specific analysis of AKAP-Lbc, a RhoGEF that is controlled by its interaction with 14-3-3.     

14-3-3 proteins: eukaryotic regulatory proteins with many functions

The enigmatically named 14-3-3 proteins have been the subject of considerable attention in recent years since they have been implicated in the regulation of diverse physiological processes, in eukaryotes ranging from slime moulds to higher plants. In plants they have roles in the regulation of the plasma membrane H⁺-ATPase and nitrate reductase, among others. Regulation of target proteins is achieved through binding of 14-3-3 to short, often phosphorylated motifs in the target, resulting either in its activation (e.g. H⁺-ATPase), inactivation (e.g. nitrate reductase) or translocation (although this function of 14-3-3 proteins has yet to be demonstrated in plants). The native 14-3-3 proteins are homo- or heterodimers and, as each monomer has a binding site, a dimer can potentially bind two targets, promoting their association. Alternatively, target proteins may have more than one 14-3-3-binding site. In this mini review, we present a synthesis of recent results from plant 14-3-3 research and, with reference to known 14-3-3-binding motifs, suggest further subjects for research.     

Interaction of 14-3-3 with a nonphosphorylated protein ligand, exoenzyme S of Pseudomonas aeruginosa

The 14-3-3 proteins are a family of conserved, dimeric proteins that interact with a diverse set of ligands, including molecules involved in cell cycle regulation and apoptosis. It is well-established that 14-3-3 binds to many ligands through phosphoserine motifs. Here we characterize the interaction of 14-3-3 with a nonphosphorylated protein ligand, the ADP-ribosyltransferase Exoenzyme S (ExoS) from Pseudomonas aeruginosa. By using affinity chromatography and surface plasmon resonance, we show that the zeta isoform of 14-3-3 (14-3-3zeta) can directly bind a catalytically active fragment of ExoS in vitro. The interaction between ExoS and 14-3-3zeta is of high affinity, with an equilibrium dissociation constant of 7 nM. ExoS lacks any known 14-3-3 binding motif, but to address the possibility that 14-3-3 binds a noncanonical phosphoserine site, we assayed ExoS for protein-bound phosphate by using mass spectrometry. No detectable phosphoproteins were found. A phosphopeptide ligand of 14-3-3, pS-Raf-259, was capable of inhibiting the binding of 14-3-3 to ExoS, suggesting that phosphorylated and nonphosphorylated ligands may share a common binding site, the conserved amphipathic groove. It is conceivable that 14-3-3 proteins may bind both phosphoserine and nonphosphoserine ligands in cells, possibly allowing kinase-dependent as well as kinase-independent regulation of 14-3-3 binding.     

Amino acids C-terminal to the 14-3-3 binding motif in CDC25B affect the efficiency of 14-3-3 binding

The phospho-site adapter protein 14-3-3 binds to target proteins at amino acid sequences matching the consensus motif Arg-X-X-Ser/Thr-X-Pro, where the serine or threonine residue is phosphorylated and X is any amino acid. The dual-specificity phosphatase CDC25B, which is involved in cell cycle regulation, contains five 14-3-3 binding motifs, but 14-3-3 preferentially binds to the motif at Ser309 in CDC25B1 (or Ser323 in CDC25B3). In the present study, we demonstrate that amino acid residues C-terminal to the 14-3-3 binding motif strongly affect the efficiency of 14-3-3 binding. Alanine substitutions at residues downstream of the Ser309 motif dramatically reduced 14-3-3 binding, although phosphorylation of Ser309 was unaffected. We also observed that binding of endogenous 14-3-3 to mutant CDC25B occurred less efficiently than to the wild type. Mutants to which 14-3-3 cannot bind efficiently tend to be located in the nucleus, although not as specifically as the alanine substitution mutant of Ser309. These results indicate that amino acid sequences C-terminal to the consensus binding site have an important role in the efficient binding of 14-3-3 to at least CDC25B, which may partly explain why some consensus sequences are inactive as 14-3-3 binding sites.     

Deciphering the role of 14-3-3 proteins

14-3-3 Proteins are found to bind to a growing number of eukaryotic proteins and evidence is accumulating that 14-3-3 proteins serve as modulators of enzyme activity. Several 14-3-3 protein recognition motifs have been identified and an increasing number of target proteins have been found to contain more than one binding site for a 14-3-3 protein. It is thus possible that 14-3-3 dimers function as clamps that simultaneously bind to two motifs within a single binding partner. Phosphorylation of a number of binding motifs has been shown to increase the affinity for 14-3-3 proteins but other mechanisms also regulate the association. It has recently been demonstrated that fusicoccin induces a tight association between 14-3-3 proteins and the plant plasma membrane H⁺-ATPase. Phorbol esters and other hydrophobic molecules may have a similar effect on the association between 14-3-3 proteins and specific binding partners.     

14-3-3 protein C-terminal stretch occupies ligand binding groove and is displaced by phosphopeptide binding

14-3-3 proteins are important regulators of numerous cellular signaling circuits. They bind to phosphorylated protein ligands and regulate their functions by a number of different mechanisms. The C-terminal part of the 14-3-3 protein is known to be involved in the regulation of 14-3-3 binding properties. The structure of this region is unknown; however, a possible location of the C-terminal stretch within the ligand binding groove of the 14-3-3 protein has been suggested. To fully understand the role of the C-terminal stretch in the regulation of the 14-3-3 protein binding properties, we investigated the physical location of the C-terminal stretch and its changes upon the ligand binding. For this purpose, we have used Forster resonance energy transfer (FRET) measurements and molecular dynamics simulation. FRET measurements between Trp242 located at the end of the C-terminal stretch and a dansyl group attached at two different cysteine residues (Cys25 or Cys189) indicated that in the absence of the ligand, the C-terminal stretch occupies the ligand binding groove of 14-3-3 protein. Our data also showed that phosphopeptide binding displaces the C-terminal stretch from the ligand binding groove. Intramolecular distances calculated from FRET measurements fit well with distances obtained from molecular dynamics simulation of full-length 14-3-3zeta protein.     

14-3-3zeta C-terminal stretch changes its conformation upon ligand binding and phosphorylation at Thr232

14-3-3 proteins are abundant binding proteins involved in many biologically important processes. 14-3-3 proteins bind to other proteins in a phosphorylation-dependent manner and function as scaffold molecules modulating the activity of their binding partners. In this work, we studied the conformational changes of 14-3-3 C-terminal stretch, a region implicated in playing a role in the regulation of 14-3-3. Time-resolved fluorescence and molecular dynamics were used to investigate structural changes of the C-terminal stretch induced by phosphopeptide binding and phosphorylation at Thr232, a casein kinase I phosphorylation site located within this region. A tryptophan residue placed at position 242 was exploited as an intrinsic fluorescence probe of the C-terminal stretch dynamics. Other tryptophan residues were mutated to phenylalanine. Time-resolved fluorescence measurements revealed that phosphopeptide binding changes the conformation and increases the flexibility of 14-3-3zeta C-terminal stretch, demonstrating that this region is directly involved in ligand binding. Phosphorylation of 14-3-3zeta at Thr232 resulted in inhibition of phosphopeptide binding and suppression of 14-3-3-mediated enhancement of serotonin N-acetyltransferase activity. Time-resolved fluorescence of Trp242 also revealed that phosphorylation at Thr232 induces significant changes of the C-terminal stretch conformation. In addition, molecular dynamic simulations suggest that phosphorylation at Thr232 induces a more extended conformation of 14-3-3zeta C-terminal stretch and changes its interaction with the rest of the 14-3-3 molecule. These results indicate that the conformation of the C-terminal stretch plays an important role in the regulation of 14-3-3 binding properties.     

Role of the 14-3-3 C-terminal loop in ligand interaction

14-3-3 proteins are a family of conserved dimeric molecules that interact with a broad range of target proteins, most of which contain phosphoserine/threonine. The amphipathic groove of 14-3-3 is the main structural feature involved in mediating its associations. We have studied another domain of 14-3-3, the C-terminal loop, to determine what role it plays in ligand interaction. A truncated form of 14-3-3zeta lacking this C-terminal loop was generated and found to bind with higher affinity than the wild-type 14-3-3zeta protein to the ligands Raf-1 and Bad. Interestingly, the truncated 14-3-3zeta also showed increased association with the 14-3-3 binding-deficient Bad/S136A mutant. Taken together, these data support a role for the C-terminal loop as a general inhibitor of 14-3-3/ligand interactions. This may provide a mechanism by which inappropriate associations with 14-3-3 are prevented.     

The proapoptotic protein Bad binds the amphipathic groove of 14-3-3zeta.

Through interaction with a multitude of target proteins, 14-3-3 proteins participate in the regulation of diverse cellular processes including apoptosis. These 14-3-3-interacting proteins include a proapoptotic Bcl-2 homolog, Bad (Bcl-2/Bcl-XL-associated death promoter). To understand how 14-3-3 interacts with Bad and modulates its function, we have identified structural elements of 14-3-3 necessary for 14-3-3/Bad association. 14-3-3 contains a conserved amphipathic groove that is required for binding to several of its ligands. We used peptides of known binding specificity as competitors to demonstrate that Bad interacts with 14-3-3zeta via its amphipathic groove. More detailed analysis revealed that several conserved residues in the groove, including Lys-49, Val-176, and Leu-220, were critical for Bad interaction. These results were applied to investigations of the ability of 14-3-3 to prevent Bad-induced cell death. When co-expressed with Akt, wild-type 14-3-3 could reduce the ability of Bad to cause death, however 14-3-3zetaK49E, which cannot bind Bad, failed to inhibit Bad. It seems that the amphipathic groove of 14-3-3 represents a general binding site for multiple ligands, raising issues related to competition of ligands for 14-3-3.     

The dimeric versus monomeric status of 14-3-3zeta is controlled by phosphorylation of Ser58 at the dimer interface

The 14-3-3 proteins play a central role in the regulation of cell growth, cycling, and apoptosis by modulating the functional activities of key signaling proteins. Through binding to a phosphoserine motif, 14-3-3 alters target proteins activities by sequestering them, relocalizing them, conformationally altering their functional activity, or by promoting interaction with other proteins. These functions of 14-3-3 are facilitated by, if not dependent on, its dimeric structure. We now show that the dimeric status of 14-3-3 is regulated by site-specific serine phosphorylation. We found that a sphingosine-dependent kinase phosphorylates 14-3-3 in vitro and in vivo on a serine residue (Ser58) located within the dimer interface. Furthermore, by developing an antibody that specifically recognizes 14-3-3zeta 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.