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.     

Identification of novel nuclear localization signals of Drosophila myeloid leukemia factor

Myeloid leukemia factor 1 (MLF1) was first identified as part of a leukemic fusion protein produced by a chromosomal translocation, and MLF family proteins are present in many animals. In mammalian cells, MLF1 has been described as mainly cytoplasmic, but in Drosophila, one of the dMLF isoforms (dMLFA) localized mainly in the nucleus while the other isoform (dMLFB), that appears to be produced by the alternative splicing, displays both nuclear and cytoplasmic localization. To investigate the difference in subcellular localization between MLF family members, we examined the subcellular localization of deletion mutants of dMLFA isoform. The analyses showed that the C-terminal 40 amino acid region of dMLFA is necessary and sufficient for nuclear localization. Based on amino acid sequences, we hypothesized that two nuclear localization signals (NLSs) are present within the region. Site-directed mutagenesis of critical residues within the two putative NLSs leads to loss of nuclear localization, suggesting that both NLS motifs are necessary for nuclear localization.     

Structural insights of the MLF1/14-3-3 interaction

Myeloid leukaemia factor 1 (MLF1) binds to 14-3-3 adapter proteins by a sequence surrounding Ser34 with the functional consequences of this interaction largely unknown. We present here the high-resolution crystal structure of this binding motif [MLF1(29-42)pSer34] in complex with 14-3-3ε and analyse the interaction with isothermal titration calorimetry. Fragment-based ligand discovery employing crystals of the binary 14-3-3ε/MLF1(29-42)pSer34 complex was used to identify a molecule that binds to the interface rim of the two proteins, potentially representing the starting point for the development of a small molecule that stabilizes the MLF1/14-3-3 protein-protein interaction. Such a compound might be used as a chemical biology tool to further analyse the 14-3-3/MLF1 interaction without the use of genetic methods. Database Structural data are available in the Protein Data Bank under the accession number(s) 3UAL [14-3-3ε/MLF1(29-42)pSer34 complex] and 3UBW [14-3-3ε/MLF1(29-42)pSer34/3-pyrrolidinol complex] Structured digital abstract ? 14-3-3 epsilon?and?MLF1?bind?by?x-ray crystallography?(View interaction) ? 14-3-3 epsilon?and?MLF1?bind?by?isothermal titration calorimetry?(View Interaction:?1,?2).     

Nuclear localization of protein kinase U-alpha is regulated by 14-3-3.

14-3-3 proteins are intracellular, dimeric molecules that bind to and modify the activity of several signaling proteins. We used human 14-3-3zeta as a bait in the yeast two-hybrid system to screen a murine embryonic cDNA library. One interacting clone was found to encode the carboxyl terminus of a putative protein kinase. The coding sequence of the human form (protein kinase Ualpha, PKUalpha) of this protein kinase was found in GenBank(TM) on the basis of sequence homology. The two-hybrid clone was also highly homologous to TOUSLED, an Arabidopsis thaliana protein kinase that is required for normal flower and leaf development. PKUalpha has been found by coimmunoprecipitation to bind to 14-3-3zeta in vivo. Our confocal laser immunofluorescence microscopic experiments revealed that PKUalpha colocalizes with the cytoplasmic intermediate filament system of cultured fibroblasts in the G(1) phase of the cell cycle. PKUalpha is found in the perinuclear area of S phase cells and in the nucleus of late G(2) cells. Transfection of cells with a dominant negative form of 14-3-3eta promotes the nuclear localization of PKUalpha. These results suggest that the subcellular localization of PKUalpha is regulated, at least in part, by its association with 14-3-3.     

A Cdc2-related protein kinase hPFTAIRE1 from human brain interacting with 14-3-3 proteins

hPFTAIRE1 （PFTK1）, a Cdc2-related protein kinase, is highly expressed in human brain. It exhibits cytoplasmic distribution in Hela cells, although it contains two nuclear localization signals （NLSs） in its N-terminus. To search for its substrates and regulatory components, we screened a two-hybrid library by using the full-length hPFTAIRE1 as a bait. Four 14-3-3 isoforms （β,ε,η,τ） were identified interacting with the hPFTAIRE1. We found a putative 14-3-3 binding consensus motif（RHSSPSS） in the hPFTAIRE 1, which overlapped with its second NLS. Deletion of the RHSSPSS motif or substitution of Ser^119 gwithAla in the conserved binding motif abolished the specific interaction between the hPFTAIRE 1 and the 14-3 -3 proteins. The mutant S 120A hPFTAIRE1 also showed a weak interaction to the 14-3-3 proteins. The results suggested that the Ser^119 is crucial for the interaction between hPFTAIREI and the 14-3-3 proteins. All the hPFTAIRE1 mutants distributed in cytoplasm of Hela cells and human neuroblastoma cells （SH-SY5Y） when fused to the C-terminus of a green fluorescent protein （GFP）, indicating that binding with the 14-3-3 proteins does not contribute to the subcellular localization of the hPFTAIRE1, although the binding may be involved in its signaling regulation.     

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.     

Interaction of the parathyroid hormone receptor with the 14-3-3 protein

The receptor for parathyroid hormone (PTH) and PTH-related protein (PTHrP) regulates calcium homeostasis, bone remodeling and skeletal development. 14-3-3 proteins bind to signaling proteins and act as molecular scaffolds and regulators of subcellular localization. We show that the parathyroid hormone receptor (PTHR) interacts with 14-3-3 and the proteins colocalize within the cell. 14-3-3 interacts with the C-terminal tail of the receptor containing a consensus 14-3-3 binding motif, but additional binding sites are also used. Protein kinase-A treatment of the receptor and especially the C-terminal tail reduces 14-3-3 binding. The expressed C-terminal tail is primarily localized in the nucleus, supporting the function of a putative nuclear localization signal that could be involved in the previously described nuclear localization of PTHR. The observed interaction between PTHR and the 14-3-3 protein implies that 14-3-3 could contribute to regulation of PTHR signaling.     

Shuttling imbalance of MLF1 results in p53 instability and increases susceptibility to oncogenic transformation

Myeloid leukemia factor 1 (MLF1) stabilizes the activity of the tumor suppressor p53 by suppressing its E3 ubiquitin ligase, COP1, through a third component of the COP9 signalosome (CSN3). However, little is known about how MLF1 functions upstream of the CSN3-COP1-p53 pathway and how its deregulation by the formation of the fusion protein nucleophosmin (NPM)-MLF1, generated by t(3;5)(q25.1;q34) chromosomal translocation, leads to leukemogenesis. Here we show that MLF1 is a cytoplasmic-nuclear-shuttling protein and that its nucleolar localization on fusing with NPM prevents the full induction of p53 by both genotoxic and oncogenic cellular stress. The majority of MLF1 was located in the cytoplasm, but the treatment of cells with leptomycin B rapidly induced a nuclear accumulation of MLF1. A mutation of the nuclear export signal (NES) motif identified in the MLF1 sequence enhanced the antiproliferative activity of MLF1. The fusion of MLF1 with NPM translocated MLF1 to the nucleolus and abolished the growth-suppressing activity. The introduction of NPM-MLF1 into early-passage murine embryonic fibroblasts allowed the cells to escape from cellular senescence at a markedly earlier stage and induced neoplastic transformation in collaboration with the oncogenic form of Ras. Interestingly, disruption of the MLF1-derived NES sequence completely abolished the growth-promoting activity of NPM-MLF1 in murine fibroblasts and hematopoietic cells. Thus, our results provide important evidence that the shuttling of MLF1 is critical for the regulation of cell proliferation and a disturbance in the shuttling balance increases the cell's susceptibility to oncogenic transformation.     

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.     

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.     

Involvement of 14-3-3 proteins in nuclear localization of telomerase

Maintenance of telomeres is implicated in chromosome stabilization and cell immortalization. Telomerase, which catalyzes de novo synthesis of telomeres, is activated in germ cells and most cancers. Telomerase activity is regulated by gene expression for its catalytic subunit, TERT, whereas several lines of evidence have suggested a post-translational regulation of telomerase activity. Here we identify the 14-3-3 signaling proteins as human TERT (hTERT)-binding partners. A dominant-negative 14-3-3 redistributed hTERT, which was normally predominant in the nucleus, into the cytoplasm. Consistent with this observation, hTERT-3A, a mutant that could not bind 14-3-3, was localized into the cytoplasm. Leptomycin B, an inhibitor of CRM1/exportin 1-mediated nuclear export, or disruption of a nuclear export signal (NES)-like motif located just upstream of the 14-3-3 binding site in hTERT impaired the cytoplasmic localization of hTERT. Compared with wild-type hTERT, hTERT-3A increased its association with CRM1. 14-3-3 binding was not required for telomerase activity either in vitro or in cell extracts. These observations suggest that 14-3-3 enhances nuclear localization of TERT by inhibiting the CRM1 binding to the TERT NES-like motif.     

A robust protocol to map binding sites of the 14-3-3 interactome: Cdc25C requires phosphorylation of both S216 and S263 to bind 14-3-3

Modern proteomic techniques have identified hundreds of proteins that bind 14-3-3s, the most widespread eukaryotic phosphoserine/threonine sensors, but accurate prediction of the target phospho-sites is difficult. Here we describe a systematic approach using synthetic peptides that tests large numbers of potential binding sites in parallel for human 14-3-3. By profiling the sequence requirements for three diverse 14-3-3 binding sites (from IRS-1, IRSp53 and GIT2), we have generated enhanced bioinformatics tools to score sites and allow more tractable testing by co-immunoprecipitation. This approach has allowed us to identify two additional sites other than Ser216 in the widely studied cell division cycle (Cdc) protein 25C, whose function depends on 14-3-3 binding. These Ser247 and Ser263 sites in human Cdc25C, which were not predicted by the existing Scansite search, are conserved across species and flank the nuclear localization region. Furthermore, we found strong interactions between 14-3-3 and peptides with the sequence Rxx[S/T]xR typical for PKC sites, and which is as abundant as the canonical Rxx[S/T]xP motif in the proteome. Two such sites are required for 14-3-3 binding in the polarity protein Numb. A recent survey of >200 reported sites identified only a handful containing this motif, suggesting that it is currently under-appreciated as a candidate binding site. This approach allows one to rapidly map 14-3-3 binding sites and has revealed alternate motifs.     

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.     

14-3-3 interacts with LKB1 via recognizing phosphorylated threonine 336 residue and suppresses LKB1 kinase function

Here we report a regulatory mechanism by which LKB1 is controlled by 14-3-3 proteins through phorsphorylation of Thr336. The results from the current study indicate that 14-3-3 ζ inhibits LKB1 from phosphorylating its substrate, AMPK (AMP-dependent protein kinase) and attenuates LKB1-mediated G1 cell cycle arrest and apoptosis by interfering with the interaction between LKB1 and its substrates. This regulation does not change either the LKB1 catalytic activity or subcellular localization of LKB1. Moreover, we demonstrate that serum starvation enhances LKB1 activity and increases the phosphorylation of Thr336. Taken together, our results suggest that autophosphorylation of Thr336 acts as an activating signal for LKB1 to recruit 14-3-3, which in turn attenuates the activation of LKB1 to keep the activity of LKB1 in check.