Phosphoprotein-binding Domains Research Articles

Human genome-wide analysis and identification of the hyperphosphorylation-elicited interactions between subarachnoid tau protein and phosphoprotein-binding domains.

Abnormally hyperphosphorylated tau can be recognized by a variety of phosphoprotein-binding domains (PBDs) to elicit downstream tau signaling in neuropathology, which has been found to have a potential association with subarachnoid hemorrhage. In this study, the genome-wide binding behavior of tau phosphorylation sites (p-sites) to PBDs involved in subarachnoid hyperphosphorylation events was systematically profiled at molecular level by integrating peptide docking, structural minimization, affinity scoring, and binding assay, from which a number of potent PBD-p-site interaction pairs were identified. It was revealed that the PBD domains exhibit distinct binding preferences for phosphotyrosine, phosphoserine, and phosphothreonine p-sites; the PBD-recognition specificity of different tau p-sites is not overlapped with each other, and their phosphorylations would therefore regulate varying biological functions in tau signaling. A number of PBD-p-site pairs were identified to have potent binding potency as compared to others. The KCIP-pS[393-399] pair was found as a strong binder, which was further optimized with a rational peptide design protocol to derive a number of affinity-improved phosphopeptides. Structural analysis revealed diverse noncovalent chemical forces across the complex interface of KCIP domain with a designed high-affinity pS[393-399]-d4, which confers both stability and specificity to the domain-peptide complex system, with affinity improved by 10.9-fold relative to the native pS[393-399].

GPS-PBS: A Deep Learning Framework to Predict Phosphorylation Sites that Specifically Interact with Phosphoprotein-Binding Domains.

Protein phosphorylation is essential for regulating cellular activities by modifying substrates at specific residues, which frequently interact with proteins containing phosphoprotein-binding domains (PPBDs) to propagate the phosphorylation signaling into downstream pathways. Although massive phosphorylation sites (p-sites) have been reported, most of their interacting PPBDs are unknown. Here, we collected 4458 known PPBD-specific binding p-sites (PBSs), considerably improved our previously developed group-based prediction system (GPS) algorithm, and implemented a deep learning plus transfer learning strategy for model training. Then, we developed a new online service named GPS-PBS, which can hierarchically predict PBSs of 122 single PPBD clusters belonging to two groups and 16 families. By comparison, GPS-PBS achieved a highly competitive accuracy against other existing tools. Using GPS-PBS, we predicted 371,018 mammalian p-sites that potentially interact with at least one PPBD, and revealed that various PPBD-containing proteins (PPCPs) and protein kinases (PKs) can simultaneously regulate the same p-sites to orchestrate important pathways, such as the PI3K-Akt signaling pathway. Taken together, we anticipate GPS-PBS can be a great help for further dissecting phosphorylation signaling networks.

BRCT domains contain an intrinsic post‐translational modification (PTM) recognition code that affects it stability

BRCA1 C‐terminal (BRCT) domains are protein binding modules that relay signals throughout various cellular pathways. The human genome encodes 23 BRCT‐domain containing proteins that are commonly found in tandem and have been characterized as phosphoprotein binding domains with functions throughout the DNA damage response. Emerging evidence suggests these domains bind other post‐translationally modified (PTM) substrates although the details are not defined. Recently, the BRCT domains from BRCA1 were shown to bind an asymmetrically dimethylated arginine (ADMA) p300 substrate to regulate the transcription of the p21 in response to DNA damage. Based on this observation, we hypothesize that the methyl‐specific binding site for the ADMA interaction is distinct from the phospho‐substrate recognition interface. To test this hypothesis, we conducted peptide binding and thermal stability experiments to demonstrate the change in stability upon binding phospho‐versus methylated substrates. Our results show that phosphorylated substrates increase the stability of BRCA1's BRCT domains. Doubly phosphorylated substrates showed the greatest increase in stability while the methylated substrate demonstrated a decrease in stability. To define the BRCT‐ADMA binding interface, we set out to determine the three‐dimensional structure. High‐throughput co‐crystallization of the BRCA1 BRCT protein with a ADMA peptide derived from the p300 substrate identified one condition for crystal growth optimization. In silico modeling of the BRCA1 BRCT structure with the ADMA molecule has highlighted two potential binding sites for the ADMA substrate, which is not conserved throughout the BRCT domain family. Our results indicate that while BRCT domains share a conserved mode of binding phosphorylated substrates, which increases their stability, and their ability to binding methylated substrates may not be conserved. These finding will be critical in understanding how BRCT domains can accommodate binding various modified substrates and regulating signals throughout the DNA damage response.Support or Funding InformationNCI K22 CA166544‐03This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

β-Hairpins as peptidomimetics of human phosphoprotein-binding domains.

Phosphoprotein-binding domains interact with cognate phosphorylated targets ruling several biological processes. The impairment of such interactions is often associated with disease development, namely cancer. The breast cancer susceptibility gene 1 (BRCA1) C-terminal (BRCT) domain is involved in the control of complex signaling networks of the DNA damage response. The capture and identification of BRCT-binding proteins and peptides may be used for the development of new diagnostic tools for diseases with abnormal phosphorylation profiles. Here we show that designed cyclic β-hairpin structures can be used as peptidomimetics of the BRCT domain, with high selectivity in binding to a target phosphorylated peptide. The amino acid residues and spatial constraints involved in the interaction between a phosphorylated peptide (GK14-P) and the BRCT domain were identified and crafted onto a 14-mer β-hairpin template in silico. Several cyclic peptides models were designed and their binding towards the target peptide and other phosphorylated peptides evaluated through virtual screening. Selected cyclic peptides were then synthesized, purified and characterized. The high affinity and selectivity of the lead cyclic peptide towards the target phosphopeptide was confirmed, and the possibility to capture it using affinity chromatography demonstrated. This work paves the way for the development of cyclic β-hairpin peptidomimetics as a novel class of affinity reagents for the highly selective identification and capture of target molecules.

IEKPD 2.0: an update with rich annotations for eukaryotic protein kinases, protein phosphatases and proteins containing phosphoprotein-binding domains.

Here, we described the updated database iEKPD 2.0 (http://iekpd.biocuckoo.org) for eukaryotic protein kinases (PKs), protein phosphatases (PPs) and proteins containing phosphoprotein-binding domains (PPBDs), which are key molecules responsible for phosphorylation-dependent signalling networks and participate in the regulation of almost all biological processes and pathways. In total, iEKPD 2.0 contained 197 348 phosphorylation regulators, including 109 912 PKs, 23 294 PPs and 68 748 PPBD-containing proteins in 164 eukaryotic species. In particular, we provided rich annotations for the regulators of eight model organisms, especially humans, by compiling and integrating the knowledge from 100 widely used public databases that cover 13 aspects, including cancer mutations, genetic variations, disease-associated information, mRNA expression, DNA & RNA elements, DNA methylation, molecular interactions, drug-target relations, protein 3D structures, post-translational modifications, protein expressions/proteomics, subcellular localizations and protein functional annotations. Compared with our previously developed EKPD 1.0 (∼0.5 GB), iEKPD 2.0 contains ∼99.8 GB of data with an ∼200-fold increase in data volume. We anticipate that iEKPD 2.0 represents a more useful resource for further study of phosphorylation regulators.

Phosphoproteomic Analysis Using the WW and FHA Domains as Biological Filters.

Protein phosphorylation plays a key role in regulating nearly all intracellular biological events. However, poorly developed phospho-specific antibodies and low phosphoprotein abundance make it difficult to study phosphoproteins. Cellular protein phosphorylation data have been obtained using phosphoproteomic approaches, but the detection of low-abundance or fast-cycling phosphorylation sites remains a challenge. Enrichment of phosphoproteins together with phosphopeptides may greatly enhance the spectrum of low-abundance but biologically important phosphoproteins. Previously, we used 14-3-3ζ to selectively enrich for HeLa cell lysate phosphoproteins. However, because 14-3-3 does not isolate phosphoproteins lacking the 14-3-3-binding motif, we looked for other domains that could complementarily enrich for phosphoproteins. We here assessed and characterized the phosphoprotein binding domains Pin1-WW, CHEK2-FHA, and DLG1-GK. Using a strategy based on affinity chromatography, phosphoproteins were collected from the lysates of HeLa cells treated with phosphatase inhibitor or cAMP activator. We identified different subsets of phosphoproteins associated with WW or FHA after calyculin A, okadaic acid, or forskolin treatment. Our Kinase-Oriented Substrate Screening (KiOSS) method, which used phosphoprotein-binding domains, showed that WW and FHA are applicable and useful for the identification of novel phospho-substrates for kinases and can therefore be used as biological filters for comprehensive phosphoproteome analysis.

Abstract 4539: Selection of non-phophorylated peptide inhibitors of BRCA1.

Abstract A growing body of literature suggests Breast Cancer-Associated Protein 1 (BRCA1) is important not only as a cause, but also as a target in the quest for cancer treatment. BRCA1 deficient cells treated with radiation as well as PARP inhibitors and other chemotherapeutics demonstrate a greater sensitivity than cells with wild type BRCA1. Inhibitors of BRCA1 would take advantage of this synthetic lethality and represent a significant advance in cancer treatment as well as an understanding of the biology of DNA repair. Despite significant study of BRCA1 protein and function, it is a large protein (208 KDa) that is still largely uncharacterized, but its N- and C-terminal domains have been described by significant structural data. The BRCT (BRCA1 C-Terminal) Domain is a phosphoprotein binding domain that is commonly mutated or lost in cancers and has a binding cleft seemingly very suitable for drug design. Small molecule screens have been conducted against this domain, but the resulting hits with moderate affinity have not been shown to induce BRCA1 deficient phenotypes. Phosphopeptides have also been studied as potential BRCA1 inhibitors, yet despite some having affinities in the mid-nanomolar range the presence of a phosphate is not without its pharmacologic challenges. We generated an mRNA display library with 1.3 x 10ˆ13 cyclized peptides covalently attached to the mRNA that encoded them. Eight rounds of selection exposing the library to a GST-BRCT fusion resulted in non-phosphorylated peptides that bind to a BRCT domain of BRCA1. The sequences resulting from the selection have common homologies and initial characterization has shown that these peptides may be the first viable non-phosphoserine containing inhibitors of BRCA1. Citation Format: E. Railey White, Zhong Ma, David E. Hacker, Jason M. Beckta, Melissa B. Huie, David C. Williams, Kristoffer Valerie, Matthew C. Hartman. Selection of non-phophorylated peptide inhibitors of BRCA1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4539. doi:10.1158/1538-7445.AM2013-4539

Structure and Functional Analysis of the RNA- and Viral Phosphoprotein-Binding Domain of Respiratory Syncytial Virus M2-1 Protein

Respiratory syncytial virus (RSV) protein M2-1 functions as an essential transcriptional cofactor of the viral RNA-dependent RNA polymerase (RdRp) complex by increasing polymerase processivity. M2-1 is a modular RNA binding protein that also interacts with the viral phosphoprotein P, another component of the RdRp complex. These binding properties are related to the core region of M2-1 encompassing residues S58 to K177. Here we report the NMR structure of the RSV M2-158–177 core domain, which is structurally homologous to the C-terminal domain of Ebola virus VP30, a transcription co-factor sharing functional similarity with M2-1. The partial overlap of RNA and P interaction surfaces on M2-158–177, as determined by NMR, rationalizes the previously observed competitive behavior of RNA versus P. Using site-directed mutagenesis, we identified eight residues located on these surfaces that are critical for an efficient transcription activity of the RdRp complex. Single mutations of these residues disrupted specifically either P or RNA binding to M2-1 in vitro. M2-1 recruitment to cytoplasmic inclusion bodies, which are regarded as sites of viral RNA synthesis, was impaired by mutations affecting only binding to P, but not to RNA, suggesting that M2-1 is associated to the holonucleocapsid by interacting with P. These results reveal that RNA and P binding to M2-1 can be uncoupled and that both are critical for the transcriptional antitermination function of M2-1.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Implications for proteasome nuclear localization revealed by the structure of the nuclear proteasome tether protein Cut8

Degradation of nuclear proteins by the 26S proteasome is essential for cell viability. In yeast, the nuclear envelope protein Cut8 mediates nuclear proteasomal sequestration by an uncharacterized mechanism. Here we describe structures of Schizosaccharomyces pombe Cut8, which shows that it contains a unique, modular fold composed of an extended N-terminal, lysine-rich segment that when ubiquitinated binds the proteasome, a dimer domain followed by a six-helix bundle connected to a flexible C tail. The Cut8 six-helix bundle shows structural similarity to 14-3-3 phosphoprotein-binding domains, and binding assays show that this domain is necessary and sufficient for liposome and cholesterol binding. Moreover, specific mutations in the 14-3-3 regions corresponding to putative cholesterol recognition/interaction amino acid consensus motifs abrogate cholesterol binding. In vivo studies confirmed that the 14-3-3 region is necessary for Cut8 membrane localization and that dimerization is critical for its function. Thus, the data reveal the Cut8 organization at the nuclear envelope. Reconstruction of Cut8 evolution suggests that it was present in the last common ancestor of extant eukaryotes and accordingly that nuclear proteasomal sequestration is an ancestral eukaryotic feature. The importance of Cut8 for cell viability and its absence in humans suggests it as a possible target for the development of specific chemotherapeutics against invasive fungal infections.

PepCyber:P PEP: a database of human protein protein interactions mediated by phosphoprotein-binding domains

Phosphoprotein-binding domains (PPBDs) mediate many important cellular and molecular processes. Ten PPBDs have been known to exist in the human proteome, namely, 14-3-3, BRCT, C2, FHA, MH2, PBD, PTB, SH2, WD-40 and WW. PepCyber:P∼PEP is a newly constructed database specialized in documenting human PPBD-containing proteins and PPBD-mediated interactions. Our motivation is to provide the research community with a rich information source emphasizing the reported, experimentally validated data for specific PPBD–PPEP interactions. This information is not only useful for designing, comparing and validating the relevant experiments, but it also serves as a knowledge-base for computationally constructing systems signaling pathways and networks. PepCyber:P∼PEP is accessible through the URL, http://www.pepcyber.org/PPEP/. The current release of the database contains 7044 PPBD-mediated interactions involving 337 PPBD-containing proteins and 1123 substrate proteins.

The DNA Damage Response Mediator MDC1 Directly Interacts with the Anaphase-promoting Complex/Cyclosome

MDC1 (NFBD1), a mediator of the cellular response to DNA damage, plays an important role in checkpoint activation and DNA repair. Here we identified a cross-talk between the DNA damage response and cell cycle regulation. We discovered that MDC1 binds the anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase that controls the cell cycle. The interaction is direct and is mediated by the tandem BRCA1 C-terminal domains of MDC1 and the C terminus of the Cdc27 (APC3) subunit of the APC/C. It requires the phosphorylation of Cdc27 and is enhanced after induction of DNA damage. We show that the tandem BRCA1 C-terminal domains of MDC1, known to directly bind the phosphorylated form of histone H2AX (gamma-H2AX), also bind the APC/C by the same mechanism, as phosphopeptides that correspond to the C termini of gamma-H2AX and Cdc27 competed with each other for the binding to MDC1. Our results reveal a link between the cellular response to DNA damage and cell cycle regulation, suggesting that MDC1, known to have a role in checkpoint regulation, executes part of this role by binding the APC/C.

Design of a Hybrid Biosensor for Enhanced Phosphopeptide Recognition Based on a Phosphoprotein Binding Domain Coupled with a Fluorescent Chemosensor

Protein-based fluorescent biosensors with sufficient sensing specificity are useful analytical tools for detection of biologically important substances in complicated biological systems. Here, we present the design of a hybrid biosensor, specific for a bis-phosphorylated peptide, based on a natural phosphoprotein binding domain coupled with an artificial fluorescent chemosensor. The hybrid biosensor consists of a phosphoprotein binding domain, the WW domain, into which has been introduced a fluorescent stilbazole having Zn(II)-dipicolylamine (Dpa) as a phosphate binding motif. It showed strong binding affinity and high sensing selectivity toward a specific bis-phosphorylated peptide in the presence of various phosphate species such as the monophosphorylated peptide, ATP, and others. Detailed fluorescence titration experiments clearly indicate that the binding-induced fluorescence enhancement and the sensing selectivity were achieved by the cooperative action of both binding sites of the hybrid biosensor, i.e., the WW domain and the Zn(II)-Dpa chemosensor unit. Thus, it is clear that the tethered Zn(II)-Dpa-stilbazole unit operated not only as a fluorescence signal transducer, but also as a sub-binding site in the hybrid biosensor. Taking advantage of its selective sensing property, the hybrid biosensor was successfully applied to real-time and label-free fluorescence monitoring of a protein kinase-catalyzed phosphorylation.

Effective Disruption of Phosphoprotein−Protein Surface Interaction Using Zn(II) Dipicolylamine-Based Artificial Receptors via Two-Point Interaction

Protein phosphorylation is ubiquitously involved in living cells, and it is one of the key events controlling protein-protein surface interactions, which are essential in signal transduction cascades. We now report that the small molecular receptors bearing binuclear Zn(II)-Dpa can strongly bind to a bis-phosphorylated peptide in a cross-linking manner under neutral aqueous conditions when the distance between the two Zn(II) centers can appropriately fit in that of the two phosphate groups of the phosphorylated peptide. The binding property was quantitatively determined by ITC (isothermal titration calorimetry), induced CD (circular dichroism), and NMR. On the basis of these findings, we demonstrated that these types of small molecules were able to effectively disrupt the phosphoprotein-protein interaction in a phosphorylated CTD peptide and the Pin1 WW domain, a phosphoprotein binding domain, at a micromolar level. The strategy based on a small molecular disruptor that directly interacts with phosphoprotein is unique and should be promising in developing a designer inhibitor for phosphoprotein-protein interaction.

DNA damage-induced cell cycle checkpoint control requires CtIP, a phosphorylation-dependent binding partner of BRCA1 C-terminal domains.

The BRCA1 C-terminal (BRCT) domain has recently been implicated as a phospho-protein binding domain. We demonstrate here that a CTBP-interacting protein CtIP interacts with BRCA1 BRCT domains in a phosphorylation-dependent manner. The CtIP/BRCA1 complex only exists in G(2) phase and is required for DNA damage-induced Chk1 phosphorylation and the G(2)/M transition checkpoint. However, the CtIP/BRCA1 complex is not required for the damage-induced G(2) accumulation checkpoint, which is controlled by a separate BRCA1/BACH1 complex. Taken together, these data not only implicate CtIP as a critical player in cell cycle checkpoint control but also provide molecular mechanisms by which BRCA1 controls multiple cell cycle transitions after DNA damage.

Risk Factors in Breast and Ovarian Cancer

Mutations in breast cancer gene 1 ( BRCA1 ) are a major cause of familial breast cancer. The BRCA1 protein functions in cellular responses to DNA damage, but its precise molecular actions are not fully understood. Two different approaches now show that the C-terminal domain of BRCA1 (BRCT), which is also found in other proteins that function in control of DNA damage, specifically binds to target sequences when serine or threonine residues in the target domain are phosphorylated. Yu et al. (see the Perspective by Caldecott) demonstrate the phosphorylation-dependent interaction of BRCA1 with BRCA1-associated C-terminal helicase and show that this interaction is required for a proper cellular response to DNA damage. Manke et al. used a proteomic strategy to identify BRCT domains in a screen for modules that would bind sequences preferentially phosphorylated by the phosphoinositide-like kinases ATM and ATR, which are activated in response to DNA damage. X. Yu, C. C. S. Chini, M. He, G. Mer, J. Chen, The BRCT domain is a phospho-protein binding domain. Science 302 , 639-642 (2003). [Abstract] [Full Text] K. W. Caldecott, The BRCT domain: Signaling with friends? Science 302 , 579-580 (2003). [Abstract] [Full Text] I. A. Manke, D. M. Lowery, A. Nguyen, M. B. Yaffe, BRCT repeats as phosphopeptide-binding modules involved in protein targeting. Science 302 , 636-639 (2003). [Abstract] [Full Text]

The BRCT domain is a phospho-protein binding domain.

The carboxyl-terminal domain (BRCT) of the Breast Cancer Gene 1 (BRCA1) protein is an evolutionarily conserved module that exists in a large number of proteins from prokaryotes to eukaryotes. Although most BRCT domain-containing proteins participate in DNA-damage checkpoint or DNA-repair pathways, or both, the function of the BRCT domain is not fully understood. We show that the BRCA1 BRCT domain directly interacts with phosphorylated BRCA1-Associated Carboxyl-terminal Helicase (BACH1). This specific interaction between BRCA1 and phosphorylated BACH1 is cell cycle regulated and is required for DNA damage-induced checkpoint control during the transition from G2 to M phase of the cell cycle. Further, we show that two other BRCT domains interact with their respective physiological partners in a phosphorylation-dependent manner. Thirteen additional BRCT domains also preferentially bind phospho-peptides rather than nonphosphorylated control peptides. These data imply that the BRCT domain is a phospho-protein binding domain involved in cell cycle control.

Looking for Like-Minded Partners

Protein kinases are regulators of many biological processes, but many of the targets of these phosphorylating enzymes remain unidentified. Some proteins have special binding domains that preferentially recognize phosphorylated amino acids, and both the kinases that modify these sites and the binding partners that recognize them look for similar sequence motifs. Elia et al. (see the Perspective by Silljé and Nigg) developed a proteomic screen to identify certain binding domains and used it to search for proteins involved in the control of cell division. The screen revealed a phosphoprotein-binding domain, the Polo-box domain (PBD), itself located in a protein kinase, Polo-like kinase 1 (Plk-1). Further experiments indicated that the PBD promotes subcellular localization of Plk1 and the interaction of Plk1 with its substrates in response to the action of mitotic kinases. A. E. H. Elia, L. C. Cantley, M. B. Yaffe, Proteomic screen finds pSer/pThr-binding domain localizing Plk1 to mitotic substrates. Science 299 , 1228-1231 (2003). [Abstract] [Full Text] H. H. W. Silljé, E. A. Nigg, Capturing Polo kinase. Science 299 , 1190-1191 (2003). [Summary] [Full Text]

The 14-3-3 Shuttle

The signaling molecule 14-3-3 binds to various phosphorylated molecules and presumably regulates their subcellular localization. However, most of the binding partners are located in the nucleus, whereas 14-3-3 is thought to reside mostly in the cytoplasm. Where then does phosphorylation and subsequent binding to 14-3-3 occur, and how is subcellular localization determined? Brunet et al. tackle this hotly debated topic by examining the nucleocytoplasmic transport of Forkhead (FKHRL1), a phosphorylated transcription factor that binds to 14-3-3. The study shows that a sequence in 14-3-3 does not act as a nuclear export signal (NES), as previously suggested, but as a phosphoprotein binding domain. 14-3-3 did not directly interact with components of the nuclear export machinery either. In fact, a mutated form of 14-3-3 that cannot bind to FKHRL1 localized to the nucleus, suggesting that unbound 14-3-3 may normally reside in the nucleus, waiting to bind to a partner protein. FRKHRL1 was phosphorylated within the nucleus, in response to growth factor stimulation of cells, facilitating subsequent interaction with 14-3-3. A NES expressed by FKHRL1 was also required to transport the complex out of the nucleus. The authors speculate that once in the cytoplasm, a nuclear localization signal present in FKHRL1 is masked by association with 14-3-3. Thus, 14-3-3 appears to enhance nuclear export, but just how it accelerates this process remains to be determined.A. Brunet, F. Kanai, J. Stehn, J. Xu, D. Sarbassova, J. V. Frangioni, S. N. Dalal, J. A. DeCaprio, M. E. Greenberg, M. B. Yaffe, 14-3-3 transits to the nucleus and participates in dynamic nucleocytoplasmic transport. J. Cell Biol. 156, 817-828 (2002).

The 14-3-3 Shuttle

The signaling molecule 14-3-3 binds to various phosphorylated molecules and presumably regulates their subcellular localization. However, most of the binding partners are located in the nucleus, whereas 14-3-3 is thought to reside mostly in the cytoplasm. Where then does phosphorylation and subsequent binding to 14-3-3 occur, and how is subcellular localization determined? Brunet et al . tackle this hotly debated topic by examining the nucleocytoplasmic transport of Forkhead (FKHRL1), a phosphorylated transcription factor that binds to 14-3-3. The study shows that a sequence in 14-3-3 does not act as a nuclear export signal (NES), as previously suggested, but as a phosphoprotein binding domain. 14-3-3 did not directly interact with components of the nuclear export machinery either. In fact, a mutated form of 14-3-3 that cannot bind to FKHRL1 localized to the nucleus, suggesting that unbound 14-3-3 may normally reside in the nucleus, waiting to bind to a partner protein. FRKHRL1 was phosphorylated within the nucleus, in response to growth factor stimulation of cells, facilitating subsequent interaction with 14-3-3. A NES expressed by FKHRL1 was also required to transport the complex out of the nucleus. The authors speculate that once in the cytoplasm, a nuclear localization signal present in FKHRL1 is masked by association with 14-3-3. Thus, 14-3-3 appears to enhance nuclear export, but just how it accelerates this process remains to be determined. A. Brunet, F. Kanai, J. Stehn, J. Xu, D. Sarbassova, J. V. Frangioni, S. N. Dalal, J. A. DeCaprio, M. E. Greenberg, M. B. Yaffe, 14-3-3 transits to the nucleus and participates in dynamic nucleocytoplasmic transport. J. Cell Biol . 156 , 817-828 (2002).