Binding of epidermal growth factor (EGF) to its receptor leads to receptor dimerization, assembly of protein complexes, and activation of signaling networks that control key cellular responses. Despite their fundamental role in cell biology, little is known about protein complexes associated with the EGF receptor (EGFR) before growth factor stimulation. We used a modified membrane yeast two-hybrid system together with bioinformatics to identify 87 candidate proteins interacting with the ligand-unoccupied EGFR. Among them was histone deacetylase 6 (HDAC6), a cytoplasmic lysine deacetylase, which we found negatively regulated EGFR endocytosis and degradation by controlling the acetylation status of alpha-tubulin and, subsequently, receptor trafficking along microtubules. A negative feedback loop consisting of EGFR-mediated phosphorylation of HDAC6 Tyr(570) resulted in reduced deacetylase activity and increased acetylation of alpha-tubulin. This study illustrates the complexity of the EGFR-associated interactome and identifies protein acetylation as a previously unknown regulator of receptor endocytosis and degradation.

Phosphoinositide-3 kinase (PI3K)/Akt signaling is activated by growth factors such as insulin and epidermal growth factor (EGF) and regulates several functions such as cell cycling, apoptosis, cell growth, and cell migration. Here, we find that Kank is an Akt substrate located downstream of PI3K and a 14-3-3-binding protein. The interaction between Kank and 14-3-3 is regulated by insulin and EGF and is mediated through phosphorylation of Kank by Akt. In NIH3T3 cells expressing Kank, the amount of actin stress fibers is reduced, and the coexpression of 14-3-3 disrupted this effect. Kank also inhibits insulin-induced cell migration via 14-3-3 binding. Furthermore, Kank inhibits insulin and active Akt-dependent activation of RhoA through binding to 14-3-3. Based on these findings, we hypothesize that Kank negatively regulates the formation of actin stress fibers and cell migration through the inhibition of RhoA activity, which is controlled by binding of Kank to 14-3-3 in PI3K-Akt signaling.

Grb2-associated binder (Gab)2 functions downstream of a variety of receptor and cytoplasmic tyrosine kinases as a docking platform for specific signal transducers and performs important functions in both normal physiology and oncogenesis. Gab2 signalling is promoted by its association with specific receptors through the adaptor Grb2. However, the molecular mechanisms that attenuate Gab2 signals have remained unclear. We now demonstrate that growth factor-induced phosphorylation of Gab2 on two residues, S210 and T391, leads to recruitment of 14-3-3 proteins. Together, these events mediate negative-feedback regulation, as Gab2(S210A/T391A) exhibits sustained receptor association and signalling and promotes cell proliferation and transformation. Importantly, introduction of constitutive 14-3-3-binding sites into Gab2 renders it refractory to receptor activation, demonstrating that site-selective binding of 14-3-3 proteins is sufficient to terminate Gab2 signalling. Furthermore, this is associated with reduced binding of Grb2. This leads to a model where signal attenuation occurs because 14-3-3 promotes dissociation of Gab2 from Grb2, and thereby uncouples Gab2 from the receptor complex. This represents a novel regulatory mechanism with implications for diverse tyrosine kinase signalling systems.

LRRK2 (leucine-rich repeat protein kinase 2) is mutated in a significant number of Parkinson's disease patients, but still little is understood about how it is regulated or functions. In the present study we have demonstrated that 14-3-3 protein isoforms interact with LRRK2. Consistent with this, endogenous LRRK2 isolated from Swiss 3T3 cells or various mouse tissues is associated with endogenous 14-3-3 isoforms. We have established that 14-3-3 binding is mediated by phosphorylation of LRRK2 at two conserved residues (Ser910 and Ser935) located before the leucine-rich repeat domain. Our results suggests that mutation of Ser910 and/or Ser935 to disrupt 14-3-3 binding does not affect intrinsic protein kinase activity, but induces LRRK2 to accumulate within discrete cytoplasmic pools, perhaps resembling inclusion bodies. To investigate links between 14-3-3 binding and Parkinson's disease, we studied how 41 reported mutations of LRRK2 affected 14-3-3 binding and cellular localization. Strikingly, we found that five of the six most common pathogenic mutations (R1441C, R1441G, R1441H, Y1699C and I2020T) display markedly reduced phosphorylation of Ser910/Ser935 thereby disrupting interaction with 14-3-3. We have also demonstrated that Ser910/Ser935 phosphorylation and 14-3-3 binding to endogenous LRRK2 is significantly reduced in tissues of homozygous LRRK2(R1441C) knock-in mice. Consistent with 14-3-3 regulating localization, all of the common pathogenic mutations displaying reduced 14-3-3-binding accumulated within inclusion bodies. We also found that three of the 41 LRRK2 mutations analysed displayed elevated protein kinase activity (R1728H, ~2-fold; G2019S, ~3-fold; and T2031S, ~4-fold). These results provide the first evidence suggesting that 14-3-3 regulates LRRK2 and that disruption of the interaction of LRRK2 with 14-3-3 may be linked to Parkinson's disease.

Overexpression and enhanced activation of the epidermal growth factor (EGF) receptor are frequent events in human cancers that correlate with poor prognosis. Anti-phosphotyrosine and anti-EGFr affinity chromatography, isotope-coded muLC-MS/MS, and immunoblot methods were combined to describe and measure signaling networks associated with EGF receptor activation and pharmacological inhibition. The squamous carcinoma cell line HN5, which overexpresses EGF receptor and displays sustained receptor kinase activation, was used as a model system, where pharmacological inhibition of EGF receptor kinase by erlotinib markedly reduced auto and substrate phosphorylation, Src family phosphorylation at EGFR Y845, while increasing total EGF receptor protein. Diverse sets of known and poorly described functional protein classes were unequivocally identified by affinity selection, comprising either proteins tyrosine phosphorylated or complexed therewith, predominantly through EGF receptor and Src family kinases, principally 1) immediate EGF receptor signaling complexes (18%); 2) complexes involved in adhesion and cell-cell contacts (34%); and 3) receptor internalization and degradation signals. Novel and known phosphorylation sites could be located despite the complexity of the peptide mixtures. In addition to interactions with multiple signaling adaptors Grb2, SHC, SCK, and NSP2, EGF receptors in HN5 cells were shown to form direct or indirect physical interactions with additional kinases including ACK1, focal adhesion kinase (FAK), Pyk2, Yes, EphA2, and EphB4. Pharmacological inhibition of EGF receptor kinase activity by erlotinib resulted in reduced phosphorylation of downstream signaling, for example through Cbl/Cbl-B, phospholipase Cgamma (PLCgamma), Erk1/2, PI-3 kinase, and STAT3/5. Focal adhesion proteins, FAK, Pyk2, paxillin, ARF/GIT1, and plakophillin were down-regulated by transient EGF stimulation suggesting a complex balance between growth factor induced kinase and phosphatase activities in the control of cell adhesion complexes. The functional interactions between IGF-1 receptor, lysophosphatidic acid (LPA) signaling, and EGF receptor were observed, both direct and/or indirectly on phospho-Akt, phospho-Erk1/2, and phospho-ribosomal S6.

Beta-arrestins are cytosolic proteins that form complexes with seven-transmembrane receptors after agonist stimulation and phosphorylation by the G protein-coupled receptor kinases. They play an essential role in receptor desensitization and endocytosis, and they also serve as receptor-regulated signaling scaffolds and adaptors. Moreover, in the past decade, a growing list of protein-protein interactions of beta-arrestins pertinent to these functions has been documented. The discovery of several novel functions of beta-arrestins stimulated us to perform a global proteomics analysis of beta-arrestin-interacting proteins (interactome) as modulated by a model seven-transmembrane receptor, the angiotensin II type 1a receptor, in an attempt to assess the full range of functions of these versatile molecules. As determined by LC tandem MS, 71 proteins interacted with beta-arrestin 1, 164 interacted with beta-arrestin 2, and 102 interacted with both beta-arrestins. Some proteins bound only after agonist stimulation, whereas others dissociated. Bioinformatics analysis of the data indicates that proteins involved in cellular signaling, organization, and nucleic acid binding are the most highly represented in the beta-arrestin interactome. Surprisingly, both S-arrestin (visual arrestin) and X-arrestin (cone arrestin) were also found in heteromeric complex with beta-arrestins. The beta-arrestin interactors distribute not only in the cytoplasm, but also in the nucleus as well as other subcellular compartments. The binding of 16 randomly selected newly identified beta-arrestin partners was validated by coimmunoprecipitation assays in HEK293 cells. This study provides a comprehensive analysis of proteins that bind beta-arrestin isoforms and underscores their potentially broad regulatory roles in mammalian cellular physiology.

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 (beta, epsilon, eta, tau) were identified interacting with the hPFTAIRE1. We found a putative 14-3-3 binding consensus motif (RHSSPSS) in the hPFTAIRE1, which overlapped with its second NLS. Deletion of the RHSSPSS motif or substitution of Ser119 with Ala in the conserved binding motif abolished the specific interaction between the hPFTAIRE1 and the 14-3-3 proteins. The mutant S120A hPFTAIRE1 also showed a weak interaction to the 14-3-3 proteins. The results suggested that the Ser119 is crucial for the interaction between hPFTAIRE1 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.

To elucidate the function of keratins 8 and 18 (K8/18), major components of the intermediate filaments of simple epithelia, we searched for K8/18-binding proteins by screening a yeast two-hybrid library. We report here that human Mrj, a DnaJ/Hsp40 family protein, directly binds to K18. Among the interactions between DnaJ/Hsp40 family proteins and various intermediate filament proteins that we tested using two-hybrid methods, Mrj specifically interacted with K18. Immunostaining with anti-Mrj antibody showed that Mrj colocalized with K8/18 filaments in HeLa cells. Mrj was immunoprecipitated not only with K18, but also with the stress-induced and constitutively expressed heat shock protein Hsp/c70. Mrj bound to K18 through its C terminus and interacted with Hsp/c70 via its N terminus, which contains the J domain. Microinjection of anti-Mrj antibody resulted in the disorganization of K8/18 filaments, without effects on the organization of actin filaments and microtubules. Taken together, these results suggest that Mrj may play an important role in the regulation of K8/18 filament organization as a K18-specific co-chaperone working together with Hsp/c70.

Congenital fibrosis of the extraocular muscles type 1 (CFEOM1) is associated with heterozygous mutations in the KIF21A gene, including a major (R954W) and a minor (M947T) mutation. Kank1, which regulates actin polymerization, cell migration and neurite outgrowth, interacted with the third and fourth coiled-coil domains of KIF21A protein at its ankyrin-repeat domain. While both KIF21A(R954W) and KIF21A(M947T) enhanced the formation of a heterodimer with the wild type, KIF21A(WT), these mutants also enhanced the interaction with Kank1. Knockdown of KIF21A resulted in Kank1 predominantly occurring in the cytosolic fraction, while KIF21A(WT) slightly enhanced the translocation of Kank1 to the membrane fraction. Moreover, KIF21A(R954W) significantly enhanced the translocation of Kank1 to the membrane fraction. These results suggest that KIF21A regulates the distribution of Kank1 and that KIF21A mutations associated with CFEOM1 enhanced the accumulation of Kank1 in the membrane fraction. This might cause an abrogation of neuronal development in cases of CFEOM1 through over-regulation of actin polymerization by Kank1.

The human follitropin (follicle stimulating hormone, FSH) receptor (FSHR) is a G protein-coupled receptor (GPCR). To identify cytoplasmic proteins that may regulate FSHR function, a yeast-based interaction trap was performed. A linked construct of the first and second intracellular loops (iL1-iL2 bait) of FSHR was used as bait and a human ovarian cDNA library was used as prey. Among the proteins identified that interacted with the bait was 14-3-3tau, a member of a family of homodimeric cytoplasmic adapter proteins. Human granulosa cells, the site of FSHR expression in the ovary, were found to contain 14-3-3tau. Importantly, 14-3-3tau co-immunoprecipitated with FSHR stably expressed in HEK 293 cells. Its association with FSHR was follitropin-dependent. Over-expression of 14-3-3tau resulted in a modest decrease of follitropin-induced cAMP accumulation. Collectively, these data support a role for 14-3-3tau in follitropin action. The finding that 14-3-3tau interacts with FSHR is novel and should lead to new insights into the regulation of GPCR in general and FSHR specifically.

ADAM22 is one of three catalytically inactive ADAM family members highly expressed in the brain. ADAM22 has numerous splice variants, all with considerable cytoplasmic tails of up to 148 amino acids. ADAM22 can act to inhibit cell proliferation, however, it has been suggested that it also acts as an adhesion protein. We identified three 14-3-3 protein members by a yeast two-hybrid screen and show by co-immunoprecipitation that the cytoplasmic domain of ADAM22 can interact with all six 14-3-3 proteins expressed in the brain. In addition, we show that 14-3-3 proteins interact preferentially with the serine phosphorylated precursor form of ADAM22. ADAM22 has two 14-3-3 protein binding consensus motifs; the first binding site, spanning residues 831-834, was shown to be the most crucial for 14-3-3 binding to occur. The interaction between ADAM22 and 14-3-3 proteins is dependent on phosphorylation of ADAM22, but not of 14-3-3 proteins. ADAM22 point mutants lacking functional 14-3-3 protein binding motifs could no longer accumulate efficiently at the cell surface. Deletion of both 14-3-3 binding sites and newly identified ER retention motifs restored localization of ADAM22 at the cell surface. These results reveal a role for 14-3-3 proteins in targeting ADAM22 to the membrane by masking ER retention signals.

BACKGROUND:Chediak-Higashi syndrome (CHS) is an inherited immunodeficiency disease characterized by giant lysosomes and impaired leukocyte degranulation. CHS results from mutations in the lysosomal trafficking regulator (LYST) gene, which encodes a 425-kD cytoplasmic protein of unknown function. The goal of this study was to identify proteins that interact with LYST as a first step in understanding how LYST modulates lysosomal exocytosis. MATERIALS AND METHODS: Fourteen cDNA fragments, covering the entire coding domain of LYST, were used as baits to screen five human cDNA libraries by a yeast two-hybrid method, modified to allow screening in the activation and the binding domain, three selectable markers, and more stringent confirmation procedures. Five of the interactions were confirmed by an in vitro binding assay. RESULTS: Twenty-one proteins that interact with LYST were identified in yeast two-hybrid screens. Four interactions, confirmed directly, were with proteins important in vesicular transport and signal transduction (the SNARE-complex protein HRS, 14-3-3, and casein kinase II). CONCLUSIONS:On the basis of protein interactions, LYST appears to function as an adapter protein that may juxtapose proteins that mediate intracellular membrane fusion reactions. The pathologic manifestations observed in CHS patients and in mice with the homologous mutation beige suggest that understanding the role of LYST may be relevant to the treatment of not only CHS but also of diseases such as asthma, urticaria, and lupus, as well as to the molecular dissection of the CHS-associated cancer predisposition.

The Williams-Beuren syndrome (WBS) is a contiguous gene syndrome caused by chromosomal rearrangements at chromosome band 7q11.23. Several endocrine phenotypes, in particular impaired glucose tolerance and silent diabetes, have been described for this clinically complex disorder. The WBSCR14 gene, one of the genes mapping to the WBS critical region, encodes a member of the basic-helix-loop-helix leucine zipper family of transcription factors, which dimerizes with the Max-like protein, Mlx. This heterodimeric complex binds and activates, in a glucose-dependent manner, carbohydrate response element (ChoRE) motifs in the promoter of lipogenic enzymes. We identified five novel WBSCR14-interacting proteins, four 14-3-3 isotypes and NIF3L1, which form a single polypeptide complex in mammalian cells. Phosphatase treatment abrogates the association between WBSCR14 and 14-3-3, as shown previously for multiple 14-3-3 interactors. WBSCR14 is exported actively from the nucleus through a CRM1-dependent mechanism. This translocation is contingent upon the ability to bind 14-3-3. Through this mechanism the 14-3-3 isotypes directly affect the WBSCR14:Mlx complexes, which activate the transcription of lipogenic genes.