Downregulation of 14-3-3sigma prevents clonal evolution and leads to immortalization of primary human keratinocytes

In human epidermal keratinocytes, replicative senescence, is determined by a progressive decline of clonogenic and dividing cells. Its timing is controlled by clonal evolution, that is, by the continuous transition from stem cells to transient amplifying cells. We now report that downregulation of 14-3-3sigma, which is specifically expressed in human stratified epithelia, prevents keratinocyte clonal evolution, thereby forcing keratinocytes into the stem cell compartment. This allows primary human keratinocytes to readily escape replicative senescence. 14-3-3sigma-dependent bypass of senescence is accompanied by maintenance of telomerase activity and by downregulation of the p16(INK4a) tumor suppressor gene, hallmarks of keratinocyte immortalization. Taken together, these data therefore suggest that inhibition of a single endogenous gene product fosters immortalization of primary human epithelial cells without the need of exogenous oncogenes and/or oncoviruses.     

The N terminus of the transmembrane protein BP180 interacts with the N-terminal domain of BP230, thereby mediating keratin cytoskeleton anchorage to the cell surface at the site of the hemidesmosome

In epidermal cells, the keratin cytoskeleton interacts with the elements in the basement membrane via a multimolecular junction called the hemidesmosome. A major component of the hemidesmosome plaque is the 230-kDa bullous pemphigoid autoantigen (BP230/BPAG1), which connects directly to the keratin-containing intermediate filaments of the cytoskeleton via its C terminus. A second bullous pemphigoid antigen of 180 kDa (BP180/BPAG2) is a type II transmembrane component of the hemidesmosome. Using yeast two-hybrid technology and recombinant proteins, we show that an N-terminal fragment of BP230 can bind directly to an N-terminal fragment of BP180. We have also explored the consequences of expression of the BP230 N terminus in 804G cells that assemble hemidesmosomes in vitro. Unexpectedly, this fragment disrupts the distribution of BP180 in transfected cells but has no apparent impact on the organization of endogenous BP230 and alpha6beta4 integrin. We propose that the BP230 N terminus competes with endogenous BP230 protein for BP180 binding and inhibits incorporation of BP180 into the cell surface at the site of the hemidesmosome. These data provide new insight into those interactions of the molecules of the hemidesmosome that are necessary for its function in integrating epithelial and connective tissue types.     

The zeta isoform of 14-3-3 proteins interacts with the third intracellular loop of different alpha2-adrenergic receptor subtypes

The alpha2-adrenergic receptors (alpha2ARs) are localized to and function on the basolateral surface in polarized renal epithelial cells via a mechanism involving the third cytoplasmic loop. To identify proteins that may contribute to this retention, [35S]Met-labeled Gen10 fusion proteins with the 3i loops of the alpha2AAR (Val217-Ala377), alpha2BAR (Lys210-Trp354), and alpha2CAR (Arg248-Val363) were used as ligands in gel overlay assays. A protein doublet of approximately 30 kDa in Madin-Darby canine kidney cells or pig brain cytosol (alpha2B >/= alpha2C> alpha2A) was identified. The interacting protein was purified by sequential DEAE and size exclusion chromatography, and subsequent microsequencing revealed that they are the zeta isoform of 14-3-3 proteins. [35S]Met-14-3-3zeta binds to all three native alpha2AR subtypes, assessed using a solid phase binding assay (alpha2A>/=alpha2B> alpha2C), and this binding depends on the presence of the 3i loops. Attenuation of the alpha2AR-14-3-3 interactions in the presence of a phosphorylated Raf-1 peptide corresponding to its 14-3-3 interacting domain (residues 251-266), but not by its non-phosphorylated counterpart, provides evidence for the functional specificity of these interactions and suggests one potential interface for the alpha2AR and 14-3-3 interactions. These studies represent the first evidence for G protein-coupled receptor interactions with 14-3-3 proteins and may provide a mechanism for receptor localization and/or coordination of signal transduction.     

An essential phosphorylation-site domain of human cdc25C interacts with both 14-3-3 and cyclins

Human cdc25C is a dual-specificity phosphatase involved in the regulation of cell cycle progression in both unperturbed cells and in cells subject to DNA damage or replication checkpoints. In this study, we describe the structure-function relationship of an essential domain of human cdc25C that interacts with 14-3-3 proteins. We show that this domain is a bi-functional interactive motif that interacts with cyclins primarily through their P-box motif in addition to 14-3-3 proteins. Characterization of the structural features of this domain by NMR and circular dichroism reveals two distinct alpha helical moieties interconnected by a loop carrying the 14-3-3 binding site. Moreover, the helical folding is induced upon binding to 14-3-3, suggestive of a conformational regulation of this domain of cdc25C through interactions with partner proteins in vivo. Combining our structural and biochemical data, we propose a detailed model of the molecular mechanism of cdc25C regulation by differential association with 14-3-3 and cdc2-cyclin B.     

The cystic fibrosis transmembrane conductance regulator interacts with and regulates the activity of the HCO3- salvage transporter human Na+-HCO3- cotransport isoform 3

Cystic fibrosis transmembrane conductance regulator (CFTR) regulates both HCO(3)(-) secretion and HCO(3)(-) salvage in secretory epithelia. At least two luminal transporters mediate HCO(3)(-) salvage, the Na(+)/H(+) exchanger (NHE3) and the Na(+)-HCO(3)(-) cotransport (NBC3). In a previous work, we show that CFTR interacts with NHE3 to regulate its activity (Ahn, W., Kim, K. W., Lee, J. A., Kim, J. Y., Choi, J. Y., Moe, O. M., Milgram, S. L., Muallem, S., and Lee, M. G. (2001) J. Biol. Chem. 276, 17236-17243). In this work, we report that transient or stable expression of human NBC3 (hNBC3) in HEK cells resulted in a Na(+)-dependent, DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid)- and 5-ethylisopropylamiloride-insensitive HCO(3)(-) transport. Stimulation of CFTR with forskolin markedly inhibited NBC3 activity. This inhibition was prevented by the inhibition of protein kinase A. NBC3 and CFTR could be reciprocally coimmunoprecipitated from transfected HEK cells and from the native pancreas and submandibular and parotid glands. Precipitation of NBC3 or CFTR from transfected HEK293 cells and from the pancreas and submandibular gland also coimmunoprecipitated EBP50. Glutathione S-transferase-EBP50 pulled down CFTR and hNBC3 from cell lysates when expressed individually and as a complex when expressed together. Notably, the deletion of the C-terminal PDZ binding motifs of CFTR or hNBC3 prevented coimmunoprecipitation of the proteins and inhibition of hNBC3 activity by CFTR. We conclude that CFTR and NBC3 reside in the same HCO(3)(-)-transporting complex with the aid of PDZ domain-containing scaffolds, and this interaction is essential for regulation of NBC3 activity by CFTR. Furthermore, these findings add additional evidence for the suggestion that CFTR regulates the overall trans-cellular HCO(3)(-) transport by regulating the activity of all luminal HCO(3)(-) secretion and salvage mechanisms of secretory epithelial cells.     

The cyclin-dependent kinase 11 interacts with 14-3-3 proteins

Cyclin-dependent kinase 11 isoforms (CDK11) are members of the p34(cdc2) superfamily. They have been shown to play a role in RNA processing and apoptosis. In the present study, we investigate whether CDK11 interacts with 14-3-3 proteins. Our study shows that the putative 14-3-3 binding site (113-RHRSHS-118) within the N-terminal domain of CDK11(p110) is functional. Endogenous CDK11(p110) binds directly to 14-3-3 proteins and phosphorylation of the serine 118 within the RHRSHS motif seems to be required for the binding. Besides, CDK11(p110) is capable of interacting with several different isoforms of 14-3-3 proteins both in vitro and in vivo. The interaction of 14-3-3 gamma with CDK11(p110) occurs throughout the entire cell cycle and reaches maximum at the G2/M phase. Interestingly, 14-3-3 gamma shows strong interaction with N-terminal portion of caspase-cleaved CDK11(p110) (CDK11(p60)) product at 48 h after Fas treatment, which correlates with the maximal cleavage level of CDK11(p110) and the maximum activation level of CDK11 kinase activity during apoptosis. Collectively, these results suggest that CDK11 kinases could be regulated by interaction with 14-3-3 proteins during cell cycle and apoptosis.     

Rat inositol 1,4,5-trisphosphate receptor isoform 2 interacts with itself in its C-terminal portion and upstream of the first transmembrane domain.

In response to stimulation at the plasma membrane, hepatocellular Ca(2+) signals are fast and precise and lead to rapid local changes in cytoplasmic free Ca(2+) concentration. These changes result from the opening of the inositol 1,4,5-trisphosphate receptor (InsP(3)R), which is a four-subunit intracellular InsP(3)-gated channel that releases Ca(2+) from the stores. To investigate the molecular mechanism underlying interactions between the InsP(3)R subunits, we cloned the predominant hepatocellular isoform, InsP(3)R isoform 2 (InsP(3)R2), and screened for interactions using the yeast two-hybrid assay. We found that the C-terminal domain of rat InsP(3)R2 interacts with itself, and that the cytoplasmic part preceding the first transmembrane domain, a region near a Ca(2+)-binding site, also interacts with itself. These interactions were confirmed by pull-down experiments. The C-terminal domain of InsP(3)R2 is also able to interact with the C-termini of rat InsP(3)R1 and InsP(3)R3. These results advance our understanding of the molecular mechanisms that underlie the oligomerization and interactions of the InsP(3)R subunits during the opening/closing of the Ca(2+) channel.     

14-3-3sigma is down-regulated in human prostate cancer

The 14-3-3sigma is a negative regulator of the cell cycle, which is induced by p53 in response to DNA damage. It has been characterized as an epithelium-specific marker and down-regulation of the protein has been shown in breast cancers, suggesting its tumor-suppressive activity in epithelial cells. Here we demonstrate that 14-3-3sigma protein is down-regulated in human prostate cancer cell lines, LNCaP, PC3, and DU145 compared with normal prostate epithelial cells. Immunohistochemical analysis of primary prostate cells shows that the expression of 14-3-3sigma protein is epithelial cell-specific. Among prostate pathological specimens, > 95% of benign hyperplasia samples show significant and diffuse immunostaining of 14-3-3sigma in the cytoplasm whereas < 20% of carcinoma samples show positive staining. In terms of mechanisms for the down-regulation of 14-3-3sigma in prostate cancer cells, hypermethylation of the gene promoter plays a causal role in LNCaP cells as 14-3-3sigma mRNA level was elevated by 5-aza-2'-deoxycytidine demethylating treatment. Intriguingly, the proteasome-mediated proteolysis is responsible for 14-3-3sigma reduction in DU145 and PC3 cells, as 14-3-3sigma protein expression was increased by treatment with a proteasome inhibitor MG132. Furthermore, tumor necrosis factor-related apoptosis-inducing ligand enhances 14-3-3sigma gene and protein expression in DU145 and PC3 cells. These data suggest that 14-3-3sigma expression is down-regulated during the neoplastic transition of prostate epithelial cells.     

The microtubule-binding protein Hook3 interacts with a cytoplasmic domain of scavenger receptor A

The class A scavenger receptor (SR-A) is a multifunctional transmembrane glycoprotein that is implicated in atherogenesis, innate immunity, and cell adhesion. Despite extensive structure-function studies of the receptor, intracellular molecules that directly interact with SR-A and regulate the receptor trafficking have not been determined. In the current study, we have identified a microtubule-binding protein, Hook3, as a novel interacting partner of SR-A. The association between a rat Hook3 isoform and SR-A was suggested by yeast two-hybrid screening and mass spectrometry analysis of SR-A-cytoplasmic domain-bound proteins in rat alveolar macrophages. The binding of overexpressed and endogenous human Hook3 to SR-A was demonstrated by pull-down assay and co-immunoprecipitations. Furthermore, endogenous murine SR-A and HK3 co-sedimented from cell lysates isolated from Raw264.7 murine macrophage cells. The interaction of Hook3 with SR-A was significantly stimulated after SR-A had recognized the extracellular ligand. Studies using truncations demonstrated that the positively charged C-terminal Val614-Ala717 region of human Hook3 was required for the interaction with the negatively charged residues, Glu12, Asp13, and Asp15 in the human SR-A cytoplasmic domain. By transfecting small interfering RNA targeting Hook3, total and surface expression, receptor-mediated ligand uptake and protein stability of SR-A were significantly promoted, whereas the protein synthesis and maturation were not altered. We propose for the first time that Hook3 may participate in the turnover of the endocytosed scavenger receptor.     

FTICR-MS analysis of 14-3-3 isoform substrate selection

The 14-3-3s are a ubiquitous class of eukaryotic proteins that participate in a second regulatory step in many phosphorylation-based signal transduction systems. The Arabidopsis family of 14-3-3 proteins represents a rather large 14-3-3 gene family. The biological motive for such diversity within a single protein family is not yet completely understood. The work presented here utilizes 14-3-3 micro-affinity chromatography in conjunction with Fourier transform ion cyclotron resonance mass spectrometry to survey the substrate sequence selectivity of two Arabidopsis 14-3-3 isoforms that represent the two major subclasses of this protein family. A method was developed to compare the relative binding of eight synthetic phosphopeptide sequences. The degree to which each phosphopeptide bound to either isoform was assigned a relative value, defined here as the binding ratio. The method provided a simple means for visualizing differences in substrate sequence selection among different 14-3-3 isoforms. A reproducible preference for specific phosphopeptide sequences was measured for both isoforms. This binding preference was consistent among the two classes of isoforms, suggesting that any pressure for isoform selectivity must reside outside the central core that interacts with the phosphopeptide sequence of the client.     

14-3-3 protein directly interacts with the kinase domain of calcium/calmodulin-dependent protein kinase kinase (CaMKK2)

Background

Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a member of the Ca²⁺/calmodulin-dependent kinase (CaMK) family involved in adiposity regulation, glucose homeostasis and cancer. This upstream activator of CaMKI, CaMKIV and AMP-activated protein kinase is inhibited by phosphorylation, which also triggers an association with the scaffolding protein 14-3-3. However, the role of 14-3-3 in the regulation of CaMKK2 remains unknown.

Evolution and isoform specificity of plant 14-3-3 proteins

The 14-3-3 proteins, once thought of as obscure mammalian brain proteins, are fast becoming recognized as major regulators of plant primary metabolism and of other cellular processes. Their presence as large gene families in plants underscores their essential role in plant physiology. We have examined the Arabidopsis thaliana 14-3-3 gene family, which currently is the largest and most complete 14-3-3 family with at least 12 expressed members and 15 genes from the now completed Arabidopsis thaliana genome project. The phylogenetic branching of this family serves as the prototypical model for comparison with other large plant 14-3-3 families and as such may serve to rationalize clustering in a biological context. Equally important for ascribing common functions for the various 14-3-3 isoforms is determining an isoform-specific correlation with localization and target partnering. A summary of localization information available in the literature is presented. In an effort to identify specific 14-3-3 isoform location and participation in cellular processes, we have produced a panel of isoform-specific antibodies to Arabidopsis thaliana 14-3-3s and present initial immunolocalization studies that suggest biologically relevant, discriminative partnering of 14-3-3 isoforms.     

Human Dectin-1 isoform E is a cytoplasmic protein and interacts with RanBPM

Human Dectin-1, a type II transmembrane receptor, is alternatively spliced, generating eight isoforms. Of these isoforms, the isoform E (hDectin-1E) is structurally unique, containing a complete C-type lectin-like domain as well as an ITAM-like sequence. So far, little is known about its function. In the present study, we demonstrated that hDectin-1E was not secreted and it mainly resided in the cytoplasm. Using yeast two-hybrid screening, we identified a Ran-binding protein, RanBPM, as an interacting partner of hDectin-1E. GST pull-down assays showed that RanBPM interacted directly with hDectin-1E and the region containing SPRY domain was sufficient for the interaction. The binding of hDectin-1E and RanBPM was further confirmed in vivo by co-immunoprecipitation assay and confocal microscopic analysis. Taken together, our data provide a clue to the understanding of the function about hDectin-1E.     

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.     

Specific 14-3-3 isoform detection and immunolocalization in prion diseases

14-3-3 proteins are involved in signalling processes in neuronal cells. Using isoform-specific antibodies we have examined the variation in 14-3-3 isoform neurolocation in normal and scrapie-infected murine brain and show that in defined areas of the brain there are significant changes associated with the pathology of the disease process. The appearance of 14-3-3 proteins in the cerebrospinal fluid (CSF) is a consequence of neuronal disease and the detection of specific isoforms of the 14-3-3 proteins in the CSF is characteristic of some neurodegenerative diseases. In this study, monitoring specifically for the gamma 14-3-3 isoform in the CSF by both Western-blot analysis and ELISA we can show a level of correlation between the assays.