Gradient of increasing affinity of importin beta for nucleoporins along the pathway of nuclear import

Nuclear import and export signals on macromolecules mediate directional, receptor-driven transport through the nuclear pore complex (NPC) by a process that is suggested to involve the sequential binding of transport complexes to different nucleoporins. The directionality of transport appears to be partly determined by the nucleocytoplasmic compartmentalization of components of the Ran GTPase system. We have analyzed whether the asymmetric localization of discrete nucleoporins can also contribute to transport directionality. To this end, we have used quantitative solid phase binding analysis to determine the affinity of an importin beta cargo complex for Nup358, the Nup62 complex, and Nup153, which are in the cytoplasmic, central, and nucleoplasmic regions of the NPC, respectively. These nucleoporins are proposed to provide progressively more distal binding sites for importin beta during import. Our results indicate that the importin beta transport complex binds to nucleoporins with progressively increasing affinity as the complex moves from Nup358 to the Nup62 complex and to Nup153. Antibody inhibition studies support the possibility that importin beta moves from Nup358 to Nup153 via the Nup62 complex during import. These results indicate that nucleoporins themselves, as well as the nucleocytoplasmic compartmentalization of the Ran system, are likely to play an important role in conferring directionality to nuclear protein import.     

Using peptide arrays to define nuclear carrier binding sites on nucleoporins

In the peptide SPOT array technique, an array of different peptides are synthesized on, and covalently linked to, cellulose membranes. In one usage of this technique, these peptides are screened in an overlay assay to determine which short sequence(s) contains a binding site for an interacting protein. By preparing overlapping peptides that cover the entire sequence of a protein, all of the binding domains on the protein for a second protein can be identified. We have utilized the peptide SPOT array technique to identify the short amino acid sequences within nuclear pore complex proteins (also known as nucleoporins or Nups) that bind the nuclear carrier importin-beta. Crystallization studies by others have indicated that nuclear carriers such as importin-beta bind to phenylalanine-glycine (FG) repeats present in numerous copies in the sequences of a family of nucleoporins. Consistent with this, we found that most (but not all) of the Nup binding sites for importin-beta identified by this technique contain Fx, FG, FxFG, FxFx, or GLFG sequences, although not all such sequences bound importin-beta. Peptide SPOT array substitution studies confirmed a crucial role for the phenylalanine in FG repeats and identified a lysine residue flanking some repeats that is crucial for importin-beta binding to those repeats. In addition to these expected binding sequences for importin-beta, we found multiple instances of a peptide lacking a canonical FG repeat that strongly bound importin-beta, indicating that additional Nup sequences may form binding sites for importin-beta.     

WNT protein-independent constitutive nuclear localization of beta-catenin protein and its low degradation rate in thalamic neurons

Nuclear localization of β-catenin is a hallmark of canonical Wnt signaling, a pathway that plays a crucial role in brain development and the neurogenesis of the adult brain. We recently showed that β-catenin accumulates specifically in mature thalamic neurons, where it regulates the expression of the Ca(v)3.1 voltage-gated calcium channel gene. Here, we investigated the mechanisms underlying β-catenin accumulation in thalamic neurons. We report that a lack of soluble factors produced either by glia or cortical neurons does not impair nuclear β-catenin accumulation in thalamic neurons. We next found that the number of thalamic neurons with β-catenin nuclear localization did not change when the Wnt/Dishevelled signaling pathway was inhibited by Dickkopf1 or a dominant negative mutant of Dishevelled3. These results suggest a WNT-independent cell-autonomous mechanism. We found that the protein levels of APC, AXIN1, and GSK3β, components of the β-catenin degradation complex, were lower in the thalamus than in the cortex of the adult rat brain. Reduced levels of these proteins were also observed in cultured thalamic neurons compared with cortical cultures. Finally, pulse-chase experiments confirmed that cytoplasmic β-catenin turnover was slower in thalamic neurons than in cortical neurons. Altogether, our data indicate that the nuclear localization of β-catenin in thalamic neurons is their cell-intrinsic feature, which was WNT-independent but associated with low levels of proteins involved in β-catenin labeling for ubiquitination and subsequent degradation.     

Multiple Cos2/Ci interactions regulate Ci subcellular localization through microtubule dependent and independent mechanisms

The Hedgehog (Hh) family of secreted proteins governs many developmental processes in both vertebrates and invertebrates. In Drosophila, Hh acts by blocking the formation of a truncated repressor form of Cubitus interruptus (Ci) and by stimulating the nuclear translocation and activity of full-length Ci (Ci155). In the absence of Hh, Ci155 is sequestered in the cytoplasm by forming protein complexes with Costal2 (Cos2), Fused (Fu) and Suppressor of Fused [Su(fu)]. How complex formation regulates Ci155 subcellular localization is not clear. We find that Cos2 interacts with two distinct domains of Ci155, an amino (N)-terminal domain (CDN) and a carboxyl (C)-terminal domain (CORD), and Cos2 competes with Su(fu) for binding to the N-terminal region of Ci155. We provide evidence that both N- and C-terminal Cos2 binding domains are involved in the cytoplasmic retention of Ci155 in imaginal discs. Treating imaginal discs with microtubule-destabilizing reagent nocodazole promotes nuclear translocation of Ci155, suggesting that the microtubule network plays an important role in the cytoplasmic retention of Ci155. In addition, we find that adding a nuclear localization signal (NLS) to exposed regions of Ci155 greatly facilitates its nuclear translocation, suggesting that the cytoplasmic retention of Ci155 may also depend on NLS masking.     

Nuclear import by karyopherin-βs: Recognition and inhibition

Proteins in the karyopherin-β family mediate the majority of macromolecular transport between the nucleus and the cytoplasm. Eleven of the 19 known human karyopherin-βs and 10 of the 14 S. cerevisiae karyopherin-βs mediate nuclear import through recognition of nuclear localization signals or NLSs in their cargos. This receptor-mediated process is essential to cellular viability as proteins are translated in the cytoplasm but many have functional roles in the nucleus. Many known karyopherin-β-cargo interactions were discovered through studies of the individual cargos rather than the karyopherins, and this information is thus widely scattered in the literature. We consolidate information about cargos that are directly recognized by import-karyopherin-βs and review common characteristics or lack thereof among cargos of different import pathways. Knowledge of karyopherin-β-cargo interactions is also critical for the development of nuclear import inhibitors and the understanding of their mechanisms of inhibition. This article is part of a Special Issue entitled: Regulation of Signaling and Cellular Fate through Modulation of Nuclear Protein Import.     

An optimization approach to predicting protein structural class from amino acid composition.

Proteins are generally classified into four structural classes: all-alpha proteins, all-beta proteins, alpha + beta proteins, and alpha/beta proteins. In this article, a protein is expressed as a vector of 20-dimensional space, in which its 20 components are defined by the composition of its 20 amino acids. Based on this, a new method, the so-called maximum component coefficient method, is proposed for predicting the structural class of a protein according to its amino acid composition. In comparison with the existing methods, the new method yields a higher general accuracy of prediction. Especially for the all-alpha proteins, the rate of correct prediction obtained by the new method is much higher than that by any of the existing methods. For instance, for the 19 all-alpha proteins investigated previously by P.Y. Chou, the rate of correct prediction by means of his method was 84.2%, but the correct rate when predicted with the new method would be 100%! Furthermore, the new method is characterized by an explicable physical picture. This is reflected by the process in which the vector representing a protein to be predicted is decomposed into four component vectors, each of which corresponds to one of the norms of the four protein structural classes.     

Carbonic anhydrase IX reduces E-cadherin-mediated adhesion of MDCK cells via interaction with beta-catenin

Carbonic anhydrase IX (CA IX) is a cancer-associated transmembrane isoform of zinc metalloenzymes that catalyse interconversion between carbon dioxide and bicarbonate. CA IX is strongly induced by tumor hypoxia and has been proposed to participate in acidification of tumor microenvironment and in cell adhesion. To elucidate the cell adhesion-related role of CA IX, we investigated its subcellular localization and relationship to E-cadherin, a key adhesion molecule whose loss or destabilization is linked to tumor invasion. For this purpose, we generated MDCK cells with constitutive expression of human CA IX protein. During the monolayer formation, CA IX was localized to cell-cell contacts and its distribution in lateral membranes overlapped with E-cadherin. Calcium switch-triggered disruption and reconstitution of cell contacts resulted in relocalization of both CA IX and E-cadherin to cytoplasm and back to plasma membrane. A similar phenomenon was observed in hypoxia-treated and reoxygenated cells. Moreover, CA IX-expressing MDCK cells exhibited reduced cell adhesion capacity and lower levels of Triton-insoluble E-cadherin. Finally, CA IX was found to coprecipitate with beta-catenin. We conclude that CA IX has a capacity to modulate E-cadherin-mediated cell adhesion via interaction with beta-catenin, which could be of potential significance in hypoxia-induced tumor progression.     

Plasma 11beta-hydroxy-4-androstene-3,17-dione: comparison of a time-resolved fluoroimmunoassay using a biotinylated tracer with a radioimmunoassay using a tritiated tracer

The plasma concentration of 11β-hydroxy-4-androstene-3,17-dione (11β) is very high in 21-hydroxylase deficiency, Cushing’s syndrome, and hyperandrogenism of adrenal origin, and very low in congenital 11-hydroxylase deficiency and adrenal insufficiency. Thus, when plasma 4-androstenedione is elevated, it is useful to measure the plasma 11β level in order to determine the adrenal or ovarian origin of the hyperandrogenism.

To eliminate disadvantages related to the 11β radioimmunoassay (RIA), which uses a tritiated tracer, as well as the high cost associated with scintillation proximity assay (SPA), we developed a non-isotopic 11β assay that utilizes an 11β-biotin conjugate synthesized in our laboratory to measure time-resolved fluorescence after addition of streptavidin-europium to microtitration wells.

The analytical qualities of this assay are very similar to those of the radioimmunoassay using a tritiated tracer, and an extraction step followed by celite chromatography (which separates 11β from interfering plasma steroids) prior to a final radioimmuno-competition step. The correlation coefficient between 11β levels measured by time-resolved plasma 11β fluoroimmunoassay (TR-FIA) and RIA was 0.965.

Finally, the TR-FIA technique was more sensitive and of greater precision than the RIA method.     

A WNT/beta-catenin signaling activator, R-spondin, plays positive regulatory roles during skeletal myogenesis

R-spondins (RSPOs) are a recently characterized family of secreted proteins that activate WNT/β-catenin signaling. In this study, we investigated the potential roles of the RSPO proteins during myogenic differentiation. Overexpression of the Rspo1 gene or administration of recombinant RSPO2 protein enhanced mRNA and protein expression of a basic helix-loop-helix (bHLH) class myogenic determination factor, MYF5, in both C2C12 myoblasts and primary satellite cells, whereas MYOD or PAX7 expression was not affected. RSPOs also promoted myogenic differentiation and induced hypertrophic myotube formation in C2C12 cells. In addition, Rspo2 and Rspo3 gene knockdown by RNA interference significantly compromised MYF5 expression, myogenic differentiation, and myotube formation. Furthermore, Myf5 expression was reduced in the developing limbs of mouse embryos lacking the Rspo2 gene. Finally, we demonstrated that blocking of WNT/β-catenin signaling by DKK1 or a dominant-negative form of TCF4 reversed MYF5 expression, myogenic differentiation, and hypertrophic myotube formation induced by RSPO2, indicating that RSPO2 exerts its activity through the WNT/β-catenin signaling pathway. Our results provide strong evidence that RSPOs are key positive regulators of skeletal myogenesis acting through the WNT/β-catenin signaling pathway.     

Cyclin-dependent kinase 2 regulates the interaction of Axin with beta-catenin

Axin, a negative regulator of Wnt, forms a complex with glycogen synthase kinase 3beta, beta-catenin, and adenomatous polyposis coli and promotes GSK3beta-dependent phosphorylation of beta-catenin, thereby stimulating degradation of the beta-catenin. An essential step in that process is the phosphorylation of Axin. Examination of Axin's amino acid sequence revealed it to contain six arginine-X-leucine (RXL) sequences, the cyclin-dependent kinase 2 (CDK2) binding motif, and 10 CDK2 consensus phosphorylation sequences. We also found that cyclin A/CDK2 phosphorylates Axin, thereby enhancing its association with beta-catenin. This suggests that cyclin A/CDK2 is a negative regulator of beta-catenin-mediated signal transduction, which exerts its effects through phosphorylation of Axin.     

The transmembrane nucleoporin NDC1 is required for targeting of ALADIN to nuclear pore complexes

NDC1 is a transmembrane nucleoporin that is required for NPC assembly and nucleocytoplasmic transport. We show here that NDC1 directly interacts with the nucleoporin ALADIN, mutations of which are responsible for triple-A syndrome, and that this interaction is required for targeting of ALADIN to nuclear pore complexes (NPCs). Furthermore, we show that NDC1 is required for selective nuclear import. Our findings suggest that NDC1-mediated localization of ALADIN to NPCs is essential for selective nuclear protein import, and that abrogation of the interaction between ALADIN and NDC1 may be important for the development of triple-A syndrome.     

Absolute β-catenin concentrations in Wnt pathway-stimulated and non-stimulated cells

The intracellular level of the proto-oncoprotein β-catenin is a parameter for the activity of the Wnt pathway, which has been linked to carcinogenesis. The paper introduces a novel sandwich-based ELISA for the determination of the β-catenin concentration in lysates from cells or tissues. The advantages of the method were proven by determining β-catenin levels in cell lines and in cells after activation of the Wnt pathway. Analysis revealed high β-catenin concentrations in the cell lines HeLa, KB, HT1080, MCF-7, U-87 and U-373, which had not been described before. β-Catenin concentrations were compared in HEK293 and C57MG cells after activation of the Wnt pathway. The β-catenin concentrations increased by different factors depending on whether the Wnt pathway was activated by incubation with LiCl or with Wnt-3a-conditioned medium. This finding indicated that the β-catenin level depends on the way and level of Wnt pathway activation. The quantitative analysis of β-catenin in colorectal tumours revealed high β-catenin levels in tumours with truncating mutations in the APC gene.     

The LIM-only protein FHL2 interacts with beta-catenin and promotes differentiation of mouse myoblasts

FHL2 is a LIM-domain protein expressed in myoblasts but down-regulated in malignant rhabdomyosarcoma cells, suggesting an important role of FHL2 in muscle development. To investigate the importance of FHL2 during myoblast differentiation, we performed a yeast two-hybrid screen using a cDNA library derived from myoblasts induced for differentiation. We identified beta-catenin as a novel interaction partner of FHL2 and confirmed the specificity of association by direct in vitro binding tests and coimmunoprecipitation assays from cell lysates. Deletion analysis of both proteins revealed that the NH2-terminal part of beta-catenin is sufficient for binding in yeast, but addition of the first armadillo repeat is necessary for binding FHL2 in mammalian cells, whereas the presence of all four LIM domains of FHL2 is needed for the interaction. Expression of FHL2 counteracts beta-catenin-mediated activation of a TCF/LEF-dependent reporter gene in a dose-dependent and muscle cell-specific manner. After injection into Xenopus embryos, FHL2 inhibited the beta-catenin-induced axis duplication. C2C12 mouse myoblasts stably expressing FHL2 show increased myogenic differentiation reflected by accelerated myotube formation and expression of muscle-specific proteins. These data imply that FHL2 is a muscle-specific repressor of LEF/TCF target genes and promotes myogenic differentiation by interacting with beta-catenin.     

The interaction of CK2alpha and CK2beta, the subunits of protein kinase CK2, requires CK2beta in a preformed conformation and is enthalpically driven

The protein kinase CK2 (former name: "casein kinase 2") predominantly occurs as a heterotetrameric holoenzyme composed of two catalytic chains (CK2alpha) and two noncatalytic subunits (CK2beta). The CK2beta subunits form a stable dimer to which the CK2alpha monomers are attached independently. In contrast to the cyclins in the case of the cyclin-dependent kinases CK2beta is no on-switch of CK2alpha; rather the formation of the CK2 holoenzyme is accompanied with an overall change of the enzyme's profile including a modulation of the substrate specificity, an increase of the thermostability, and an allocation of docking sites for membranes and other proteins. In this study we used C-terminal deletion variants of human CK2alpha and CK2beta that were enzymologically fully competent and in particular able to form a heterotetrameric holoenzyme. With differential scanning calorimetry (DSC) we confirmed the strong thermostabilization effect of CK2alpha on CK2beta with an upshift of the CK2alpha melting temperature of more than 9 degrees . Using isothermal titration calorimetry (ITC) we measured a dissociation constant of 12.6 nM. This high affinity between CK2alpha and CK2beta is mainly caused by enthalpic rather than entropic contributions. Finally, we determined a crystal structure of the CK2beta construct to 2.8 A resolution and revealed by structural comparisons with the CK2 holoenzyme structure that the CK2beta conformation is largely conserved upon association with CK2alpha, whereas the latter undergoes significant structural adaptations of its backbone.