Recruitment of adenomatous polyposis coli and beta-catenin to axin-puncta.

The adenomatous polyposis coli (APC) tumour suppressor is a multifunctional protein involved in the regulation of Wnt signalling and cytoskeletal dynamics. Little is known about how APC controls these disparate functions. In this study, we have used APC- and axin-fluorescent fusion proteins to examine the interactions between these proteins and show that the functionally distinct populations of APC are also spatially separate. Axin-RFP forms cytoplasmic punctate structures, similar to endogenous axin puncta. Axin-RFP recruits beta-catenin destruction complex proteins, including APC, beta-catenin, glycogen synthase kinase-3-beta (GSK3-beta) and casein kinase-1-alpha (CK1-alpha). Recruitment into axin-RFP puncta sequesters APC from clusters at cell extensions and this prevents its microtubule-associated functions. The interaction between APC-GFP and axin-RFP within the cytoplasmic puncta is direct and dramatically alters the dynamic properties of APC-GFP. However, recruitment of APC to axin puncta is not absolutely required for beta-catenin degradation. Instead, formation of axin puncta, mediated by the DIX domain, is required for beta-catenin degradation. An axinDeltaDIX mutant did not form puncta, but still mediated recruitment of destruction complex proteins and phosphorylation of beta-catenin. We conclude that there are distinct pools of APC and that the formation of axin puncta, rather than the axin/APC complex, is essential for beta-catenin destruction.

A simple method allowing DIC imaging in conjunction with confocal microscopy.

Current optical methods to collect Nomarski differential interference contrast (DIC) or phase images with a transmitted light detector (TLD) in conjunction with confocal laser scanning microscopy (CLSM) can be technically challenging and inefficient. We describe for the first time a simple method that combines the use of the commercial product QPm (Iatia, Melbourne Australia) with brightfield images collected with the TLD of a CLSM, generating DIC, phase, Zernike phase, dark-field or Hoffman modulation contrast images. The brightfield images may be collected at the same time as the confocal images. This method also allows the calculation of contrast-enhanced images from archival data. The technique described here allows for the creation of contrast-enhanced images such as DIC or phase, without compromising the intensity or quality of confocal images collected simultaneously. Provided the confocal microscope is equipped with a motorized z-drive and a TLD, no hardware or optical modifications are required. The contrast-enhanced images are calculated with software using the quantitative phase-amplitude microscopy technique (Barone-Nugent et al., 2002). This technique, being far simpler during image collection, allows the microscopist to concentrate on their confocal imaging and experimental procedures. Unlike conventional DIC, this technique may be used to calculate DIC images when cells are imaged through plastic, and without the use of expensive strain-free objective lenses.

Continuous free-flow electrophoresis separation of cytosolic proteins from the human colon carcinoma cell line LIM 1215: a non two-dimensional gel electrophoresis-based proteome analysis strategy.

The conventional approach for analyzing the protein complement of a genome involves the combination of two-dimensional gel electrophoresis (2-DE) and mass spectrometric based protein identification technologies. While 2-DE is a powerful separation technique, it is severely limited by the insolubility of certain classes of proteins (e.g. hydrophobic membrane proteins), as well as the amount of protein that can be processed. Here, we describe a simple procedure for resolving complex mixtures of proteins that involves a combination of free flow electrophoresis (FFE), a liquid-based isoelectric focussing (IEF) method, and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Resolved proteins were identified by peptide fragment sequencing using capillary column reversed-phase high performance liquid chromatography (RP-HPLC)/mass spectrometry (MS). An initial demonstration of the method was performed using digitonin/ethylenediaminetetraacetic acid EDTA extracted cytosolic proteins from the human colon carcinoma cell line, LIM 1215. Cytosolic proteins were separated by liquid-based IEF (pH range 3-10) into 96 fractions, and each FFE fraction was further fractionated by SDS-PAGE. Selected protein bands were excised from the SDS-PAGE gel, digested in situ with trypsin, and subsequently identified by on-line RP-HPLC/electrospray-ionization ion trap MS. Our results indicate that FFE is: (i) an extremely powerful liquid-based IEF method for resolving proteins; (ii) not limited by the amount of sample that can be loaded onto the instrument; and (iii) capable of fractionating intact protein complexes (a potentially powerful tool for cell-mapping proteomics). An up-to-date list of cytosolic proteins from the human colorectal carcinoma cell line LIM 1215 can be found in the Joint Protein Structure Laboratory (JPSL) proteome database. This information will provide an invaluable resource for future proteomics-based biological studies of colon cancer. The JPSL proteome database can be accessed through the World Wide Web (WWW) network (http://www.ludwig.edu.au/jpsl/jpslhome.html).

Characterization of an adapter subunit to a phosphatidylinositol (3)P 3-phosphatase: identification of a myotubularin-related protein lacking catalytic activity.

The D3-phosphoinositides act as second messengers by recruiting, and thereby activating, diverse signaling proteins. We have previously described the purification of a rat phosphatidylinositol 3-phosphate [PtdIns(3)P] 3-phosphatase, comprising a heterodimer of a 78-kDa adapter subunit in complex with a 65-kDa catalytic subunit. Here, we have cloned and characterized the cDNA encoding the human 3-phosphatase adapter subunit (3-PAP). Sequence alignment showed that 3-PAP shares significant sequence similarity with the protein and lipid 3-phosphatase myotubularin, and with several other members of the myotubularin gene family including SET-binding factor 1. However, unlike myotubularin, 3-PAP does not contain a consensus HCX(5)R catalytic motif. The 3-PAP sequence contains several motifs that predict interaction with proteins containing Src homology-2 (SH2) domains, phosphotyrosine-binding (PTB) domains, members of the 14-3-3 family, as well as proteins with SET domains. Northern blot analysis identified two transcripts (5.5 kb and 2.5 kb) with highest abundance in human liver, kidney, lung, and placenta. 3-PAP immunoprecipitates isolated from platelet cytosol hydrolyzed the D3-phosphate from PtdIns(3)P and PtdIns 3,4-bisphosphate [PtdIns(3,4)P(2)]. However, insect cell-expressed 3-PAP recombinant protein was catalytically inactive, confirming our prior prediction that this polypeptide represents an adapter subunit.

Cloning and characterization of a 72-kDa inositol-polyphosphate 5-phosphatase localized to the Golgi network.

The inositol-polyphosphate 5-phosphatase enzyme family removes the 5-position phosphate from both inositol phosphate and phosphoinositide signaling molecules. We have cloned and characterized a novel 5-phosphatase, which demonstrates a restricted substrate specificity and tissue expression. The 3.9-kb cDNA predicts for a 72-kDa protein with an N-terminal proline rich domain, a central 5-phosphatase domain, and a C-terminal CAAX motif. The 3. 9-kilobase mRNA showed a restricted expression but was abundant in testis and brain. Antibodies against the sequence detected a 72-kDa protein in the testis in the detergent-insoluble fraction. Indirect immunofluorescence of the Tera-1 cell line using anti-peptide antibodies to the 72-kDa 5-phosphatase demonstrated that the enzyme is predominantly located to the Golgi. Expression of green fluorescent protein-tagged 72-kDa 5-phosphatase in COS-7 cells revealed that the enzyme localized predominantly to the Golgi, mediated by the N-terminal proline-rich domain, but not the C-terminal CAAX motif. In vitro, the protein inserted into microsomal membranes on the cytoplasmic face of the membrane. Immunoprecipitated recombinant 72-kDa 5-phosphatase hydrolyzed phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3, 5-bisphosphate, forming phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3-phosphate, respectively. We propose that the novel 5-phosphatase hydrolyzes phosphatidylinositol 3,4, 5-trisphosphate and phosphatidylinositol 3,5-bisphosphate on the cytoplasmic Golgi membrane and thereby may regulate Golgi-vesicular trafficking.

Intermolecular interactions of the p85alpha regulatory subunit of phosphatidylinositol 3-kinase.

The regulatory subunit of phosphatidylinositol 3-kinase, p85, contains a number of well defined domains involved in protein-protein interactions, including an SH3 domain and two SH2 domains. In order to investigate in detail the nature of the interactions of these domains with each other and with other binding partners, a series of deletion and point mutants was constructed, and their binding characteristics and apparent molecular masses under native conditions were analyzed. The SH3 domain and the first proline-rich motif bound each other, and variants of p85 containing the SH3 and BH domains and the first proline-rich motif were dimeric. Analysis of the apparent molecular mass of the deletion mutants indicated that each of these domains contributed residues to the dimerization interface, and competition experiments revealed that there were intermolecular SH3 domain-proline-rich motif interactions and BH-BH domain interactions mediating dimerization of p85alpha both in vitro and in vivo. Binding of SH2 domain ligands did not affect the dimeric state of p85alpha. Recently, roles for the p85 subunit have been postulated that do not involve the catalytic subunit, and if p85 exists on its own we propose that it would be dimeric.

The proinflammatory and chondral activities of leukemia inhibitory factor in goat joints are partially a function of interleukin-1.

We wished to determine if the effects of injected recombinant human leukemia inhibitory factor (LIF) are a function of endogenous goat interleukin-1 (IL-1) production and, conversely, if the effects of injected recombinant human IL-1 are a function of endogenous LIF production in goat radiocarpal joints (RCJ). In preliminary experiments, murine LIF binding protein (MuLBP) and recombinant HuIL-1RA were found to independently attenuate the cartilage proteoglycan resorbing activity of goat synovial membrane-conditioned medium (GSMCM), implying activity against goat LIF and goat IL-1, respectively. The present study shows that the proinflammatory and chondral actions of rHuLIF in goat RCJ are partially attenuated by rHuIL-1RA. This implies that a small but important component of the in vivo activity of rHuLIF is a result of IL-1 production in the synovial joint. With the exception of proteoglycan synthesis, the absence of significant effects by MuLBP on the actions of rHuIL-1alpha in goat RCJ suggests that the proinflammatory and chondral effects of IL-1alpha in vivo are probably not mediated by LIF.

Binding of a diphosphotyrosine-containing peptide that mimics activated platelet-derived growth factor receptor beta induces oligomerization of phosphatidylinositol 3-kinase.

Phosphatidylinositol 3-kinase (PI3K) is a heterodimeric enzyme comprising a p110 catalytic subunit and a p85 regulatory subunit. We have recently shown that the isolated p85 subunit exists as a dimer; therefore, we examined whether the heterodimeric enzyme was capable of further self-association. Size-exclusion chromatography demonstrated that PI3K was a 1:1 complex of p85 and p110 under native conditions. However, binding of a diphosphotyrosine-containing peptide that mimics an activated platelet-derived growth factor receptor beta induced an increase in the apparent molecular mass of PI3K. This increase was due to dimerization of PI3K and was dependent on PI3K concentration but not diphosphopeptide concentration. Dimer formation was also observed directly using fluorescence resonance energy transfer. Diphosphopeptide-induced activation of PI3K (Carpenter, C. L., Auger, K. R., Chanudhuri, M., Yoakim, M., Schaffhausen, B., Shoelson, S., and Cantley, L. C. (1993) J. Biol. Chem. 268, 9478-9483; Rordorf-Nikolic, T., Van Horn, D. J., Chen, D., White, M. F., and Backer, J. M. (1995) J. Biol. Chem. 270, 3662-3666) was not a direct result of dimerization and occurred only when phosphatidylinositol, and not phosphatidylinositol-4,5-diphosphate, was the phosphorylation substrate. Binding of the tandem SH2 domains of the p85 regulatory subunit to activated receptor tyrosine kinases therefore induces dimerization of PI3K, which may be an early step in inositol lipid-mediated signal transduction.

Human phosphoinositide 3-kinase C2beta, the role of calcium and the C2 domain in enzyme activity.

The cDNA for a human Class II phosphoinositide 3-kinase (PI 3-kinase C2beta) with a C2 domain was cloned from a U937 monocyte cDNA library and the enzyme expressed in mammalian and insect cells. Like other Class II PI 3-kinases in vitro, PI 3-kinase C2beta utilizes phosphatidylinositol (PI) and PI 4-monophosphate but not PI 4, 5-biphosphate as substrates in the presence of Mg2+. Remarkably, and unlike other PI 3-kinases, the enzyme can use either Mg-ATP or Ca-ATP to generate PI 3-monophosphate. PI 3-kinase C2beta, like the Class I PI 3-kinases, but unlike PI 3-kinase C2alpha, is sensitive to low nanomolar levels of the inhibitor wortmannin. The enzyme is not regulated by the small GTP-binding protein Ras. The C2 domain of the enzyme bound anionic phospholipids such as PI and phosphatidylserine in vitro, but did not co-operatively bind Ca2+ and phospholipids. Deletion of the C2 domain increased the lipid kinase activity suggesting that it functions as a negative regulator of the catalytic domain. Although presently it is not known whether PI 3-kinase C2beta is regulated by Ca2+ in vivo, our results suggest a novel role for Ca2+ ions in phosphate transfer reactions.

Modulation of cartilage proteoglycan metabolism by LIF binding protein.

Leukaemia inhibitory factor (LIF) and oncostatin M (OSM) exhibit pleiotropic biological activities, share many structural and genetic features and bind with high affinity to the same receptor (LIF/OSM receptor). A soluble form of the LIF-R alpha, called LIF binding protein (LBP) has been isolated from mouse serum. LIF and OSM stimulate proteoglycan (PG) release and inhibit PG synthesis in cultured pig articular cartilage explants. The aim of this study was to determine whether LBP can block PG resorption and or reverse the inhibition of PG synthesis induced by LIF and OSM. In cultured pig cartilage explants LBP was found to dose dependently inhibit LIF stimulated release of PGs and reverse the suppression of PG synthesis. LBP was found to substantially attenuate the effects of LIF. In contrast only partial inhibition of the stimulatory effect of OSM was observed at the highest concentration of LBP available. At maximal concentrations, LBP produced minimal reversal of OSM mediated inhibition of PG synthesis. When tested in combination LIF and OSM had no additive effects on PG metabolism, but the combination of LIF and IL-1 and also OSM and IL-1 did show additive effects in respect to stimulation of PG catabolism and inhibition of PG synthesis. These effects were significantly greater than those observed for LIF, OSM and IL-1 alone. The results suggest that pig articular chondrocytes possess the LIF/OSM receptor, but possibly not an independent OSM receptor. The actions of mLBP indicate that rhLBP could be a clinically useful antagonist for LIF and perhaps OSM.

The unusual species cross-reactivity of the leukemia inhibitory factor receptor alpha-chain is determined primarily by the immunoglobulin-like domain.

Human leukemia inhibitory factor (hLIF) binds to both human and mouse LIF receptors (LIFRs), while mouse LIF (mLIF) binds only to mouse LIFRs. Furthermore, hLIF binds with much higher affinity to the mouse LIFR (mLIFR) alpha-chain than does mLIF itself. To define the structural elements of the mLIFR alpha-chain conferring high affinity binding of hLIF and the species-specific interaction with mLIF, we first constructed C-terminally truncated extracellular domains of both the mLIFR and the human LIFR (hLIFR) alpha-chains, which contained only the two hemopoietin domains separated by an immunoglobulin-like domain. These recombinant truncated LIFR alpha-chains had identical binding and biological characteristics to either their naturally occurring or transfected counterparts. On the basis of this, we have generated eight interspecies receptor chimeras by combining different regions of the mouse and human LIFR sequence. Surprisingly, the immunoglobulin-like domain of the mLIFR alpha-chain played the predominant role in receptor-ligand interactions. Moreover, both high affinity binding for hLIF and the species-specific binding for mLIF mapped to the same domain of mLIFR molecule. These findings should enable the development of a "humanized" mouse LIFR that could act as a potent antagonist of hLIF biological activities in vivo.

Molecular basis of the soluble and membrane-bound forms of the murine leukemia inhibitory factor receptor alpha-chain. Expression in normal, gestating, and leukemia inhibitory factor nullizygous mice.

The murine leukemia inhibitory factor receptor alpha-chain (mLIFR) exists in a membrane-bound and a soluble form. The two major classes of mRNA transcript correspond to either the soluble or membrane-bound form of the mLIFR. In this study we have identified a complex and heterogeneous pattern of expression of mRNA transcripts for this receptor in normal mouse tissues and cell lines. In order to understand the molecular basis of these transcripts, genomic clones encompassing the region of divergence from the soluble to the membrane-bound form of the receptor were isolated. cDNAs encoding the membrane-bound form of the mLIFR were generated by an alternative splicing event where an exon that is specific to the soluble mLIFR was skipped. The membrane-bound form of the mLIFR was heterogeneously polyadenylated with at least five different sites of polyadenylation. The mRNA transcript encoding the soluble form of the mLIFR contained a region highly homologous to a murine B2 repetitive element, thus providing a possible explanation for the genesis of this transcript. The different forms of the mLIFR were analyzed in a wide range of mouse tissues in pseudopregnant mice and in mice at various stages of pregnancy. Only liver, placenta, and uterus showed an increase in the levels of mLIFR mRNA expression during pregnancy, indicating an important role for the LIFR in this process. However, somewhat surprisingly, there was no detectable difference in mLIFR mRNA levels or levels of soluble protein in leukemia inhibitory factor nullizygous mice when compared with normal mice.

Conversion of the biological specificity of murine to human leukemia inhibitory factor by replacing 6 amino acid residues.

Mouse and human leukemia inhibitory factor (mLIF and hLIF) have approximately 80% amino acid identity, but mLIF cannot bind to the hLIF receptor (hLIF-R), while hLIF binds to the alpha-chain of the mLIF receptor (mLIF-R) with a much higher affinity than does mLIF. We have previously shown that the same regions confer both these properties of hLIF and map to an area within the predicted third alpha-helix and two of the loops of hLIF (Owczarek, C. M., Layton, M. J., Metcalf, D., Lock, P., Wilson, T. A., Gough, N. M., and Nicola, N. A. (1993) EMBO J. 12, 3487-3495). The present studies, using interspecies chimeras of mLIF and hLIF, have defined 6 residues (Asp57, Ser107, His112, Ser113, Val155, and Lys158) that contribute most of the binding energy involved in the interaction of hLIF and the hLIF-R alpha-chain, and form a surface at one end of the predicted four alpha-helical bundle of the hLIF molecule. Mouse LIF is unable to bind to the hLIF-R alpha-chain or activate the cellular hLIF-R, but when these 6 residues were substituted onto an mLIF framework, they were able to reconstitute both the binding and biological activities specific to hLIF.

Homology modelling and 1H NMR studies of human leukaemia inhibitory factor.

Human leukaemia inhibitory factor (LIF) is a glycoprotein with a diverse range of activities on many cell types. A molecular model of LIF has been constructed based mainly on the structure of the related cytokine granulocyte colony-stimulating factor, and refined using simulated annealing and molecular dynamics in water. The model was stable during molecular dynamics refinement and is consistent with known stereochemical data on proteins. It has been assessed by comparison with 1H NMR data on the ionization behaviour of the six histidine residues in LIF, the imidazolium pKa values of which range from 3.6 to 7.4. These pKa values were assigned to individual histidine residues from NMR studies on a series of His-->Ala mutants. The environments of the histidine residues in the model account very well for their observed ionization behaviour. Furthermore, the model is consistent with mutagenesis studies which have defined a group of amino acid residues involved in receptor binding.

Complex binding of leukemia inhibitory factor to its membrane-expressed and soluble receptors.

The complex interaction of leukemia inhibitory factor (LIF) with its specific receptor present on the cell surface, in isolated membranes and in solution, has been examined in detail. Several aspects of this complexity have been highlighted, including the presence of high- and low-affinity murine LIF receptors, biphasic dissociation of human LIF from apparently homogeneous high- or low-affinity human LIF receptors, and unusual species cross-reactivity. The unusual species cross-reactivity observed between murine and human LIF has also been exploited to map an important receptor binding epitope on human LIF.

Cross-species receptor binding characteristics of human and mouse leukemia inhibitory factor suggest a complex binding interaction.

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine whose activities appear to be mediated through a single heterodimeric receptor complex. Human LIF (hLIF) can bind to and activate mouse LIF (mLIF) receptors but mLIF is unable to bind to hLIF receptors. Cross-species competition of mLIF and hLIF for binding to the mLIF receptor was found to be dependent on which ligand was used as the radioactive tracer (Layton, M. J., Cross, B. A., Metcalf, D., Ward, L. D., Simpson, R. J., and Nicola, N. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 8616-8620), and this phenomenon was investigated in the present study. We found that hLIF bound to the low affinity mLIF receptor with a 100-500-fold higher primary affinity and lower kinetic dissociation rate than mLIF, but both ligands displayed a single rate of ligand dissociation. In contrast, the binding of hLIF to low and high affinity hLIF receptors revealed two classes of binding site. The observed tracer-dependent phenomena suggested that both mLIF and hLIF interfere with the binding of each other to the mLIF receptor. A model is presented in which hLIF binds to two sites on mLIF and hLIF receptors, one of which interferes with the common site for mLIF. This model may reconcile some of the observed complexities of LIF/LIF receptor interactions.

NMR studies of a murine-human chimera of leukaemia inhibitory factor (LIF). Comparison with human LIF.

Leukaemia inhibitory factor (LIF) is a polyfunctional cytokine active on many cell types. We present here 1H NMR studies on the solution properties and stability of MH35, a chimera of murine and human LIF which can be expressed at high levels in Escherichia coli, thus enabling efficient labelling of the protein with the stable isotopes 13C and 15N. MH35 has 85% sequence identity with human LIF and similar activity in biological assays. The 1H chemical shifts of the 12 conserved aromatic residues and the pKa values of the five conserved histidine residues in MH35 and human LIF are very similar. Temperature dependence studies indicate that both proteins are stable, with significant conformational changes occurring only above 70 degrees C. These results show that these proteins have a similar overall structure and stability and that MH35 is therefore a suitable analogue of human LIF for structural studies in solution.

Inter-species chimeras of leukaemia inhibitory factor define a major human receptor-binding determinant.

Human leukaemia inhibitory factor (hLIF) binds to both human and mouse LIF receptors (LIF-R), while mouse LIF (mLIF) binds only to mouse LIF-R. Moreover, hLIF binds with higher affinity to the mLIF-R than does mLIF. In order to define the regions of the hLIF molecule responsible for species-specific interaction with the hLIF-R and for the unusual high-affinity binding to the mLIF-R, a series of 15 mouse/human LIF hybrids has been generated. Perhaps surprisingly, both of these properties mapped to the same region of the hLIF molecule. The predominant contribution was from residues in the loop linking the third and fourth helices, with lesser contributions from residues in the third helix and the loop connecting the second and third helices in the predicted three-dimensional structure. Since all chimeras retained full biological activity and receptor-binding activity on mouse cells, and there was little variation in the specific biological activity of the purified proteins, it can be concluded that the overall secondary and tertiary structures of each chimera were intact. This observation also implied that the primary binding sites on mLIF and hLIF for the mLIF-R were unaltered by inter-species domain swapping. Consequently, the site on the hLIF molecule that confers species-specific binding to the hLIF-R and higher affinity binding to the mLIF-R, must constitute an additional interaction site to that used by both mLIF and hLIF to bind to the mLIF-R. These studies define a maximum of 15 amino acid differences between hLIF and mLIF that are responsible for the different properties of these proteins.

A major binding protein for leukemia inhibitory factor in normal mouse serum: identification as a soluble form of the cellular receptor.

A protein that specifically binds leukemia inhibitory factor (LIF) has been isolated from normal mouse serum by using four successive fractionation steps: chromatography on a LIF affinity matrix, anion-exchange chromatography, size-exclusion chromatography, and preparative native gel electrophoresis. The purified LIF-binding protein (LBP) is a glycoprotein with an apparent molecular mass of 90 kDa that specifically binds 125I-labeled murine LIF with an affinity comparable to that of the low-affinity cellular LIF receptor (Kd = 600 pM). N-terminal sequencing has identified this protein as a soluble truncated form of the alpha chain of the cellular LIF receptor. LBP is present in normal mouse serum at high levels (1 microgram/ml) and these levels are elevated in pregnant mice and reduced in neonatal mice. Since normal serum concentrations of LBP can block the biological actions of LIF in culture, LBP may serve as an inhibitor of the systemic effects of locally produced LIF.