Far Western Blotting Research Articles

Expanding the Functional Repertoire of CTD Kinase I and RNA Polymerase II: Novel PhosphoCTD-Associating Proteins in the Yeast Proteome

CTD kinase I (CTDK-I) of Saccharomyces cerevisiae is required for normal phosphorylation of the C-terminal repeat domain (CTD) on elongating RNA polymerase II. To elucidate cellular roles played by this kinase and the hyperphosphorylated CTD (phosphoCTD) it generates, we systematically searched yeast extracts for proteins that bound to the phosphoCTD made by CTDK-I in vitro. Initially, using a combination of far-western blotting and phosphoCTD affinity chromatography, we discovered a set of novel phosphoCTD-associating proteins (PCAPs) implicated in a variety of nuclear functions. We identified the phosphoCTD-interacting domains of a number of these PCAPs, and in several test cases (namely, Set2, Ssd1, and Hrr25) adduced evidence that phosphoCTD binding is functionally important in vivo. Employing surface plasmon resonance (BIACORE) analysis, we found that recombinant versions of these and other PCAPs bind preferentially to CTD repeat peptides carrying SerPO(4) residues at positions 2 and 5 of each seven amino acid repeat, consistent with the positional specificity of CTDK-I in vitro [Jones, J. C., et al. (2004) J. Biol. Chem. 279, 24957-24964]. Subsequently, we used a synthetic CTD peptide with three doubly phosphorylated repeats (2,5P) as an affinity matrix, greatly expanding our search for PCAPs. This resulted in identification of approximately 100 PCAPs and associated proteins representing a wide range of functions (e.g., transcription, RNA processing, chromatin structure, DNA metabolism, protein synthesis and turnover, RNA degradation, snRNA modification, and snoRNP biogenesis). The varied nature of these PCAPs and associated proteins points to an unexpectedly diverse set of connections between Pol II elongation and other processes, conceptually expanding the role played by CTD phosphorylation in functional organization of the nucleus.

Src Homology 2 Domain Binding as a Phosphoproteomic Approach to Signaling in Acute Lymphoblastic Leukemia.

A general theme of orchestrated signal transduction is played by activated receptor phosphotyrosine kinases (PTK) and receptor PTK targets which propagate signals via recognition of sequence-specific phoshorylated tyrosines by so-called Src homology 2 (SH2) domains. SH2 domain interactions are used as a means of recruiting target proteins to activated PTKs and to translocate them to the plasma membrane where many effector proteins activated by receptor PTKs such as phospholipase C-γ or PI-3 kinase have their substrates. SH2 domains make up the most prevalent type of phosphotyrosine binding domains involved in signaling downstream of activated PTKs. SH2 domains are not only present in proteins with intrinsic enzymatic activity but also in adaptor proteins which shuttle effector enzymes to target signaling complexes. Increasing numbers of diseases are known to involve phosphotyrosine specific kinases and/or phosphatases going awry exemplified by the notorious ErbB2 receptor PTK in breast cancer or the Bcr-Abl PTK in CML. Currently, the tyrosine phosphorylation state in most acute lymphoblastic leukemias is undefined which is predicted to differ among the various subgroups and to be distinct from the signaling state of normal hematopoietic cells. To identify aberrant tyrosine kinase or phosphatase activity in the various types of acute lymphoblastic leukemia is of great interest since enzymes in general make good targets for drugs. A novel SH2 domain binding approach is presented which can detect distinctive profiles of tyrosine-phosphorylated proteins in complex mixtures of cellular proteins. A battery of SH2 domains is employed as probes in a competitive far-Western blot based assay to identify specific tyrosine-phosphorylated sites which reflect active signaling pathways in a cell. A further refinement of this technology is under way with DNA-tagged probes being developed which allow for multiplexing and high throughput quantitative assessment of SH2-domain binding by quantitative PCR or microarray technologies.

Evidence for conservation of architecture and physical properties of Omp85-like proteins throughout evolution.

Omp85-like proteins represent a family of proteins involved in protein translocation, and they are present in all domains of life, except archaea. In eukaryotes, Omp85-like proteins have been demonstrated to form tetrameric pore-forming complexes that interact directly with their substrate proteins. Studies performed with bacterial Omp85-like proteins have demonstrated pore-forming activity but no evidence of multimerization. In this article, we characterize the Haemophilus influenzae HMW1B protein, an Omp85-like protein that has been demonstrated to be critical for secretion of the H. influenzae HMW1 adhesin. Analysis of purified protein by biochemical and electron microscopic techniques revealed that HMW1B forms a tetramer. Examination using liposome-swelling assays demonstrated that HMW1B has pore-forming activity, with a pore size of approximately equal to 2.7 nm. Far-Western blot analysis established that HMW1B interacts with the N terminus of HMW1. These results provide evidence that a bacterial Omp85-like protein forms a tetramer and interacts directly with a substrate protein, suggesting that the architecture and physical properties of Omp85-like proteins have been conserved throughout evolution.

Phosphorylation of calmodulin fragments by protein kinase CK2. Mechanistic aspects and structural consequences.

Calmodulin is phosphorylated in vivo and in vitro by protein kinase CK2 in a manner that is unique among CK2 substrates for being inhibited by the regulatory beta-subunit of the kinase and dramatically enhanced by polybasic peptides. Using synthetic fragments of calmodulin variably encompassing the CK2 phosphorylation sites here we show that individual phosphorylation of Thr79, Ser81, Ser101, and Thr117 is critically influenced by the size and composition of the peptides and that the C-terminal domain of calmodulin is implicated both in down-regulation of calmodulin phosphorylation by the beta-subunit and in its abnormal responsiveness to polylysine. A far-Western blot analysis discloses polylysine-dependent interaction between calmodulin and the N-terminal domain of the beta-subunit. We also show that phosphorylation of Ser81 hampers subsequent phosphorylation of Thr79 and by itself promotes the unfolding of the central helix, whose flexibility is instrumental to the interaction with calmodulin-dependent enzymes. Collectively taken, our data are consistent with a multifaceted regulation of calmodulin phosphorylation through the concerted action of distinct CaM domains, the catalytic and regulatory subunits of CK2, and polycationic effectors mimicking in vivo the effect of polylysine.

Growth-promoting effects of lactoferrin on L. acidophilus and Bifidobacterium spp.

We investigated the effects of lactoferrin on the growth of L. acidophilus CH-2, Bifidobacterium breve ATCC 15700, B. longum ATCC 15707, B. infantis ATCC 15697, and B. bifidum ATCC 15696. The growth of L. acidophilus was stimulated by bovine holo-lactoferrin but not by apo-lactoferrin. With bifidobacteria, bovine lactoferrin stimulated growth of three strains: B. breve, B. infantis and B. bifidum under certain conditions. Both apoprotein and holoprotein had similar effects. However, B. longum growth was not affected by lactoferrin. Thus, the mechanism of stimulating growth of bifidobacteria may be different from that of L. acidophilus. By far-western blotting using biotinylated lactoferrin and horseradish peroxidase-conjugated streptavidin, lactoferrin-binding proteins were detected in the membrane protein fraction of L. acidophilus, B. bifidum, B. infantis and B. breve. The molecular weights of lactoferrin-binding proteins of L. acidophilus were estimated from SDS-polyacrylamide gel electrophoresis to be 27, 41 and 67 kDa, and those of the three bifidobacterial strains were estimated to be 67-69 kDa. However, no such lactoferrin-binding components were detected in the membrane fraction of B. longum. It is interesting that the appearance of lactoferrin-binding proteins in the membrane fraction of these species corresponds to their growth stimulation by lactoferrin.

The central and C-terminal domains of VPg of Clover yellow vein virus are important for VPg-HCPro and VPg-VPg interactions.

Interactions between the major proteins of Clover yellow vein virus (ClYVV) were investigated using a GAL4 transcription activator-based yeast two-hybrid system (YTHS). Self-interactions manifested by VPg and HCPro and an interaction between NIb and NIaPro were observed in ClYVV. In addition, a strong HCPro-VPg interaction was detected by both YTHS and by in vitro far-Western blot analysis in ClYVV. A potyvirus HCPro-VPg interaction has not been reported previously. Using YTHS, domains in ClYVV for the VPg self-interaction and the HCPro-VPg interaction were mapped. The VPg C-terminal region (38 amino acids) was important for the VPg-VPg interaction and the central 19 amino acids were needed for the HCPro-VPg interaction.

Protein kinase CK2 and protein kinase D are associated with the COP9 signalosome

The COP9 signalosome (CSN) purified from human erythrocytes possesses kinase activity that phosphoryl ates proteins such as c-Jun and p53 with consequence for their ubiquitin (Ub)-dependent degradation. Here we show that protein kinase CK2 (CK2) and protein kinase D (PKD) co-purify with CSN. Immunoprecipitation and far-western blots reveal that CK2 and PKD are in fact associated with CSN. As indicated by electron microscopy with gold-labeled ATP, at least 10% of CSN particles are associated with kinases. Kinase activity, most likely due to CK2 and PKD, co-immuno precipitates with CSN from HeLa cells. CK2 binds to DeltaCSN3(111-403) and CSN7, whereas PKD interacts with full-length CSN3. CK2 phosphorylates CSN2 and CSN7, and PKD modifies CSN7. Both CK2 and PKD phosphorylate c-Jun as well as p53. CK2 phosphoryl ates Thr155, which targets p53 to degradation by the Ub system. Curcumin, emodin, DRB and resveratrol block CSN-associated kinases and induce degradation of c-Jun in HeLa cells. Curcumin treatment results in elevated amounts of c-Jun-Ub conjugates. We conclude that CK2 and PKD are recruited by CSN in order to regulate Ub conjugate formation.

Identification of a Lactoferrin-derived Peptide Possessing Binding Activity to Hepatitis C Virus E2 Envelope Protein

Bovine and human lactoferrins (LF) prevent hepatitis C virus (HCV) infection in cultured human hepatocytes; the preventive mechanism is thought to be the direct interaction between LF and HCV. To clarify this hypothesis, we have characterized the binding activity of LF to HCV E2 envelope protein and have endeavored to determine which region(s) of LF are important for this binding activity. Several regions of human LF have been expressed and purified as thioredoxin-fused proteins in Escherichia coli. Far-Western blot analysis using these LF fragments and the E2 protein, expressed in Chinese hamster ovary cells, revealed that the 93 carboxyl amino acids of LF specifically bound to the E2 protein. The 93 carboxyl amino acids of LFs derived from bovine and horse cells also possessed similar binding activity to the E2 protein. In addition, the amino acid sequences of these carboxyl regions appeared to show partial homology to CD81, a candidate receptor for HCV, and the binding activity of these carboxyl regions was also comparable with that of CD81. Further deletion analysis identified 33 amino acid residues as the minimum binding site in the carboxyl region of LF, and the binding specificity of these 33 amino acids was also confirmed by using 33 maltose-binding protein-fused amino acids. Furthermore, we demonstrated that the 33 maltose-binding protein-fused amino acids prevented HCV infection in cultured human hepatocytes. In addition, the site-directed mutagenesis to an Ala residue in both terminal residues of the 33 amino acids revealed that Cys at amino acid 628 was determined to be critical for binding to the E2 protein. These results led us to consider the development of an effective anti-HCV peptide. This is the first identification of a natural protein-derived peptide that specifically binds to HCV E2 protein and prevents HCV infection.

Identification and characterization of a novel calcineurin-binding protein in scallop testis.

Calcineurin has been inferred to function in meiosis and spermiogenesis in testis. Here, we identified a calcineurin-binding protein in scallop testis by Far-Western blot analysis using purified calcineurin as a probe. The molecular mass of the binding protein estimated on the blot was 75 kDa. The isolated cDNA clone encoded a novel 474-residue protein, named CaNBP75. The region between T6 and A210 of CaNBP75 was responsible for the interaction with calcineurin. CaNBP75 was predominantly expressed in testis and ovary of scallop. Thus, CaNBP75 may modulate the physiological function of calcineurin in the testis and ovary of scallop, such as in spermiogenesis or meiosis.

Rice grassy stunt tenuivirus nonstructural protein p5 interacts with itself to form oligomeric complexes in vitro and in vivo.

We investigated the interaction of Rice grassy stunt tenuivirus (RGSV) nonstructural protein p5, a protein of 22 kDa encoded on vRNA 5, with all 12 RGSV proteins by using a GAL4 transcription activator-based yeast two-hybrid system. The p5 protein interacted only with itself and not with any other viral protein; the interacting domains were localized within the N-terminal 96 amino acids of p5. The p5-p5 interaction was reproduced in an Sos recruitment-mediated yeast two-hybrid system as well in by far-Western blots. Native p5 protein extracted from RGSV-infected rice tissue was detected in a large complex with a molecular mass of approximately 260 kDa composed of 12 molecules of p5 or a p5 oligomer with an unidentified host factor(s).

Remarkable stabilization of neutrophil NADPH oxidase using RacQ61L and a p67phox-p47phox fusion protein.

Activation of the phagocyte NADPH oxidase occurs via assembly of cytosolic p47(phox), p67(phox), and Rac with the membrane-bound flavocytochrome b(558). Recently, we have found that p67(phox)-(1-210) (p67N) fused with p47(phox)-(1-286) (p47N) or with Rac efficiently stabilizes the oxidase in a cell-free reconstitution system. In an attempt to further stabilize the oxidase, we herein used a constitutively active Rac, RacQ61L, and examined its effect on the oxidase stability. The half-life (t(1/2)) of the activity reconstituted with wild-type Rac was 12 min at 37 degrees C, which was extended 6-fold by RacQ61L. Also, the stability of the oxidase without p47(phox) increased 8-fold using RacQ61L. RacQ61L had a higher affinity for the complex than wild-type Rac and increased the affinity of p67N for the complex. Far-western blotting showed an enhanced binding between RacQ61L and p67N. The oxidase was stabilized by nanomolar FAD, and RacQ61L lowered the FAD concentration required. The combination of RacQ61L and a fusion protein consisting of p67N and p47N produced an extremely stable enzyme (t(1/2) = 184 min at 37 degrees C). The effectiveness of RacQ61L and fusion proteins on stabilization was in the following order: p67N-Rac < p67N + RacQ61L < or = p67N-RacQ61L << p67N-p47N + RacQ61L. These results indicate that a tightly bound ternary complex of p67(phox), Rac, and p47(phox) is very effective in maintaining the oxidase and confirm that the longevity of the activated state requires continuous association of these components. This simple and efficient method of stabilization may provide a useful tool to elucidate the nature of the activated oxidase.

Profiling the global tyrosine phosphorylation state by Src homology 2 domain binding.

Reversible tyrosine phosphorylation plays a crucial role in signal transduction, regulating many biological functions including proliferation, differentiation, and motility. The comprehensive characterization of the tyrosine phosphorylation state of a cell is of great interest for understanding the mechanisms that underlie signaling; however, current methods for analyzing tyrosine-phosphorylated proteins in crude protein extracts provide limited information, or are laborious and require relatively large amounts of protein. We have developed a simple, rapid, and flexible competitive binding assay based on the far-Western blot technique, in which a battery of Src homology 2 domain probes is used to detect patterns of specific tyrosine-phosphorylated sites. We demonstrate that distinct profiles of tyrosine phosphorylation can be detected with high sensitivity and specificity and low background. This proteomic approach can be used to rapidly profile the global tyrosine phosphorylation state of any cell of interest and has obvious applications as a molecular diagnostic tool, for example in the classification of tumors. The general strategy we describe here is not limited to Src homology 2 domains and could be used to profile the binding sites for any class of protein interaction domain.

Borna disease virus phosphoprotein binds a neurite outgrowth factor, amphoterin/HMG-1.

The Borna disease virus (BDV) p24 phosphoprotein is an abundant protein in BDV-infected cultured cells and animal brains. Therefore, there is a possibility that binding of the p24 protein to cellular factor(s) induces functional alterations of infected neural cells in the brain. To identify a cellular protein(s) that interacts with BDV p24 protein, we performed far-Western blotting with extracts from various cell lines. Using recombinant p24 protein as a probe, we detected a 30-kDa protein in all cell lines examined. Binding between the 30-kDa and BDV p24 proteins was also demonstrated using BDV p24 affinity and ion-exchange chromatography columns. Microsequence analysis of the purified 30-kDa protein revealed that its N terminus showed complete homology with rat amphoterin protein, which is a neurite outgrowth factor abundant in the brain during development. Mammalian two-hybrid and immunoprecipitation analyses also confirmed that amphoterin is a specific target for the p24 protein in vivo. Furthermore, we showed that infection by BDV, as well as purified p24 protein in the medium, significantly decreased cell process outgrowth of cells grown on laminin, indicating the functional inhibition of amphoterin by interaction with the p24 protein. Immunohistochemical analysis revealed decreased levels of amphoterin protein at the leading edges of BDV-infected cells. Moreover, the expression of the receptor for advanced glycation end products, of which the extracellular moiety is a receptor for amphoterin, was not significantly activated in BDV-infected cells during the process of extension, suggesting that the secretion of amphoterin from the cell surface is inhibited by the binding of the p24 protein. These results suggested that BDV infection may cause direct damage in the developing brain by inhibiting the function of amphoterin due to binding by the p24 phosphoprotein.

Functional Interaction of bZIP Proteins and the Large Subunit of Replication Factor C in Liver and Adipose Cells

The transcription factor CCAAT/enhancer-binding protein-alpha (C/EBPalpha) has a vital role in cell growth and differentiation. To delineate further a mechanism for C/EBPalpha-mediated differentiation, we screened C/EBPalpha-interacting proteins through far-Western screening. One of the strongest interactions was with RFC140, the large subunit of the replication factor C complex. C/EBPalpha specifically interacted with RFC140 from rat liver nuclear extract as determined by a combination of affinity chromatography and co-immunoprecipitation. Subsequent far-Western blotting showed that the bZIP domain of C/EBPalpha interacted with the DNA-binding region of RFC140. Overexpression of RFC140 in mammalian cells increased the transactivation activity of C/EBPalpha on both minimal and native promoters. Consistent with the enhanced transactivation, a complex of C/EBPalpha and RFC140 proteins with the cognate DNA element was detected in vitro. The specific interaction between C/EBPalpha and RFC140 was detected in the terminal differentiation of 3T3-L1 preadipocytes to adipocytes. The synergistic transcription effect of these two proteins increased the promoter activity and protein expression of peroxisome proliferator-activated receptor-gamma, which is a main regulator of adipocyte differentiation. Our results demonstrate that the specific transcription factor C/EBPalpha and the general DNA replication factor RFC140 interact functionally and physically. This observation highlights a unique mechanism by which the levels of the general replication factor can strongly modulate the functional activity of the specific transcription factor as a coactivator.

HRRN3 is essential in the SL1-mediated recruitment of RNA Polymerase I to rRNA gene promoters.

A crucial step in transcription is the recruitment of RNA polymerase to promoters. In the transcription of human rRNA genes by RNA Polymerase I (Pol I), transcription factor SL1 has a role as the essential core promoter binding factor. Little is known about the mechanism by which Pol I is recruited. We provide evidence for an essential role for hRRN3, the human homologue of a yeast Pol I transcription factor, in this process. We find that whereas the bulk of human Pol I complexes (I alpha) are transcriptionally inactive, hRRN3 defines a distinct subpopulation of Pol I complexes (I beta) that supports specific initiation of transcription. Human RRN3 interacts directly with TAF(I)110 and TAF(I)63 of promoter-selectivity factor SL1. Blocking this connection prevents recruitment of Pol I beta to the rDNA promoter. Furthermore, hRRN3 can be found in transcriptionally autonomous Pol I holoenzyme complexes. We conclude that hRRN3 functions to recruit initiation-competent Pol I to rRNA gene promoters. The essential role for hRRN3 in linking Pol I to SL1 suggests a mechanism for growth control of Pol I transcription.

Expression of FLRG, a novel activin A ligand, is regulated by TGF-beta and during hematopoiesis [corrected

The human gene FLRG, identified from a B-cell chronic lymphocytic leukemia bearing a t(11;19) translocation, encodes a secreted glycoprotein highly homologous with follistatin. Activin A is a TGF-beta family member involved in the regulation of growth and differentiation of various types of cells, such as those of the hematopoietic system. Its biological activity is antagonized by binding with follistatin. We investigated the binding of FLRG to activin A and the expression pattern of FLRG, follistatin, and activin A during hematopoiesis. The binding of FLRG with activin A was investigated by immunoprecipitation and Far-Western blot analysis. Gene expression was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) and Northern Blot in purified hematopoietic populations. We demonstrate that FLRG, like follistatin, is able to bind to activin A. In bone marrow stromal cells, both mRNA and protein FLRG levels were found to be dramatically increased by TGF-beta. FLRG and activin A are expressed in the same cells, with a higher level of expression in the myeloid cells compared with the erythroid and megakaryocytic cells. FLRG and follistatin expression were different in the hematopoietic subpopulations tested. Moreover, we observed that FLRG and activin A expression was up-regulated during hematopoiesis. FLRG and activin A are expressed in the same hematopoietic cells and regulated by TGF-beta. Moreover, FLRG interacts with activin A, suggesting that FLRG, like follistatin, participates in the diverse regulatory functions of activin A, such as those in hematopoiesis.

Confirming yeast two-hybrid protein interactions using in vitro glutathione-S-transferase pulldowns.

Protein interactions play a vital physiologic role in mediating many important aspects of cell biology including cell division, transcriptional regulation, and signal transduction. The biologic significance of protein interactions is highlighted by the important role that they play in nuclear receptor-mediated transcription. Nuclear hormone receptors, which include the receptors for thyroid hormone, vitamin D3, estrogen, and progesterone, are ligand-inducible transcription factors that activate and repress transcription through a complex array of protein interactions (, , , ). In an effort to understand the mechanism by which nuclear receptors regulate the transcription of specific target genes, many recent studies have focused on the identification of proteins that bind nuclear receptors. The concerted efforts of a number of investigators have led to the isolation and characterization of a growing list of proteins that interact with nuclear hormone receptors. The majority of these proteins were identified through the yeast two-hybrid system, while others were identified by Far-Western blotting or conventional chromatography.

Bovine prion protein as a modulator of protein kinase CK2

On the basis of far-Western blot and plasmon resonance (BIAcore) experiments, we show here that recombinant bovine prion protein (bPrP) (25–242) strongly interacts with the catalytic α/α´ subunits of protein kinase CK2 (also termed ‘casein kinase 2’). This association leads to increased phosphotransferase activity of CK2α, tested on calmodulin or specific peptides as substrate. We also show that bPrP counteracts the inhibition of calmodulin phosphorylation promoted by the regulatory β subunits of CK2. A truncated form of bPrP encompassing the C-terminal domain (residues 105–242) interacts with CK2 but does not affect its catalytic activity. The opposite is found with the N-terminal fragment of bPrP (residues 25–116), although the stimulation of catalysis is less efficient than with full-size bPrP. These results disclose the potential of the PrP to modulate the activity of CK2, a pleiotropic protein kinase that is particularly abundant in the brain.

Bovine prion protein as a modulator of protein kinase CK2

On the basis of far-Western blot and plasmon resonance (BIAcore) experiments, we show here that recombinant bovine prion protein (bPrP) (25-242) strongly interacts with the catalytic alpha/alpha' subunits of protein kinase CK2 (also termed 'casein kinase 2'). This association leads to increased phosphotransferase activity of CK2alpha, tested on calmodulin or specific peptides as substrate. We also show that bPrP counteracts the inhibition of calmodulin phosphorylation promoted by the regulatory beta subunits of CK2. A truncated form of bPrP encompassing the C-terminal domain (residues 105-242) interacts with CK2 but does not affect its catalytic activity. The opposite is found with the N-terminal fragment of bPrP (residues 25-116), although the stimulation of catalysis is less efficient than with full-size bPrP. These results disclose the potential of the PrP to modulate the activity of CK2, a pleiotropic protein kinase that is particularly abundant in the brain.

Direct Interaction of Actin with p47phox of Neutrophil NADPH Oxidase

The cell-free activation of human neutrophil NADPH oxidase is enhanced by actin, and actin filaments formed during activation are suggested to stabilize the oxidase. In an attempt to elucidate the mechanism, we examined the protein–protein interactions between actin and cytosolic components of the oxidase. Far-Western blotting using recombinant phox proteins showed that both α- and β-actin interacted with p47phox and rac1, and weakly with rac2. A deletion mutant of p47phox proved that its C-terminal region was essential for the interaction. The dissociation constant (Kd) for interaction between actin and p47phox was estimated to be 0.45 μM by surface plasmon resonance, and that between actin and rac1 or rac2 was 1.7 or 4.6 μM, respectively. Far-Western blotting using cytosol as a target showed an interaction between actin and endogenous p47phox and rac proteins. These results suggest that actin can directly interact with p47phox and possibly with rac in the cells.