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B K Kay

Publications and source records attributed to B K Kay.

At least 55 records · Page 3Linked to original sources
PubMed1997

Examining the specificity of Src homology 3 domain--ligand interactions with alkaline phosphatase fusion proteins.

Sixteen-amino-acid-long peptides, corresponding to the optimal ligand preferences of the Src homology 3 (SH3) domains of Abl, Cortactin, Crk, p53BP2, and Src, were fused to the N-terminus of Escherichia coli alkaline phosphatase (AP). These secreted fusion proteins have been used as one-step detection probes of peptide ligand-SH3 domain interactions on microtiter plates and membranes. The binding of both the class I and II SH3 ligand-AP fusion proteins to their targets is robust and specific in comparison to chemically synthesized biotinylated peptides, used either in monovalent or tetravalent formats. p53BP2 and Cortactin SH3 ligand-AP fusions have been used to screen a mouse embryo lambda cDNA expression library and resulted in the cloning of p53BP2 and several known proteins with SH3 domains similar to that of Cortactin, respectively. In addition, the approximately 60-amino-acid-long SH3 domains of Src and Abl were fused to AP and the resulting fusion proteins were found to bind specifically to their respective peptide ligands in microtiter plates and proteins containing proline-rich regions in screens of a lambda cDNA expression library. Thus, SH3 peptide ligand- and SH3 domain-AP fusion proteins are convenient and sensitive reagents for examining the specificity of SH3 domain-ligand interactions, identifying potentially interacting proteins, and establishing high-throughput screens of combinatorial chemical libraries.

Alkaline Phosphatase↗
PubMed1997

Using molecular repertoires to identify high-affinity peptide ligands of the WW domain of human and mouse YAP.

The WW domain is a globular protein domain that is involved in mediating protein-protein interaction and that ultimately participates in various intracellular signaling events. The domain binds to polyproline ligands containing the xPPxY consensus (where x signifies any amino acid, P is proline and Y is tyrosine). One of the first WW domain-ligand links that was characterized in vitro was the WW domain of Yes-Associated Protein (YAP) and its WBP-1 ligand. To further characterize this molecular interaction, we used two independent approaches, both of which focused on the mutational analysis of the WBP-1 ligand. We screened repertoires of synthetic decamer peptides containing the xPPxY core of WBP-1 in which all ten positions were sequentially replaced with the remaining amino acids. In addition, we screened decamer repertoires with all permutations of the amino acids which individually increased the binding to the WW domain of YAP, as compared to the wild type. In a parallel approach, we used a phage-displayed combinatorial peptide library biased for the presence of two consecutive prolines to study ligand preferences for the WW domain of YAP. Interestingly, these two lines of investigation converged and yielded the core sequence PPPPYP, which is preferred by the YAP-WW domain. This sequence was found within the p53 (tumor suppressor) binding protein-2, a probable cognate or alternative ligand interacting with YAP.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Isolation of a NCK-associated kinase, PRK2, an SH3-binding protein and potential effector of Rho protein signaling.

The NCK adapter protein is comprised of three consecutive Src homology 3 (SH3) protein-protein interaction domains and a C-terminal SH2 domain. Although the association of NCK with activated receptor protein-tyrosine kinases, via its SH2 domain, implicates NCK as a mediator of growth factor-induced signal transduction, little is known about the pathway(s) downstream of NCK recruitment. To identify potential downstream effectors of NCK we screened a bacterial expression library to isolate proteins that bind its SH3 domains. Two molecules were isolated, the Wiskott-Aldrich syndrome protein (WASP, a putative CDC42 effector) and a serine/threonine protein kinase (PRK2, closely related to the putative Rho effector PKN). Using interspecific backcross analysis the Prk2 gene was mapped to mouse chromosome 3. Unlike WASP, which bound the SH3 domains of several signaling proteins, PRK2 specifically bound to the middle SH3 domain of NCK and (weakly) that of phospholipase Cgamma. PRK2 also specifically bound to Rho in a GTP-dependent manner and cooperated with Rho family proteins to induce transcriptional activation via the serum response factor. These data suggest that PRK2 may coordinately mediate signal transduction from activated receptor protein-tyrosine kinases and Rho and that NCK may function as an adapter to connect receptor-mediated events to Rho protein signaling.

3T3 Cells↗
PubMed1996

Increasing the distance between the snRNA promoter and the 3' box decreases the efficiency of snRNA 3'-end formation.

Chimeric genes which contained the mouse U1b snRNA promoter, portions of the histone H2a or globin coding regions and the U1b 3'-end followed by a histone 3'-end were constructed. The distance between the U1 promoter and the U1 3' box was varied between 146 and 670 nt. The chimeric genes were introduced into CHO cells by stable transfection or into Xenopus oocytes by microinjection. The efficiency of utilization of the U1 3' box, as measured by the relative amounts of transcripts that ended at the U1 3' box and the histone 3'-end, was dependent on the distance between the promoter and 3'-end box. U1 3'-ends were formed with >90% efficiency on transcripts shorter than 200 nt, with 50-70% efficiency on transcripts of 280-400 nt and with only 10-20% efficiency on transcripts >500 nt. Essentially identical results were obtained after stable transfection of CHO cells or after injecting the genes into Xenopus oocytes. The distance between the U1 promoter and the U1 3' box must be <280 nt for efficient transcription termination at the U1 3' box, regardless of the sequence transcribed.

Animals↗
PubMed1996

Inhibition of human complement by a C3-binding peptide isolated from a phage-displayed random peptide library.

We have screened a phage-displayed random peptide library for binding to C3b, the proteolytically activated form of complement component C3, and have identified a novel peptide that suppresses complement activation. This phage-displayed peptide bound to C3, C3b, and C3c, but not to C3d, indicating that it binds to the C3c region of C3. A synthetic 27-amino acid peptide corresponding to the phage-displayed peptide also bound to C3 and C3 fragments and inhibited both the classical and alternative pathways of complement activation. The inhibition of complement activation was reversible. Studies with overlapping peptides indicated that the functional activity was located in the cyclic 13-amino acid N-terminal region (ICVVQDWGHHRCT) of the parent peptide. Reduction and alkylation of this 13-residue synthetic peptide destroyed its inhibitory activity. Analysis of the mechanism of inhibition revealed that the peptide inhibited C3 cleavage in normal human serum as well as when the alternative pathway was reconstituted with purified complement components, and the observed inhibition was not due to sterically hindered access to the C3a/C3b cleavage site. Further, the peptide did not inhibit the cleavage of factor B, indicating that it did not affect the interaction of CA with factor B or the formation of C3b,Bb. The peptide also had no effect on the binding of properdin to C3, demonstrating that the observed inhibition of C3 cleavage in normal human serum was not due in part to its effect on the properdin-stabilized C3 convertase, C3b,Bb,P. These results indicate that the peptide we have identified interacts with C3 to inhibit its activation.

Amino Acid Sequence↗
PubMed1996

Identification of a biologically significant DNA-binding peptide motif by use of a random phage display library.

A peptide library approach was used to identify peptides that could bind to different DNA structures. A 23-mer random peptide library was displayed in the context of the pIII protein of M13 filamentous phage. Double-stranded (ds) oligodeoxyribonucleotides (oligos) were immobilized in 96-well plates using either chemical conjugation or a biotin-avidin linking method. Individual phage clones capable of binding to immobilized oligos were selected from the phage library. Using a plaque dilution assay for rapid screening of binding preferences, four groups of oligo-binding (OB) phage were tentatively identified as showing preference for: (1) single-stranded (ss) oligos irrespective of sequence; (2) ds oligos irrespective of sequence; (3) sequence-specific binding to ss oligos; and (4) weak non-specific binding to all types of oligos tested. A quantitative solution-phase competition assay was used to confirm the ability of certain phage to discriminate ss from ds oligos. A consensus motif, FGRA, was found in those phage clones that preferentially bound ss oligos; this motif has previously been noted in the binding domains of several ribonucleoproteins and ss DNA-binding proteins. Peptides based on the FGRA motif, but not scrambled controls, were able to inhibit the binding of appropriate phage clones or of Escherichia coli ss DNA-binding protein to oligos. This suggests that amino acid sequences that are capable of affecting biologically significant protein-DNA interactions can be identified from random peptide libraries using phage display techniques.

Amino Acid Sequence↗
PubMed1996

Identification of calmodulin-binding peptide consensus sequences from a phage-displayed random peptide library.

The calcium-binding protein, calmodulin (CaM), was used to screen a phage library displaying random peptides 26 amino acids (aa) in length. Twenty CaM-binding peptides were identified, 17 of which contained one of three consensus sequence motifs: + W-OlambdaR, WRAAV or WRXXAAAL, where +, -, O, lambda and X are positively charged, negatively charged, hydrophobic, leucine or valine, and any residue, respectively. The Trp residue in these motifs is located within 14 aa of the N-terminus of the displayed peptide. Previous studies [Dedman et al., J. Biol. Chem. 268 (1993) 23025-23030] using a library displaying random peptides 15 aa in length identified CaM-binding peptides which contained a Trp-Pro dipeptide motif. These results suggest that the type of CaM-binding motif identified can vary between different types of combinatorial peptides.

Amino Acid Sequence↗
PubMed1996

Distinct ligand preferences of Src homology 3 domains from Src, Yes, Abl, Cortactin, p53bp2, PLCgamma, Crk, and Grb2.

Src homology 3 (SH3) domains are conserved protein modules 50-70 amino acids long found in a variety of proteins with important roles in signal transduction. These domains have been shown to mediate protein-protein interactions by binding short proline-rich regions in ligand proteins. However, the ligand preferences of most SH3 domains and the role of these preferences in regulating SH3-mediated protein-protein interactions remain poorly defined. We have used a phage-displayed library of peptides of the form X6PXXPX6 to identify ligands for eight different SH3 domains. Using this approach, we have determined that each SH3 domain prefers peptide ligands with distinct sequence characteristics. Specifically, we have found that the Src SH3 domain selects peptides sharing the consensus motif LXXRPLPXpsiP, whereas Yes SH3 selects psiXXRPLPXLP, Abl SH3 selects PPXthetaXPPPpsiP, Cortactin SH3 selects +PPpsiPXKPXWL, p53bp2 SH3 selects RPXpsiPpsiR+SXP, PLCgamma SH3 selects PPVPPRPXXTL, Crk N-terminal SH3 selects psiPpsiLPpsiK, and Grb2 N-terminal SH3 selects +thetaDXPLPXLP (where psi, theta, and + represent aliphatic, aromatic, and basic residues, respectively). Furthermore, we have compared the binding of phage expressing peptides related to each consensus motif to a panel of 12 SH3 domains. Results from these experiments support the ligand preferences identified in the peptide library screen and evince the ability of SH3 domains to discern subtle differences in the primary structure of potential ligands. Finally, we have found that most known SH3-binding proteins contain proline-rich regions conforming to the ligand preferences of their respective SH3 targets.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Identification of cyclized calmodulin antagonists from a phage display random peptide library.

To isolate peptide ligands that bound calmodulin (CaM) specifically, we screened an M13 phage library displaying cyclized octamer random peptides with immobilized bovine CaM. Isolates were recovered, sequenced, and deduced to express nine independent peptides, five of which contained the sequence Trp-Gly-Lys (WGK). Four of the nine peptide sequences were synthesized in cyclized, biotinylated form. All of the peptides required Ca2+ to bind CaM. The cyclized, disulfide-bonded form of one such peptide, SCLRWGKWSNCGS, bound CaM better than its reduced form or an analogue in which the cysteine residues were replaced by serine. The cyclized peptide also exhibited the ability to inhibit CaM-dependent kinase activity. Systematic alanine substitution of residues in this peptide sequence implicate the tryptophan residue as being critical for binding, with other residues contributing to binding to varying degrees. Cloning of ligand targets (COLT) confirmed the specificity of one of the cyclized peptides, yielding full-length and C-terminal CaM clones, in addition to a full-length clone of troponin C, a CaM-related protein. This study has demonstrated that conformationally constrained peptides isolated from a phage library acted as specific, Ca(2+)-dependent CaM ligands.

Amino Acid Sequence↗
PubMed1996

Binding properties of SH3 peptide ligands identified from phage-displayed random peptide libraries.

Combinatorial libraries have yielded high-affinity ligands for SH3 domains of a number of different proteins. We have shown that synthetic peptides containing these SH3 ligand sequences serve as specific probes of SH3 domains. Direct binding of the N-terminal biotinylated peptide ligands was conveniently detected in ELISA, filter-blotting, and dot-blotting experiments with the use of streptavidin-conjugated enzymes. In some cases, detection of peptide-SH3 interactions required that the biotinylated peptides first were preconjugated with streptavidin to form a multivalent complex. Interestingly, these nominally tetravalent SH3 peptide ligands cross-react to varying degrees with different SH3 domains. We have used such complexes to screen lambda cDNA expression libraries and have isolated clones that encode both known and novel SH3-domain-containing proteins. Based on the success of this methodology, we propose a general strategy by which ligands of a modular domain-containing protein can be isolated from random peptide libraries and used to screen cDNA expression libraries systematically for novel modular domain-containing proteins.

Amino Acid Sequence↗
PubMed1996

Identification of epitope recognized by an anti-c-myc monoclonal antibody that cross-reacts with E. coli sigma factor using phage display libraries.

BACKGROUND: During the epitope mapping of monoclonal antibodies specific for myc proteins, two E. coli proteins cross-reactive with an anti-c-myc monoclonal antibody (MYC-X-5/1) were identified. One of the proteins is approximately 90 kDa and the other is over 150 kDa in apparent molecular mass. The molecular masses of these cross-reactive proteins suggested that they may be subunits of E. coli RNA polymerase. OBJECTIVES: We have investigated whether or not the proteins cross-reactive with MYC-X-5/1 are subunits of E. coli RNA polymerase. In addition, we have attempted to determine the epitope of MYC-X-5/1. STUDY DESIGN: The reactivity of MYC-X-5/1 antibody was tested against highly purified E. coli RNA polymerase holo-enzyme preparations and the cell lysate made from E. coli carrying a multi-copy plasmid with an insert of the rpoD gene, the structural gene for the E. coli sigma subunit. The epitope of MYC-X-5/1 was determined by use of phage display of random peptide libraries. RESULTS: On immunoblotting assays, MYC-X-5/1 reacted with the 90-kDa protein in the E. coli RNA polymerase preparations and with the 90-kDa protein over-expressed in E. coli carrying the plasmid with the rpoD insert. In addition, we have deduced the epitope of the MYC-X-5/1 antibody to be residues 235-245 of the human c-myc protein. A highly similar sequence to this was also identified in residues 62-72 of the sigma subunit of E. coli RNA polymerase. CONCLUSION: These data demonstrated that the 90-kDa protein cross-reactive with MYC-X-5/1 is the sigma subunit of E. coli RNA polymerase. Furthermore, this study shows that random peptide libraries displayed on filamentous phage are useful tools for epitope mapping and defining cross-reactivities of monoclonal antibodies.

Amino Acid Sequence↗
PubMed1996

Cloning of ligand targets: systematic isolation of SH3 domain-containing proteins.

Based on the prevalence of modular protein domains, such as Src homology domain 3 and 2 (SH3 and SH2), among important signaling molecules, we have sought to identify new SH3 domain-containing proteins. However, modest sequence similarity among these domains restricts the use of DNA-based methods for this purpose. To circumvent this limitation, we have developed a functional screen that permits the rapid cloning of modular domains based on their ligand-binding activity. Using operationally defined SH3 ligands from combinatorial peptide libraries, we screened a series of mouse and human cDNA expression libraries. We found that 69 of the 74 clones isolated encode at least one SH3 domain. These clones encode 18 different SH3-containing proteins, 10 of which have not been described previously. The isolation of entire repertoires of modular domain-containing proteins will prove invaluable in genome analysis and in bringing new targets into drug discovery programs.

Amino Acid Sequence↗
PubMed1995

Characterization of phage that bind plastic from phage-displayed random peptide libraries.

During routine screenings of random peptide libraries displayed at the N terminus of the pIII coat protein of M13 bacteriophage, clones were isolated that bound directly to the polystyrene (PS) surface used to immobilize the target protein. The plastic-binding phage (P-b phi) bind to both unblocked plastic (PS and polyvinyl chloride, PVC) and plastic blocked with bovine serum albumin (BSA) but require non-ionic detergent to bind to plastic blocked with milk. Comparison of the P-b phi to antibody-binding phage (Ab-b phi) indicates that similar numbers of phage particles are bound, but fewer P-b phi the recovered by acid elution. Sequence determination of the displayed peptides reveals they lack amino-acid sequence similarity yet are highly enriched for the Tyr and Trp residues. However, because not all phage that display peptides rich in Tyr and Trp residues bind to plastic, and other methods of screening random peptide libraries have identified different classes of plastic-binding peptides, the relative abundance of Tyr and Trp residues should not be considered diagnostic of plastic-binding. In summary, these results help characterize one of the most common methods used to screen random peptide libraries and suggest strategies to avoid isolating P-b phi. Furthermore, while it is generally believed that proteins bind to plastic by non-specific interactions, these results show that a bias in aa composition can exist.

Amino Acid Sequence↗
PubMed1995

Molecular basis of human cardiac troponin T isoforms expressed in the developing, adult, and failing heart.

Cardiac troponin T (cTnT), a protein essential for calcium-regulated myofibrillar ATPase activity, is expressed in the human heart as four isoforms (cTnT1 through cTnT4, numbered in the order of decreasing molecular size). The expression of these isoforms at the protein level has previously been found by us to differ in the normal and failing adult and fetal human heart. In the present study, we have cloned and sequenced four full-length cDNAs corresponding to the four native cTnT protein isoforms and have expressed these cDNAs in an in vitro transcription and translation system. The cDNAs differ by the variable inclusion of a 15- and a 30-nt exon in the 5' half of the coding region. These cDNAs yielded proteins that comigrate with the native isoforms, cTnT1 through cTnT4. Polyclonal antisera, raised against a synthetic peptide corresponding to the 10-residue peptide encoded by the 30-nt exon, reacted with the two human isoforms largest in molecular size (cTnT1 and cTnT2) and the two largest cTnT isoforms of the rabbit and rat. The isoforms cTnT1 and cTnT2, containing either both peptides encoded by the 30- and 15-nt exons or the peptide encoded by the 30-nt exon alone, are expressed in the fetal heart, with cTnT2 being expressed at a very low level. cTnT4, lacking both of these sequences, is expressed in the fetal heart and is reexpressed in the failing adult heart, whereas cTnT3, containing the 5-residue peptide, is the dominant isoform in the adult heart.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1995

Formation and metabolism of histone mRNAs with mutant 3' ends formed by snRNA termination signals.

Histone mRNAs are the only non-polyadenylated mRNAs, ending in a conserved 26 nt sequence which can form a stem-loop. It has not been possible to make histone mRNAs with mutant stem-loops since most mutations in the stem-loop interfere with the 3' processing reaction. The snRNA genes transcribed by RNA polymerase II form their 3' ends by transcription termination directed by a signal which is located entirely 3' of the snRNA coding sequence. Chimeric genes which express RNAs ending in a histone 3' end or in mutant histone 3' ends formed by snRNA termination signals were constructed. The mRNAs from these genes were efficiently transported from the nucleus after injection of the genes into frog oocytes. This was true even for RNAs which end in mutant stem-loops suggesting that the snRNA termination signals promote transport of the transcripts.

Animals↗
PubMed1994

Changes in the stem-loop at the 3' terminus of histone mRNA affects its nucleocytoplasmic transport and cytoplasmic regulation.

The stem-loop structure at the 3' end of replication-dependent histone mRNA is required for efficient pre-mRNA processing, localization of histone mRNA to the polyribosomes, and regulation of histone mRNA degradation. A protein, the stem-loop binding protein (SLBP), binds the 3' end of histone mRNA and is thought to mediate some or all of these processes. A mutant histone mRNA with two nucleotide changes in the loop was constructed and found to be transported inefficiently to the cytoplasm. The mutant histone mRNA, unlike the wild-type histone mRNA, was not rapidly degraded when DNA synthesis is inhibited, and was not stabilized upon inhibition of protein synthesis. The stem-loop binding protein (SLBP) has between a 20-50 fold greater affinity for the wild type histone stem-loop structure than for the mutant stem-loop structure, suggesting that the alteration in the efficiency of transport and the normal degradation pathway in histone mRNA may be due to the reduced affinity of the mutant stem-loop for the SLBP.

Animals↗