Andrew B. Sparks

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 Cγ. 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.

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.

CHAPTER 13 – Screening Phage-Displayed Random Peptide Libraries

This chapter describes screening phage-displayed random peptide libraries. Screening of phage-displayed random peptide libraries represents a powerful means of identifying peptide ligands for targets of interest. Fundamentally, the screening of phage-displayed random peptide libraries for binding peptides includes the steps such as obtaining a suitable library and source of target, and incubating the library with target and capturing binding phage. It is found that in addition to being used as a vector for accessible expression of peptide libraries, M 13 has been used to display mutant libraries of entire proteins, including antibodies and growth hormone. To minimize problems arising from instability of phage-displayed peptides, phage should be protected from proteolytic degradation. Displayed peptides are susceptible to degradation during phage propagation in a bacterial culture. Elution of phage from the target:phage complex is an important aspect of screening phage-displayed random peptide libraries. One round of affinity purification typically affects the recovery of approximately 1% of PFU representing any given binding clone. The number of phage particles representing any given binding clone that are recovered from the first round is reduced to the point that binding clones may be lost if subjected to a second round of purification without intervening amplification. It is found that enrichment of binding phage may be assessed by doping a library aliquot with an equal number of nonbinding phage which is distinguishable from library phage.

Construction of Random Peptide Libraries in Bacteriophage M13

This chapter discusses construction of random peptide libraries in bacteriophage M I3. Bacteriophage M 13 has been adapted for the expression of diverse populations of peptides in a manner that affords the rapid purification of active peptides by affinity selection. A variety of strategies have been used to produce clonable degenerate DNA fragments that are appropriate for encoding random peptides. Synthesis of long oligonucleotides often results in a crude product containing a large fraction of contaminating n-I and smaller products. In addition to preparing an adequate quantity of vector DNA, steps should be taken to limit the number of parental clones among the recombinants in the library. Several strategies have been employed to minimize the recovery of parental clones by vector reclosure, including the use of two different restriction enzymes that produce noncompatible cohesive ends, the treatment of digested vector with alkaline phosphatase, and the gel purification of digested vector from undigested vector and parental insert fragments. Electroporation produces high efficiencies of transformation by subjecting a cell/DNA mixture to a brief but intense electrical field of exponential decay. It is suggested that because of the strong electrical fields used in this procedure, the cell/DNA mixture must be free of salt to avoid arcing during electroporation.

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

SummaryCombinatorial 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 λcDNA 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.

CHAPTER 4 – Microbiological Methods

This chapter discusses various aspects of microbiological methods. Most aspects of phage display require the use of basic microbiological methods. Heat sterilizes a platinum inoculating loop and it is cooled by waving the loop in the air or plunging it into sterile culture medium. The glass spreader can be sterilized by dipping it into a beaker containing 95% ethanol and then holding it in the flame of a Bunsen burner to ignite the ethanol. In some cases, lacZ carrying viral or phagemid vectors can be followed by monitoring the expression of 13-galactosidase activity. The number of plates should be equal to the number of dilutions that will be tested, although a few extra plates can be added to the incubator in order to anticipate any ad hoc increases in the size of the experiment. For many phage-display experiments, phagemid vectors are used in place of phage. One important difference between the two vector systems is that quantitation of the number of phage particles is done by monitoring their ability to form colonies in the presence of an antibiotic. In some cases, it is advantageous to purify phage from the polyethylene glycol using CsCI equilibrium gradients. It is suggested that trace amounts of PEG can interfere with the binding of phage to various targets.

CHAPTER 5 – Construction of Random Peptide Libraries in Bacteriophage M13

This chapter discusses construction of random peptide libraries in bacteriophage M I3. Bacteriophage M 13 has been adapted for the expression of diverse populations of peptides in a manner that affords the rapid purification of active peptides by affinity selection. A variety of strategies have been used to produce clonable degenerate DNA fragments that are appropriate for encoding random peptides. Synthesis of long oligonucleotides often results in a crude product containing a large fraction of contaminating n-I and smaller products. In addition to preparing an adequate quantity of vector DNA, steps should be taken to limit the number of parental clones among the recombinants in the library. Several strategies have been employed to minimize the recovery of parental clones by vector reclosure, including the use of two different restriction enzymes that produce noncompatible cohesive ends, the treatment of digested vector with alkaline phosphatase, and the gel purification of digested vector from undigested vector and parental insert fragments. Electroporation produces high efficiencies of transformation by subjecting a cell/DNA mixture to a brief but intense electrical field of exponential decay. It is suggested that because of the strong electrical fields used in this procedure, the cell/DNA mixture must be free of salt to avoid arcing during electroporation.

Chapter 5 - Construction of Random Peptide Libraries in Bacteriophage MI3

This chapter discusses construction of random peptide libraries in bacteriophage M I3. Bacteriophage M 13 has been adapted for the expression of diverse populations of peptides in a manner that affords the rapid purification of active peptides by affinity selection. A variety of strategies have been used to produce clonable degenerate DNA fragments that are appropriate for encoding random peptides. Synthesis of long oligonucleotides often results in a crude product containing a large fraction of contaminating n-I and smaller products. In addition to preparing an adequate quantity of vector DNA, steps should be taken to limit the number of parental clones among the recombinants in the library. Several strategies have been employed to minimize the recovery of parental clones by vector reclosure, including the use of two different restriction enzymes that produce noncompatible cohesive ends, the treatment of digested vector with alkaline phosphatase, and the gel purification of digested vector from undigested vector and parental insert fragments. Electroporation produces high efficiencies of transformation by subjecting a cell/DNA mixture to a brief but intense electrical field of exponential decay. It is suggested that because of the strong electrical fields used in this procedure, the cell/DNA mixture must be free of salt to avoid arcing during electroporation.