title: Identification and characterization of peptide probes directed against PKCalpha conformations.
abstract: Phage display is a powerful technology that allows identification of high affinity peptides that bind specifically to a given molecular target. Using a highly complex peptide display library, we have identified separate classes of peptides that bind to protein kinase C alpha (PKCalpha) only under activation conditions. Furthermore, peptide binding was specific to PKCalpha and not to any of the other closely related PKC isoforms. The conformational and isoform specificity of the peptide binding was demonstrated using surface plasmon resonance as well as time-resolved fluorescence assays. Kinase assays showed that these peptides were not direct substrates for PKC nor did they inhibit phosphorylation of PKC substrates. These peptides are most likely directed against protein-protein interaction sites on PKC. The data presented here offers another example of application of phage display technology to identify conformation-dependent peptide probes against therapeutically important drug targets. These peptides are ideally suited to be used as surrogate ligands to identify compounds that bind specifically to PKCalpha, as well as conformational probes to detect activated forms of PKCalpha.
authors.affiliation: Karo Bio USA Inc., 4222 Emperor Blvd, Ste 560, Durham, NC 27703, USA. salman.ashraf@karobio.com
authors.forename: S S
authors.initials: SS
authors.lastname: Ashraf
authors.forename: E
authors.initials: E
authors.lastname: Anderson
authors.forename: K
authors.initials: K
authors.lastname: Duke
authors.forename: P T
authors.initials: PT
authors.lastname: Hamilton
authors.forename: Z
authors.initials: Z
authors.lastname: Fredericks
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D015335:Molecular Probes / Q000737:chemistry / Q000378:metabolism; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011493:Protein Kinase C / Q000737:chemistry* / Q000378:metabolism; D051571:Protein Kinase C-alpha
chemical_list: D007527:Isoenzymes; D015335:Molecular Probes; D019151:Peptide Library; D010455:Peptides; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha