title: Development of a peptidase-resistant substrate for single-cell measurement of protein kinase B activation.
abstract: An iterative design strategy using three criteria was utilized to develop a peptidase-resistant substrate peptide for protein kinase B. Libraries of peptides possessing non-native amino acids were screened for time to 50% phosphorylation, degradation half-life within a lysate, and appearance of a dominant fragment. The lead peptide possessed a half-life of 92 ± 7 and 16 ± 2 min in HeLa and LNCaP cytosolic lysates, respectively, representing a 4.6- and 2.7-fold lifetime improvement over that of the starting peptide. The redesigned peptide possessed a 4.5-fold improvement in phosphorylation efficiency compared to the starting peptide. The same peptide fragments were formed when the lead peptide was incubated in a lysate or loaded into single cells although the fragments formed in significantly different ratios suggesting that distinct peptidases metabolized the peptide in the two preparations. The rate of peptide degradation and phosphorylation was on average 0.1 ± 0.2 zmol pg(-1) s(-1) and 0.04 ± 0.08 zmol pg(-1) s(-1), respectively, for single LNCaP cells loaded with 4 ± 8 μM of peptide. Peptidase-resistant kinase substrates should find widespread utility in both lysate-based and single-cell assays of kinase activity.
authors.affiliation: Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
authors.forename: Angela
authors.initials: A
authors.lastname: Proctor
authors.forename: Qunzhao
authors.initials: Q
authors.lastname: Wang
authors.forename: David S
authors.initials: DS
authors.lastname: Lawrence
authors.forename: Nancy L
authors.initials: NL
authors.lastname: Allbritton
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001120:Arginine; D003600:Cytosol / Q000378:metabolism; D015195:Drug Design; D004789:Enzyme Activation / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D010447:Peptide Hydrolases / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D059748:Proteolysis; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism*; D059010:Single-Cell Analysis
chemical_list: D009842:Oligopeptides; D047428:Protein Kinase Inhibitors; D001120:Arginine; D051057:Proto-Oncogene Proteins c-akt; D010447:Peptide Hydrolases