title: Peptide microarray analysis of substrate specificity of the transmembrane Ser/Thr kinase KPI-2 reveals reactivity with cystic fibrosis transmembrane conductance regulator and phosphorylase.
abstract: Human lemur (Lmr) kinases are predicted to be Tyr kinases based on sequences and are related to neurotrophin receptor Trk kinases. This study used homogeneous recombinant KPI-2 (Lmr2, LMTK2, Cprk, brain-enriched protein kinase) kinase domain and a library of 1,154 peptides on a microarray to analyze substrate specificity. We found that KPI-2 is strictly a Ser/Thr kinase that reacts with Ser either preceded by or followed by Pro residues but unlike other Pro-directed kinases does not strictly require an adjacent Pro residue. The most reactive peptide in the library corresponds to Ser-737 of cystic fibrosis transmembrane conductance regulator, and the recombinant R domain of cystic fibrosis transmembrane conductance regulator was a preferred substrate. Furthermore the KPI-2 kinase phosphorylated peptides corresponding to the single site in phosphorylase and purified phosphorylase b, making this only the second known phosphorylase b kinase. Phosphorylase was used as a specific substrate to show that KPI-2 is inhibited in living cells by addition of nerve growth factor or serum. The results demonstrate the utility of the peptide library to probe specificity and discover kinase substrates and offer a specific assay that reveals hormonal regulation of the activity of this unusual transmembrane kinase.
authors.affiliation: Center for Cell Signaling, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.
authors.forename: Hong
authors.initials: H
authors.lastname: Wang
authors.forename: David L
authors.initials: DL
authors.lastname: Brautigan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000032:analysis*; D006005:Phosphorylases / Q000378:metabolism*; D040081:Protein Array Analysis / Q000379:methods*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D008565:Membrane Proteins; D019151:Peptide Library; D010455:Peptides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D006005:Phosphorylases; C470798:LMTK2 protein, human; D017346:Protein Serine-Threonine Kinases