title: Discovery of cellular substrates for protein kinase A using a peptide array screening protocol.
abstract: Post-translational modification of proteins is a universal form of cellular regulation. Phosphorylation on serine, threonine, tyrosine or histidine residues by protein kinases is the most widespread and versatile form of covalent modification. Resultant changes in activity, localization or stability of phosphoproteins drives cellular events. MS and bioinformatic analyses estimate that ~30% of intracellular proteins are phosphorylated at any given time. Multiple approaches have been developed to systematically define targets of protein kinases; however, it is likely that we have yet to catalogue the full complement of the phosphoproteome. The amino acids that surround a phosphoacceptor site are substrate determinants for protein kinases. For example, basophilic enzymes such as PKA (protein kinase A), protein kinase C and calmodulin-dependent kinases recognize basic side chains preceding the target serine or threonine residues. In the present paper we describe a strategy using peptide arrays and motif-specific antibodies to identify and characterize previously unrecognized substrate sequences for protein kinase A. We found that the protein kinases PKD (protein kinase D) and MARK3 [MAP (microtubule-associated protein)-regulating kinase 3] can both be phosphorylated by PKA. Furthermore, we show that the adapter protein RIL [a product of PDLIM4 (PDZ and LIM domain protein 4)] is a PKA substrate that is phosphorylated on Ser(119) inside cells and that this mode of regulation may control its ability to affect cell growth.
authors.affiliation: Howard Hughes Medical Institute, Department of Pharmacology, University of Washington School of Medicine, 1959 Pacific Avenue NE, Seattle, WA 98195, USA. smithdon@uw.edu
authors.forename: F Donelson
authors.initials: FD
authors.lastname: Smith
authors.forename: Bret K
authors.initials: BK
authors.lastname: Samelson
authors.forename: John D
authors.initials: JD
authors.lastname: Scott
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D015153:Blotting, Western; D049109:Cell Proliferation; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D060588:LIM Domain Proteins; D008297:Male; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011471:Prostatic Neoplasms / Q000378:metabolism* / Q000473:pathology; D040081:Protein Array Analysis; D011493:Protein Kinase C / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012694:Serine; D013379:Substrate Specificity; D013912:Threonine; D014407:Tumor Cells, Cultured
chemical_list: D004268:DNA-Binding Proteins; D060588:LIM Domain Proteins; C486247:PDLIM4 protein, human; D013912:Threonine; D012694:Serine; C111833:MARK3 protein, human; C089212:protein kinase D; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C