Discovery of cellular substrates for protein kinase A using a peptide array screening protocol.

NIH Public Access Author Manuscript Biochem J. Author manuscript; available in PMC 2012 March 2. NIH-PA Author Manuscript Published in final edited form as: Biochem J. 2011 August 15; 438(1): 103–110. doi:10.1042/BJ20110720. Discovery of cellular substrates for protein kinase A using a peptide array screening protocol F. Donelson SMITH1, Bret K. SAMELSON, and John D. SCOTT1 Howard Hughes Medical Institute, Department of Pharmacology, University of Washington School of Medicine, 1959 Pacific Avenue NE, Seattle, WA 98195, U.S.A. Abstract NIH-PA Author Manuscript 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 Ser119 inside cells and that this mode of regulation may control its ability to affect cell growth. Keywords kinase; peptide array; phosphoproteome; protein kinase A (PKA) NIH-PA Author Manuscript INTRODUCTION Protein kinases regulate all aspects of cellular behaviour. These key signal transduction enzymes act by phosphorylating target sequences in substrates. This results in modulation of protein function or stability, alterations in association with other proteins, or the movement from one cellular compartment to another [1]. The evolutionary advantage of protein phosphorylation as a versatile and simple form of post-translational modification is supported by evidence that protein kinase genes represent approximately 2 % of most vertebrate genomes. The human ‘kinome’ contains nearly 500 protein kinases, most of which target serine/threonine or tyrosine residues [2]. Historically, one of the first protein © The Authors 1 Correspondence may be addressed to either of these authors ( or ). AUTHOR CONTRIBUTION Donelson Smith, Bret Samelson and John Scott designed the experiments. Donelson Smith and Bret Samelson performed the experiments. Donelson Smith and John Scott wrote the paper. SMITH et al. Page 2 NIH-PA Author Manuscript kinases to be investigated at the molecular level was PKA (cAMP-dependent protein kinase/ protein kinase A). Peptide-based elucidation of the substrate specificity and X-ray crystallography of the catalytic subunit of PKA have provided a framework for understanding the molecular and cellular roles of this abundant enzyme [3]. Although some enzymes such as RAF and MEK [MAPK (mitogen-activated protein kinase)/ ERK (extracellular-signal-regulated kinase) kinase] exhibit exquisite substrate specificities and generally phosphorylate single substrates, most protein kinases are promiscuous and modify a broad spectrum of proteins. Furthermore, many kinases share overlapping substrate specificity. Thus the same residues can be targeted by multiple kinases in vivo. For example, PKA phosphorylates sites that conform loosely to serine or threonine residues found just distal to basic residues. However, there are a number of other basophilic kinases that target similar sites, including Akt, PAK (p21-activated kinase) and RSK (ribosomal S6 kinase). Therefore a major challenge in deciphering the kinome is determining substrate specificity for particular kinases and identifying targets that may mediate the physiological effects of these enzymes. NIH-PA Author Manuscript Several technologies are currently available to facilitate the identification of phosphorylation sites in proteins [4]. In addition to advances in MS that have allowed optimized proteomelevel characterization of phosphorylation events, peptide and protein arrays have become popular for determining substrate preferences and finding new potential target proteins [5,6]. Finally, phospho-specific antibodies directed against phosphorylation site motifs or individual phosphorylation sites are powerful tools for examining phosphorylation events [7,8]. In the present paper we describe a strategy for protein kinase substrate identification that combines solid-phase phosphorylation and blotting with phospho-specific motif-selective antibodies. These studies led us to further characterize phosphorylation sites in several proteins, including the PDZ-LIM protein RIL [encoded by PDLIM4 (PDZ and LIM domain protein 4)]. RIL is known to be associated with the actin cytoskeleton. RIL is phosphorylated on Ser119, just distal to the PDZ domain, both in vitro and inside cells. Finally, expression of a phosphorylation-site mutant of RIL in PC-3 prostate adenocarcinoma cells increases cell growth as compared with wild-type RIL. MATERIALS AND METHODS Reagents and antibodies NIH-PA Author Manuscript Reagents for SPOT synthesis were purchased from Intavis. The polyclonal anti-phosphoPKA substrate antibody was from Cell Signaling Technology. The anti-phospho-RIL antibody was generated in rabbits against the peptide PATSRRS[pS]ISGISLE. Other chemicals and reagents were from Sigma–Aldrich, EMD Biosciences or New England Biolabs. Database searching A number of different motif scanners available online were used to identify R-X-X-S/T motifs, including Scansite (http://scansite.mit.edu/) [9], eMotif (http://motif.stanford.edu/distributions/emotif/index.html) [10] and GenomeNet Motif (http://www.genome.jp/tools/motif/). Searches were performed using several different motifs that were variations on the basic R/K-R/K-X-S/T PKA phosphorylation-site motif. Among these were the following: R-X-R-R-X-S-Φ (where Φ is a hydrophobic residue), RR-X-S-Φ and R-R-X-S/T. When using the Scansite motif scanner, we used both the PKA substrate motif residing in the program as well as the feature for creating new input motifs for uses by the scanner. Typically results from each database largely overlapped, although Biochem J. Author manuscript; available in PMC 2 (...truncated)