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.
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Research Article|
July 27 2011
Discovery of cellular substrates for protein kinase A using a peptide array screening protocol
F. Donelson Smith;
F. Donelson Smith
1
1Correspondence may be addressed to either of these authors (email smithdon@uw.edu or scottjdw@uw.edu).
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Bret K. Samelson;
Bret K. Samelson
1Howard Hughes Medical Institute, Department of Pharmacology, University of Washington School of Medicine, 1959 Pacific Avenue NE, Seattle, WA 98195, U.S.A.
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John D. Scott
John D. Scott
1
1Howard Hughes Medical Institute, Department of Pharmacology, University of Washington School of Medicine, 1959 Pacific Avenue NE, Seattle, WA 98195, U.S.A.
1Correspondence may be addressed to either of these authors (email smithdon@uw.edu or scottjdw@uw.edu).
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Publisher: Portland Press Ltd
Received:
April 20 2011
Revision Received:
June 03 2011
Accepted:
June 07 2011
Accepted Manuscript online:
June 07 2011
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2011 Biochemical Society
2011
Biochem J (2011) 438 (1): 103–110.
Article history
Received:
April 20 2011
Revision Received:
June 03 2011
Accepted:
June 07 2011
Accepted Manuscript online:
June 07 2011
Citation
F. Donelson Smith, Bret K. Samelson, John D. Scott; Discovery of cellular substrates for protein kinase A using a peptide array screening protocol. Biochem J 15 August 2011; 438 (1): 103–110. doi: https://doi.org/10.1042/BJ20110720
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