Substrate and inhibitor recognition of protein kinases: what is known about the catalytic subunit of phosphorylase kinase?
Although much can be learned about the specificity of protein kinases from studies with peptide substrates, the question remains, how do kinases recognize their three-dimensional protein substrates? Information derived from such studies provides further understanding of substrate recognition and can facilitate the design of specific protein kinase inhibitors. Phosphorylase kinase (PhK) catalyzes the phosphorylation of phosphorylase b (phos. b) to form the active phosphorylase a. No other protein kinase can duplicate this reaction. Why? To probe this question and establish what features in the protein are important for substrate binding and product release, mutants of phos. b have been studied. This report shows how mutations change the properties of the protein substrate and the ability of these mutants to be phosphorylated by PhK and other kinases. Action of protein kinases on their substrates is often regulated by autoinhibitory segments. The C-terminus of the catalytic gamma-subunit of PhK contains two inhibitory sites overlapping two calmodulin-binding regions. These two peptide segments resemble sequences in phos. b and may explain why peptides of these regions are potent inhibitors of PhK. We will show results with peptide inhibitors, using various expressed forms of the catalytic subunit, which describe their modes of interaction and mechanisms of inhibition. Metal ions can change molecular interactions. With PhK, Mn2+ facilitates the use of GTP as a phosphoryl group donor and greatly increases phosphorylation of a tyrosine residue in angiotensin II. This implies that the spatial arrangement of specificity determinants can be manipulated so that PhK can utilize other substrates.
Graves
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Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames 50011, USA.
Bartleson
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C
Biorn
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A
Pete
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M
D000818:Animals; D020134:Catalytic Domain / Q000502:physiology; D005544:Forecasting; D006801:Humans; D008345:Manganese / Q000737:chemistry; D009154:Mutation / Q000502:physiology*; D010764:Phosphorylase Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D006005:Phosphorylases / Q000378:metabolism*; D047428:Protein Kinase Inhibitors
D047428:Protein Kinase Inhibitors; D008345:Manganese; D006005:Phosphorylases; D010764:Phosphorylase Kinase