Protein kinases are key enzymes in cellular regulation: cyclic AMP-dependent kinases mediate hormone actin; the isulin and epidermal growth factor receptors also possess kinase activity; Rous-sarcoma virus induced cellular transformation requires a tyrosine protein kinase; and protein kinases are thought to regulate pathways involved in cellular differentiation. Our ultimate goal is to develop methodologies for the specific inhibition of the various protein kinases which will allow unprecedented control of cellular function. Through our own efforts and those of other laboratories the substrate specificity of the cAMP-dependent protein kinase has been defined. We have used this knowledge to develop the first peptide based affinity label for this enzyme and are proposing to develop new affinity labels, photoaffinity labels, and tight binding competitive inhibitors for the kinase. Recently, it was discovered in our laboratory that cAMP-dependent protein kinase will not phosphorylate peptide substrates containing certain N-methylated amide bonds. We plan to determine whether cGMP-dependent protein kinase, which has very similar substrate specificity requirements to the cAMP-dependent enzyme, might differ in these specific peptide substrate requirements. This would mean that inhibitors could be adapted to interact exclusively with one of the two kinases. We intend to continue to investigate the chemistry underlying the mechanism of the catalytic action of cAMP-dependent protein kinase. Those active site residues which are modified by affinity labels or photoaffinity labels will be identified. Additionally, NMR studies in collaboration with Prof. A. S. Mildvan focussed on defining the steric relationship between enzyme bound functional groups and substrates will be performed, as will experiments in collaboration with Prof. J. R. Knowles on the stereochemistry of protein kinase catalyzed phosphoryl group transfer.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM032204-06
Application #
3280835
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1983-01-01
Project End
1989-01-31
Budget Start
1988-07-01
Budget End
1989-01-31
Support Year
6
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Rockefeller University
Department
Type
Graduate Schools
DUNS #
071037113
City
New York
State
NY
Country
United States
Zip Code
10065
Mobashery, S; Doughty, M; Kaiser, E T (1990) Inactivation of the catalytic subunit of bovine cAMP-dependent protein kinase by a peptide-based affinity inactivator. Biopolymers 29:131-8
Radziejewski, C; Miller, W T; Mobashery, S et al. (1989) Purification of recombinant pp60v-src protein tyrosine kinase and phosphorylation of peptides with different secondary structure preference. Biochemistry 28:9047-52
Miller, W T; Kaiser, E T (1988) Probing the peptide binding site of the cAMP-dependent protein kinase by using a peptide-based photoaffinity label. Proc Natl Acad Sci U S A 85:5429-33
Mobashery, S; Kaiser, E T (1988) Identification of amino acid residues involved in substrate recognition by the catalytic subunit of bovine cyclic AMP dependent protein kinase: peptide-based affinity labels. Biochemistry 27:3691-6
Bramson, H N; Thomas, N E; Miller, W T et al. (1987) Conformation of Leu-Arg-Arg-Ala-Ser-Leu-Gly bound in the active site of adenosine cyclic 3',5'-phosphate dependent protein kinase. Biochemistry 26:4666-70
Thomas, N E; Bramson, H N; Miller, W T et al. (1987) Role of enzyme-peptide substrate backbone hydrogen bonding in determining protein kinase substrate specificities. Biochemistry 26:4461-6
Kaiser, E T (1987) Studies on the mechanism of action of phosphoryl transferase enzymes. Biochem Soc Trans 15:1187-8
Thomas, N E; Bramson, H N; Nairn, A C et al. (1987) Distinguishing among protein kinases by substrate specificities. Biochemistry 26:4471-4
Mildvan, A S; Rosevear, P R; Fry, D C et al. (1985) NMR studies of the mechanism of action and regulation of protein kinase. Curr Top Cell Regul 27:133-44
Bramson, H N; Thomas, N E; Kaiser, E T (1985) The use of N-methylated peptides and depsipeptides to probe the binding of heptapeptide substrates to cAMP-dependent protein kinase. J Biol Chem 260:15452-7