Investigation of the catalytic and regulatory properties of cardiac muscle protein kinase will help to illuminate the nature of the molecular interaction of cyclic AMP with isolated enzymes. Becuase cyclic AMP appears to affect the activity of many enzymes and because it may be the key intermediate in the mechanisms of action of numerous hormones, the potential importance of understanding in depth the chemistry underlying the catalytic action and regulatory behavior of cyclic AMP-activated enzymes like protein kinase may be very great. Our work on cyclic AMP-dependent protein kinase has the following specific aims for the next project period: (a) identification of curcial functional groups in the catalytic site; (b) further elucidation of the process by which the gamma-phosphoryl group of ATP is transferred to a peptide substrate; (c) determination of the conformation of peptide substrates bound to the catalytic subunit; and (d) determination of the environment at the sites of binding of cyclic AMP to the type II regulatory subunit and of the nature of the interactions between the regulatory and catalytic subunits. Additionally, we propose to extend our investigations of protein kinase action to the cyclic GMP-dependent enzyme. The approaches to be used are similar to those being successfully employed with cyclic AMP-dependent protein kinase and should lead to the formulation of a mechanism of action for the cyclic GMP-dependent enzyme.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM032204-03
Application #
3280833
Study Section
Biochemistry Study Section (BIO)
Project Start
1983-01-01
Project End
1985-12-31
Budget Start
1985-01-01
Budget End
1985-12-31
Support Year
3
Fiscal Year
1985
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