A novel activating effect of the regulatory subunit of protein kinase A on catalytic subunit activity.
The strength of the interaction between the catalytic and regulatory subunits in protein kinase A differs among species. The linker region from regulatory subunits is non-conserved. To evaluate the participation of this region in the interaction with the catalytic subunit, we have assayed its effect on the enzymatic properties of the catalytic subunit. Protein kinase A from three fungi, Mucor rouxii, Mucor circinelloides and Saccharomyces cerevisiae have been chosen as models. The R-C interaction is explored by using synthetic peptides of 8, 18 and 47 amino acids, corresponding to the R subunit autophosphorylation site plus a variable region toward the N terminus (0, 10, or 39 residues). The K(m) of the catalytic subunits decreased with the length of the peptide, while the V(max) increased. Viscosity studies identified product release as the rate limiting step for phosphorylation of the longer peptides. Pseudosubstrate derivatives of the 18 residue peptides did not display a competitive inhibition behavior toward either kemptide or a bona fide protein substrate since, at low relative pseudosubstrate/substrate concentration, stimulation of kemptide or protein substrate phosphorylation was observed. The behavior was mimicked by intact R. We conclude that in addition to its negative regulatory role, the R subunit stimulates C activity via distal interactions.
Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, Piso 4, 1428 Buenos Aires, Argentina.
Jimena
J
Rinaldi
Josefina
J
Ocampo
Silvia
S
Rossi
Silvia
S
Moreno
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D002384:Catalysis; D020134:Catalytic Domain; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation; D015966:Gene Expression Regulation, Fungal; D007700:Kinetics; D008969:Molecular Sequence Data; D009089:Mucor / Q000201:enzymology* / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D000596:Amino Acids; D010455:Peptides; D017868:Cyclic AMP-Dependent Protein Kinases