The catalytic subunit of Dictyostelium cAMP-dependent protein kinase -- role of the N-terminal domain and of the C-terminal residues in catalytic activity and stability.
The C subunit of Dictyostelium cAMP-dependent protein kinase (PKA) is unusually large (73 kDa) due to the presence of 330 amino acids N-terminal to the conserved catalytic core. The sequence following the core, including a C-terminal -Phe-Xaa-Xaa-Phe-COOH motif, is highly conserved. We have characterized the catalytic activity and stability of C subunits mutated in sequences outside the catalytic core and we have analyzed their ability to interact with the R subunit and with the heat-stable protein-kinase inhibitor PKI. Mutants carrying deletions in the N-terminal domain displayed little difference in their kinetic properties and retained their capacity to be inhibited by R subunit and by PKI. In contrast, the mutation of one or both of the phenylalanine residues in the C-terminal motif resulted in a decrease of catalytic activity and stability of the proteins. Inhibition by the R subunit or by PKI were however unaffected. Sequence-comparison analysis of other protein kinases revealed that a -Phe-Xaa-Xaa-Phe- motif is present in many Ser/Thr protein kinases, although its location at the very end of the polypeptide is a particular feature of the PKA family. We propose that the presence of this motif may serve to identify isoforms of protein kinases.
Etchebehere
L C
LC
Unité de Régulation Enzymatique des Activités Cellulaires, CNRS UMR 321, Institut Pasteur, Paris, France.
Van Bemmelen
M X
MX
Anjard
C
C
Traincard
F
F
Assemat
K
K
Reymond
C
C
Véron
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004023:Dictyostelium / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006358:Hot Temperature; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D014508:Urea
D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D011994:Recombinant Proteins; C015482:protein kinase modulator; D014508:Urea; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases