Functional malleability of the carboxyl-terminal tail in protein kinase A.
The catalytic (C) subunit of protein kinase A (PKA) is regarded as a framework for the protein kinase family. Its sequence is composed of a conserved core (residues 40 300) between two segments at the amino and carboxyl termini of the protein. Since the various protein kinases differ in their specificity, it seems reasonable to assume that these nonhomologous segments may be involved in endowing each kinase with its individual specificity. Here we present data to show that the cluster of acidic amino acids (328DDYEEEE334) at the carboxyl-terminal "tail" of the C subunit, specifically Tyr330, contributes to its substrate recognition. This is based on three complementary lines of evidence: (i) on a conformation-sensitive cleavage of the C subunit by a kinase-splitting membranal proteinase that specifically recognizes this cluster, to demonstrate the occurrence in solution of "open" (cleavable) and "closed" (noncleavable) conformations of the C subunit; (ii) on analysis of the three-dimensional structures of the open and closed conformations of the C subunit, showing an approximately 7-A movement of the phenolic hydroxyl of Tyr330 to reach (in the closed conformation) an approximately 3-A distance from the nitrogen atoms of the Arg residue at position p-3 of the PKA consensus sequence; and (iii) on single-site mutations of the C subunit (e.g. Y330A) that show a significant contribution of Tyr330 to the Km of PKA for its substrates/inhibitors and to its catalytic efficacy (Vmax/Km).
Chestukhin
A
A
Department of Biological Regulation, Weizmann Institute of Science, Rehovot 76100, Israel.
Litovchick
L
L
Schourov
D
D
Cox
S
S
Taylor
S S
SS
Shaltiel
S
S
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009038:Motion; D009842:Oligopeptides / Q000737:chemistry; D011487:Protein Conformation; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014443:Tyrosine / Q000737:chemistry
D008024:Ligands; D009842:Oligopeptides; D011994:Recombinant Proteins; D014443:Tyrosine; C037642:kemptide; D017868:Cyclic AMP-Dependent Protein Kinases; D010450:Endopeptidases; C059784:kinase-splitting membranal proteinase