Mapping the residues of protein kinase CK2 alpha subunit responsible for responsiveness to polyanionic inhibitors.
The quadruple mutation of the whole basic cluster, K74KKK77 conserved in the catalytic subunits of protein kinase CK2 and implicated in substrate recognition, not only abolishes inhibition by heparin but even induces with some peptide substrates an up to 5-fold stimulation by heparin in the 0.5-5 micrograms/ml concentration range. Two other mutants defective in substrate recognition, R191, 195K198A and K79R80K83A, display either a 100-fold reduction or no alteration at all in heparin inhibition, respectively. In contrast sensitivity to heparin inhibition is increased 30-fold by a single mutation affecting Arg-228 while it is not altered by a triple mutation in the small insert of subdomain XI (mutant R278K279R280A). The effect of the same mutations on inhibition by pseudosubstrate EEEEEYEEEEEEE is different, the mutant displaying the most reduced sensitivity being R191,195K198A, followed by K74-77A and K79R80K83A; the other mutants are almost indistinguishable from CK2 wild type. Substantial reduction of inhibition by poly(Glu,Tyr)4:1 is only observable with mutant R191,195K198A, whereas R228A is significantly more sensitive to inhibition. These data show that the mode of inhibition of CK2 by polyanionic compounds occurs through substantially different mechanisms involving residues that are variably concerned with substrate recognition.
Vaglio
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P
Dipartimento di Chimica Biologica, Universita di Padova, Italy.
Sarno
S
S
Marin
O
O
Meggio
F
F
Issinger
O G
OG
Pinna
L A
LA
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000502:physiology; D047390:Casein Kinase II; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006493:Heparin / Q000494:pharmacology*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D013379:Substrate Specificity
D000596:Amino Acids; D004791:Enzyme Inhibitors; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; C027606:L-glutamic acid-L-tyrosine copolymer; D006493:Heparin; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases