Protein kinase CK2 is a ubiquitous pleiotropic serine/threonine protein kinase whose holoenzyme is comprised of two catalytic (alpha and/or alpha') and two non-catalytic, beta-subunits. The beta-subunit possesses antagonist functions that can be physically dissected by generating synthetic fragments encompassing its N-terminal and C-terminal domains. Here we show that by mutating basic residues in the 74-77 and in the 191-198 regions of the alpha-subunit, the negative regulation by the beta-subunit and by its N-terminal synthetic fragment CK2beta-(1-77), which is observable using calmodulin as a substrate for phosphorylation, is drastically reduced. In contrast, the positive regulation by a C-terminal, CK2beta-(155-215)-peptide is unaffected or even increased. Moreover, the basal activity of alpha mutants K74-77A, K79R80K83A, and R191R195K198A toward specific peptide substrates is stimulated by the beta-subunit many fold more than that of alpha wild type, while extrastimulation by beta mutant D55L56E57A, observable with alpha wild type, is abolished with these mutants. These data support the conclusion that down regulation by the acidic residues clustered in the N-terminal moiety of beta is mediated by basic residues in the 74-83 and in the 191-198 sequences of the alpha-subunit. These are also implicated in substrate recognition consistent with the concept that the N-terminal acidic region of the beta subunit operates as a pseudosubstrate. In contrast, another CK2alpha mutant, V66A, is more sensitive to inhibition by either beta-subunit or its N-terminal, CK2beta-(1-77)-peptide, while its stimulation by the C-terminal peptide, CK2beta-(155-215), is comparable to that of alpha wild type. These observations suggest an indirect role of Val66 in conferring to the alpha-subunit a conformation less sensitive to down regulation by beta-subunit.

Basic residues in the 74-83 and 191-198 segments of protein kinase CK2 catalytic subunit are implicated in negative but not in positive regulation by the beta-subunit

SARNO, STEFANIA;MARIN, ORIANO;MEGGIO, FLAVIO;PINNA, LORENZO
1997

Abstract

Protein kinase CK2 is a ubiquitous pleiotropic serine/threonine protein kinase whose holoenzyme is comprised of two catalytic (alpha and/or alpha') and two non-catalytic, beta-subunits. The beta-subunit possesses antagonist functions that can be physically dissected by generating synthetic fragments encompassing its N-terminal and C-terminal domains. Here we show that by mutating basic residues in the 74-77 and in the 191-198 regions of the alpha-subunit, the negative regulation by the beta-subunit and by its N-terminal synthetic fragment CK2beta-(1-77), which is observable using calmodulin as a substrate for phosphorylation, is drastically reduced. In contrast, the positive regulation by a C-terminal, CK2beta-(155-215)-peptide is unaffected or even increased. Moreover, the basal activity of alpha mutants K74-77A, K79R80K83A, and R191R195K198A toward specific peptide substrates is stimulated by the beta-subunit many fold more than that of alpha wild type, while extrastimulation by beta mutant D55L56E57A, observable with alpha wild type, is abolished with these mutants. These data support the conclusion that down regulation by the acidic residues clustered in the N-terminal moiety of beta is mediated by basic residues in the 74-83 and in the 191-198 sequences of the alpha-subunit. These are also implicated in substrate recognition consistent with the concept that the N-terminal acidic region of the beta subunit operates as a pseudosubstrate. In contrast, another CK2alpha mutant, V66A, is more sensitive to inhibition by either beta-subunit or its N-terminal, CK2beta-(1-77)-peptide, while its stimulation by the C-terminal peptide, CK2beta-(155-215), is comparable to that of alpha wild type. These observations suggest an indirect role of Val66 in conferring to the alpha-subunit a conformation less sensitive to down regulation by beta-subunit.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11577/145802
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