Effects of Autophosphorylation on Casein Kinase II Activity: Evidence from Mutations in the .beta. Subunit

Lin, Wey Jinq; Sheu, Gwo‐Tarng; Traugh, Jolinda A.

doi:10.1021/bi00188a032

Cited by 26 publications

(19 citation statements)

References 41 publications

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“…The effects of such mutations on the functions of CK2 in cells have not yet been examined. Muta-tions of the autophosphorylation site do not effect the ability of CK2b to form complexes with CK2a when recombinant subunits are reconstituted in vitro Lin et al, 1994]. In a similar vein, our data now suggest that neither kinase activity nor autophosphorylation of CK2b is required to form stable complexes between CK2b and the catalytic subunits of CK2.…”

Section: Discussionsupporting

confidence: 63%

“…In the present study, we now present evidence that suggests that the autophosphorylation of CK2 also occurs through an intramolecular process in cells. In vitro studies with mutants lacking the autophosphorylation site(s) suggest that autophosphorylation modulates the activity of CK2 towards some but not all substrates [Lin et al, 1994]. The effects of such mutations on the functions of CK2 in cells have not yet been examined.…”

Section: Discussionmentioning

confidence: 99%

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Expression and localization of epitope-tagged protein kinase CK2

1997

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Protein kinase CK2, formerly known as casein kinase II, is a ubiquitous protein serine/threonine kinase. The enzyme exists in tetrameric complexes composed of two catalytic (CK2 alpha and/or CK2 alpha') subunits and two subunits (CK2 beta) that appear to have a role in modulating the activity of the catalytic subunits. With the exception of their unrelated carboxy-terminal domains, the two isozymic forms of mammalian CK2 display extensive sequence identity. Furthermore, CK2 alpha and CK2 alpha' exhibit remarkable conservation between species, suggesting that they may have unique functions. In the present study, the cDNAs encoding CK2 alpha and CK2 alpha' were modified by addition of the hemagglutinin tag of the influenza virus at the amino terminus of the respective proteins. The epitope-tagged proteins were transfected into Cos-7 cells and the localization of the expressed proteins determined by indirect immunofluorescence using monoclonal antibodies specific for the epitope tag. The use of transfection favors the formation of homotetrameric complexes (i.e., alpha 2 beta 2, alpha' 2 beta 2) instead of heterotetrameric complexes (i.e., alpha alpha' beta 2) that are present in many cells. Epitope-tagged CK2 alpha and CK2 alpha' displayed kinase activity and the ability to form complexes with CK2 beta. The results of these studies also indicate definitively that CK2 alpha and CK2 alpha' are both localized predominantly within the nucleus. Mutation of conserved lysine residues within the ATP binding domains of CK2 alpha and CK2 alpha' resulted in loss of kinase activity. However, examination of these mutants indicates that kinase activity is not essential for formation of complexes between subunits of CK2 and is not required for nuclear localization of CK2.

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Section: Discussionsupporting

confidence: 63%

Section: Discussionmentioning

confidence: 99%

Expression and localization of epitope-tagged protein kinase CK2

1997

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“…In fact, despite the remarkable conserva- tion of these autophosphorylation sites between species, a precise understanding of its functions has remained elusive. Prior to this study, the only potential functional e ect of autophosphorylation was a modest (*30%) e ect on the catalytic activity of CK2 when examined in vitro (Lin et al, 1994;Bodenbach et al, 1994). The relationship of this latter observation to the physiological functions or regulation of CK2 in cells was not investigated.…”

Section: Discussionmentioning

confidence: 99%

Phosphorylation regulates the stability of the regulatory CK2β subunit

et al. 2002

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Protein kinase CK2 is a protein serine/threonine kinase that exhibits elevated expression in a number of cancers and displays oncogenic activity in mice. The regulatory CK2b subunit has a central role in assembly of functional tetrameric CK2 complexes where it participates in modulation of catalytic activity and substrate speci®city. Since overexpression of CK2b results in elevated levels of CK2 activity, we investigated the molecular mechanisms that control its degradation since perturbations in these pathways could contribute to elevated CK2 in cancer. In this study, we demonstrate that CK2b is degraded by a proteasome-dependent pathway and that it is ubiquitinated. We have also investigated the role of phosphorylation and a putative destruction box in regulating its stability in cells. Importantly, replacement of three serine residues within the autophosphorylation site of CK2b with glutamic acid residues resulted in a signi®cant decrease in its degradation indicating that autophosphorylation is involved in regulating its stability. Notably, although the autophosphorylation site of CK2b is remarkably conserved between species, this is the ®rst functional role ascribed to this site. Furthermore, based on these results, we speculate that alterations in the phosphorylation or dephosphorylation of the regulatory CK2b subunit could underlie the elevated expression of CK2 that is observed in cancer cells.

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“…down regulates CK2a by acting as a pseudosubstrate whose acidic residues are clustered in the 55 -64 region and probably at the autophosphorylation site as well (Boldyreff et a]., 1994;Lin et al, 1994) mimic the specificity determinants of a bona fide phosphoacceptor site. It is conceivable, therefore that, as shown in Fig.…”

Section: Discussionmentioning

confidence: 99%

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 β‐Subunit

Sarno¹,

Vaglio²,

Marin³

et al. 1997

European Journal of Biochemistry

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Protein kinase CK2 is a ubiquitous pleiotropic serine/threonine protein kinase whose holoenzyme is comprised of two catalytic (a and/or a') and two non-catalytic, P-subunits. The B-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 a-subunit, the negative regulation by the P-subunit and by its N-terminal synthetic fragment CK2P-( 1 -77), which is observable using calmodulin as a substrate for phosphorylation, is drastically reduced. In contrast, the positive regulation by a C-terminal, CK2B-(155 -215)-peptide is unaffected or even increased. Moreover, the basal activity of (x mutants K74-77A, K79R80K83A, and R191R195K198A toward specific peptide substrates is stimulated by the P-subunit many fold more than that of a wild type, while extrastimulation by p mutant DSSLShE57A, observable with a 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 P is mediated by basic residues in the 74-83 and in the 191 -198 sequences of the a-subunit. These are also implicated in substrate recognition consistent with the concept that the N-terminal acidic region of the p subunit operates as a pseudosubstrate. In contrast, another CK2rx mutant, V66A, is more sensitive to inhibition by either P-subunit or its N-terminal, CK2P-(1 -77)-peptide, while its stiinulation by the C-terminal peptide, CK2P-(ISS-21 S), is comparable to that of rx wild type. These observations suggest an indirect role of Val66 in conferring to the a-subunit a conformation less sensitive to down regulation by B-subunit.Keywords: protein kinase CK2 ; casein kinase 2 ; mutant; protein phosphorylation.Protein kinase CK2 (also termed casein kinase-2 or IL) is an essential and ubiquitous Ser/Thr protein kinare implicated in the phosphorylation of many proteins that take part in a variety of cellular functions, with special reference to signal transduction, gene expression, and cell proliferation (reviewed by Pinna, 1990;Tuazon and Traugh, 1991 ;Allende and Allende, 1995). The minimum consensus sequence for CK2 is as follows: S/T-X-X-D/E/Sp/Yp, but additional acidic/phosphorylated residues, besides the crucial one at position n + 3 are found in most phosphoacceptor sites affected by CK2 and they are also required for efficient phosphorylation of artificial peptide substrates sual predilection for very acidic sites have been investigated by mutational analysis of CK2 catalytic a-subunit (Sarno et al., 1995). These studies disclosed the role of basic residues clustered in the 74-83 and 191 -198 regions in recognizing the specificity determinants at various positions down-stream from the target amino acid (Sarno et al., 1996).The level of CK2 is invariably elevated in proliferating cells and neoplastic tissues (Issinger, 1993) and the catalytic subunit of CK2 causes ...

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Effects of Autophosphorylation on Casein Kinase II Activity: Evidence from Mutations in the .beta. Subunit

Cited by 26 publications

References 41 publications

Expression and localization of epitope-tagged protein kinase CK2

Expression and localization of epitope-tagged protein kinase CK2

Phosphorylation regulates the stability of the regulatory CK2β 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 β‐Subunit

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