Kinome profiling of myxoid liposarcoma reveals NF-kappaB-pathway kinase activity and Casein Kinase II inhibition as a potential treatment option

The NF-kappaB/Rel transcription factors participate in the activation of immune system regulatory genes and viral early genes including the human immunodeficiency virus type 1 long terminal repeat. NF-kappaB/Rel proteins are coupled to inhibitory molecules, collectively termed IkappaB, which are responsible for cytoplasmic retention of NF-kappaB. Cell activation leads to the phosphorylation and degradation of IkappaBalpha, permitting NG-kappaB/Rel translocation to the nucleus and target gene activation. To further characterize the signaling events that contribute to IkappaBalpha phosphorylation, a kinase activity was isolated from Jurkat T cells that specifically interacted with IkappaBalpha in an affinity chromatography step and phosphorylated IkappaBalpha with high specificity in vitro. By using an in-gel kinase assay with recombinant IkappaBalpha as substrate, two forms of the kinase (43 and 38 kDa) were identified. Biochemical criteria and immunological cross-reactivity identified the kinase activity as the alpha catalytic subunit of casein kinase II (CKII). Deletion mutants of IkappaBalpha delta1 to delta4) localized phosphorylation to the C-terminal PEST domain of IkappaBalpha. Point mutation of residues T-291, S-283, and T-299 dramatically reduced phosphorylation of IkappaBalpha by the kinase in vitro. NIH-3T3 cells that stably expressed wild-type IkappaBalpha (wtIkappaB), double-point-mutated IkappaBalpha (T291A, S283A), or triple-point-mutated IkappaBalpha (T291A, S283A, T299A) under the control of the tetracycline-responsive promoter were generated. Constitutive phosphorylation of the triple point mutant was eliminated in vivo, although tumor necrosis factor-inducible IkappaBalpha degradation was unaffected. In cell lines and in transiently transfected cells, mutation of the CKII sites in IkappaBalpha resulted in a protein with increased intrinsic stability. Together with results demonstrating a role for N-terminal sites in inducer-mediated phosphorylation and degradation of IkappaBalpha, these studies indicate that CKII sites in the C-terminal PEST domain are important for constitutive phosphorylation and intrinsic stability of IkappaBalpha.

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Independent role of phosphoinositol-3-kinase (PI3K) and casein kinase II (CK-2) in EGFR and Her-2-mediated constitutive NF-kappaB activation in prostate cancer cells

The Prostate ◽

10.1002/pros.20291 ◽

2005 ◽

Vol 65 (4) ◽

pp. 306-315 ◽

Cited By ~ 24

Author(s):

Cécile Le Page ◽

Ismael Hervé Koumakpayi ◽

Laurent Lessard ◽

Fred Saad ◽

Anne-Marie Mes-Masson

Keyword(s):

Prostate Cancer ◽

Cancer Cells ◽

Casein Kinase Ii ◽

Casein Kinase ◽

Prostate Cancer Cells ◽

Her 2 ◽

Nf Kappab

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The Human Cytomegalovirus Virion Possesses an Activated Casein Kinase II That Allows for the Rapid Phosphorylation of the Inhibitor of NF-κB, IκBα

Journal of Virology ◽

10.1128/jvi.02382-06 ◽

2007 ◽

Vol 81 (10) ◽

pp. 5305-5314 ◽

Cited By ~ 24

Author(s):

Maciej T. Nogalski ◽

Jagat P. Podduturi ◽

Ian B. DeMeritt ◽

Liesl E. Milford ◽

Andrew D. Yurochko

Keyword(s):

Human Cytomegalovirus ◽

Molecular Mechanisms ◽

Kinase Activity ◽

Human Fibroblasts ◽

Casein Kinase Ii ◽

Casein Kinase ◽

Temporal Regulation ◽

Protein Kinase Ckii

ABSTRACT We documented that the NF-κB signaling pathway was rapidly induced following human cytomegalovirus (HCMV) infection of human fibroblasts and that this induced NF-κB activity promoted efficient transactivation of the major immediate-early promoter (MIEP). Previously, we showed that the major HCMV envelope glycoproteins, gB and gH, initiated this NF-κB signaling event. However, we also hypothesized that there were additional mechanisms utilized by the virus to rapidly upregulate NF-κB. In this light, we specifically hypothesized that the HCMV virion contained IκBα kinase activity, allowing for direct phosphorylation of IκBα following virion entry into infected cells. In vitro kinase assays performed on purified HCMV virion extract identified bona fide IκBα kinase activity in the virion. The enzyme responsible for this kinase activity was identified as casein kinase II (CKII), a cellular serine-threonine protein kinase. CKII activity was necessary for efficient transactivation of the MIEP and IE gene expression. CKII is generally considered to be a constitutively active kinase. We suggest that this molecular characteristic of CKII represents the biologic rationale for the viral capture and utilization of this kinase early after infection. The packaging of CKII into the HCMV virion identifies that diverse molecular mechanisms are utilized by HCMV for rapid NF-κB activation. We propose that HCMV possesses multiple pathways to increase NF-κB activity to ensure that the correct temporal regulation of NF-κB occurs following infection and that sufficient threshold levels of NF-κB are reached in the diverse array of cells, including monocytes and endothelial cells, infected in vivo.

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Constitutive phosphorylation of IkappaBalpha by casein kinase II occurs preferentially at serine 293: requirement for degradation of free IkappaBalpha.

Molecular and Cellular Biology ◽

10.1128/mcb.16.7.3554 ◽

1996 ◽

Vol 16 (7) ◽

pp. 3554-3559 ◽

Cited By ~ 115

Author(s):

E M Schwarz ◽

D Van Antwerp ◽

I M Verma

Keyword(s):

Transcription Factors ◽

Half Life ◽

Phosphorylation Site ◽

Casein Kinase Ii ◽

Casein Kinase ◽

Wild Type ◽

Serine Residue ◽

Nf Kappab ◽

Pest Region ◽

Constitutive Phosphorylation

IkappaBalpha is a phosphoprotein that sequesters the NF-kappaB/Rel transcription factors in the cytoplasm by physical association. Following induction by a wide variety of agents, IkappaBalpha is further phosphorylated and degraded, allowing NF-kappaB/Rel proteins to translocate to the nucleus and induce transcription. We have previously reported that the constitutive phosphorylation site resides in the C-terminal PEST region of IkappaBalpha and is phosphorylated by casein kinase II (CKII). Here we show that serine 293 is the preferred CKII phosphorylation site. Additionally, we show compensatory phosphorylation by CKII at neighboring serine and threonine residues. Thus, only when all five of the serine and threonine residues in the C-terminal region of IkappaBalpha are converted to alanine (MutF), is constitutive phosphorylation abolished. Finally, we show that constitutive phosphorylation is required for efficient degradation of free IkappaBalpha, in that unassociated Mutf has a half-life two times longer than wild-type IkappaBalpha. A serine residue alone at position 293, as well as aspartic acid at this position, can revert the Mutf phenotype. Therefore, the constitutive CKII phosphorylation site is an integral part of the PEST region of IkappaBalpha, and this phosphorylation is required for rapid proteolysis of the unassociated protein.

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Identification of a major maturation-activated acetyl-CoA carboxylase kinase in sea star oocytes as p44mpk

Biochemical Journal ◽

10.1042/bj2740759 ◽

1991 ◽

Vol 274 (3) ◽

pp. 759-767 ◽

Cited By ~ 15

Author(s):

S L Pelech ◽

J S Sanghera ◽

H B Paddon ◽

K A Quayle ◽

R W Brownsey

Keyword(s):

Protein Kinases ◽

Mammalian Cells ◽

Kinase Activity ◽

Minor Component ◽

Casein Kinase Ii ◽

Casein Kinase ◽

Acetyl Coa Carboxylase ◽

Sea Star ◽

Acetyl Coa ◽

Germinal Vesicle Breakdown

Maturation-activated protein-serine/threonine kinases were investigated in the high-speed supernatant fractions from sea-star oocytes harvested at the time of germinal vesicle breakdown. One of the major stimulated protein kinases able to phosphorylate acetyl-CoA carboxylase in these extracts was found to co-purify with a 44 kDa myelin basic protein kinase (p44mpk) that is activated with a similar time course during oocyte maturation. Purified sea-star oocyte p44mpk phosphorylated acetyl-CoA carboxylase (purified from rat liver) predominantly on serine and to a small extent on threonine. Furthermore, the phosphorylation of acetyl-CoA carboxylase occurred principally on a tryptic phosphopeptide which displayed electrophoretic and chromatographic properties very similar to those of the peptide that has previously been shown to undergo increased phosphorylation in response to insulin in rat adipocytes [Brownsey & Denton (1982) Biochem. J. 202, 77-86]. The acetyl-CoA carboxylase was phosphorylated at a similar rate and to a similar extent by casein kinase II, which was also purified from maturing sea-star oocytes. Although casein kinase II was also activated approximately 3-fold near the time of nuclear envelope breakdown, it was responsible for only a minor component of the total enhanced acetyl-CoA carboxylase kinase activity measured in the soluble extracts from maturing oocytes. Acetyl-CoA carboxylase was a relatively poor substrate for the major S6 peptide kinase activity that was also stimulated during resumption of meiosis in the oocytes. The properties of the p44mpk are reminiscent of those of a microtubule-associated protein 2 (MAP-2) kinase that is activated in response to insulin and other mitogens in mammalian cells [Ray & Sturgill (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 3753-3757; Hoshi, Nishida & Sakai (1988) J. Biol. Chem. 263, 5396-5401]. It is intriguing that several of the mammalian protein kinases that are acutely activated after mitogenic prompting of quiescent mouse fibroblasts (i.e. G0 to G1 transition), such as MAP-2 kinase, casein kinase II and S6 kinase II, have counterparts that are activated during M-phase in maturing sea star oocytes.

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