scholarly journals Phosphorylation of IkappaBalpha in the C-terminal PEST domain by casein kinase II affects intrinsic protein stability.

1996 ◽  
Vol 16 (4) ◽  
pp. 1401-1409 ◽  
Author(s):  
R Lin ◽  
P Beauparlant ◽  
C Makris ◽  
S Meloche ◽  
J Hiscott
Keyword(s):  
Triple Point ◽  
Cell Activation ◽  
Kinase Activity ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Kinase Assay ◽  
Intrinsic Stability ◽  
Nf Kappab ◽  
Constitutive Phosphorylation

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.

Arabidopsis MLK3, a Plant-specific Casein Kinase 1, Negatively Regulated Flowering and Phosphorylated Histone H3 in vivo

2019 ◽  
Author(s):  
Zhen Wang ◽  
Junmei Kang ◽  
Shangang Jia ◽  
Tiejun Zhang ◽  
Zhihai Wu ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Histone H3 ◽  
Casein Kinase ◽  
Kinase Assay ◽  
Wild Type ◽  
Serine Threonine Kinase ◽  
Altered Expression ◽  
Casein Kinase 1 ◽  
Threonine Kinase

Abstract Background: Casein kinase 1 (CK1) family members are highly conserved serine/threonine kinase present in most eukaryotes with multiple biological functions. Arabidopsis MUT9-like kinases ( MLKs ) belong to a clade CK1 specific to the plant kingdom and have been implicated collectively in modulating flowering related processes. Three of the four MLKs ( MLK1/2/4 ) have been characterized, however, little is known about MLK3 , the most divergent MLKs. Results: We demonstrated that compared with wild type, mlk3 , a truncated MLK3 , flowered slightly early under long day conditions and ectopic expression of MLK3 rescued the morphological defects of mlk3 , indicating that MLK3 negatively regulates flowering. GA 3 application accelerated flowering of both wild type and mlk3 , suggesting that mlk3 had normal GA response. The recombinant MLK3-GFP was localized in the nucleus exclusively. In vitro kinase assay revealed that the nuclear protein MLK3 phosphorylated histone 3 at threonine 3 (H3T3ph). Mutation of a conserved catalytic residue (Lysine 175) abolished the kinase activity and resulted in failure to complement the early flowering phenotype of mlk3 . Interestingly, the global level of H3T3 phosphorylation in mlk3 did not differ significantly from wild type, suggesting the redundant roles of MLKs in flowering regulation. The transcriptomic analysis demonstrated that 425 genes significantly altered expression level in mlk3 relative to wild type. The mlk3 mlk4 double mutant generated by crossing mlk3 with mlk4 , a loss-of-function mutant of MLK4 showing late flowering, flowered between the two parental lines, suggesting that MLK3 played an antagonistic role to MLK4 in plant transition to flowering. Conclusions: A serine/threonine kinase encoding gene MLK3 is a casein kinase 1 specific to the plant species and represses flowering slightly. MLK3 located in nucleus catalyzes the phosphorylation of histone H3 at threonine 3 in vitro and an intact lysine residue (K175) is indispensible for the kinase activity. This study sheds new light on the delicate control of flowering by the plant-specific CK1 in Arabidopsis.

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

2010 ◽  
Vol 9 (1) ◽  
pp. 257 ◽  
Author(s):  
Stefan M Willems ◽  
Yvonne M Schrage ◽  
Inge H Briaire-de Bruijn ◽  
Karoly Szuhai ◽  
Pancras CW Hogendoorn ◽  
...  
Keyword(s):  
Treatment Option ◽  
Kinase Activity ◽  
Myxoid Liposarcoma ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Potential Treatment ◽  
Nf Kappab

scholarly journals Casein kinase II phosphorylates I kappa B alpha at S-283, S-289, S-293, and T-291 and is required for its degradation.

1996 ◽  
Vol 16 (3) ◽  
pp. 899-906 ◽  
Author(s):  
J A McElhinny ◽  
S A Trushin ◽  
G D Bren ◽  
N Chester ◽  
C V Paya
Keyword(s):  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Kinase Assay ◽  
Resting Cells ◽  
Nf Kappa B ◽  
I Kappa B Alpha ◽  
Cell Extracts ◽  
Protein Kinase Ckii

The phosphoprotein I kappa B alpha exists in the cytoplasm of resting cells bound to the ubiquitous transcription factor NF-kappa B (p50-p65). In response to specific cellular stimulation, I kappa B alpha is further phosphorylated and subsequently degraded, allowing NF-kappa B to translocate to the nucleus and transactivate target genes. To identify the kinase(s) involved in I kappa B alpha phosphorylation, we first performed an I kappa B alpha in-gel kinase assay. Two kinase activities of 35 and 42 kDa were identified in cellular extracts from Jurkat T and U937 promonocytic cell lines. Specific inhibitors and immunodepletion studies identified the I kappa B alpha kinase activities as those of the alpha and alpha' subunits of casein kinase II (CKII). Immunoprecipitation studies demonstrated that CKII and I kappa B alpha physically associate in vivo. Moreover, phosphopeptide maps of I kappa B alpha phosphorylated in vitro by cellular extracts and in vivo in resting Jurkat T cells contained the same pattern of phosphopeptides as observed in maps of I kappa B alpha phosphorylated in vitro by purified CKII. Sequence analysis revealed that purified CKII and the kinase activity within cell extracts phosphorylated I kappa B alpha at its C terminus at S-283, S-288, S-293, and T-291. The functional role of CKII was tested in an in vitro I kappa B alpha degradation assay with extracts from uninfected and human immunodeficiency virus (HIV)-infected U937 cells. Immunodepletion of CKII from these extracts abrogated both the basal and enhanced HIV-induced degradation of I kappa B alpha. These studies provide new evidence that the protein kinase CKII physically associates with I kappa B alpha in vivo, induces multisite (serine/threonine) phosphorylation, and is required for the basal and HIV-induced degradation of I kappa B alpha in vitro.

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

2007 ◽  
Vol 81 (10) ◽  
pp. 5305-5314 ◽  
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.

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

1996 ◽  
Vol 16 (7) ◽  
pp. 3554-3559 ◽  
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.

scholarly journals Varicella-Zoster Virus Fc Receptor Component gI Is Phosphorylated on Its Endodomain by a Cyclin-Dependent Kinase

1999 ◽  
Vol 73 (2) ◽  
pp. 1320-1330 ◽  
Author(s):  
Ming Ye ◽  
Karen M. Duus ◽  
Junmin Peng ◽  
David H. Price ◽  
Charles Grose
Keyword(s):  
Fusion Protein ◽  
Varicella Zoster Virus ◽  
Phosphorylation Site ◽  
Cytoplasmic Tail ◽  
Casein Kinase Ii ◽  
Fc Receptor ◽  
Casein Kinase ◽  
Cyclin Dependent Kinase ◽  
Varicella Zoster

Varicella-zoster virus (VZV) glycoprotein gI is a type 1 transmembrane glycoprotein which is one component of the heterodimeric gE:gI Fc receptor complex. Like VZV gE, VZV gI was phosphorylated in both VZV-infected cells and gI-transfected cells. Preliminary studies demonstrated that a serine 343-proline 344 sequence located within the gI cytoplasmic tail was the most likely phosphorylation site. To determine which protein kinase catalyzed the gI phosphorylation event, we constructed a fusion protein, consisting of glutathione-S-transferase (GST) and the gI cytoplasmic tail, called GST-gI-wt. When this fusion protein was used as a substrate for gI phosphorylation in vitro, the results demonstrated that GST-gI-wt fusion protein was phosphorylated by a representative cyclin-dependent kinase (CDK) called P-TEFb, a homologue of CDK1 (cdc2). When serine 343 within the serine-proline phosphorylation site was replaced with an alanine residue, the level of phosphorylation of the gI fusion protein was greatly reduced. Subsequent experiments with individually immunoprecipitated mammalian CDKs revealed that the VZV gI fusion protein was phosphorylated best by CDK1, to a lesser degree by CDK2, and not at all by CDK6. Transient-transfection assays carried out in the presence of the specific CDK inhibitor roscovitine strongly supported the prior results by demonstrating a marked decrease in gI phosphorylation while gI protein expression was unaffected. Finally, the possibility that VZV gI contained a CDK phosphorylation site in its endodomain was of further interest because its partner, gE, contains a casein kinase II phosphorylation site in its endodomain; prior studies have established that CDK1 can phosphorylate casein kinase II.

scholarly journals Tyrosine phosphorylation of P-selectin in intact platelets and in a disulphide-linked complex with immunoprecipitated pp60c-src

1994 ◽  
Vol 299 (3) ◽  
pp. 613-621 ◽  
Author(s):  
P W Modderman ◽  
A E G K von dem Borne ◽  
A Sonnenberg
Keyword(s):  
Tyrosine Phosphorylation ◽  
Cell Activation ◽  
Secretory Granules ◽  
Protein S ◽  
Kinase Assay ◽  
Intact Cells ◽  
Tyrosine Residues ◽  
Reducing Conditions ◽  
Sds Page

P-selectin is a 140 kDa membrane glycoprotein found in secretory granules of platelets and endothelial cells where it is rapidly translocated to the plasma membrane upon cell activation. It then functions as a receptor for various types of leucocytes. Metabolic labelling of resting platelets with 32Pi showed that P-selectin is primarily phosphorylated on serine residues, although some tyrosine phosphorylation was observed as well. However, tyrosine phosphorylation of P-selectin was greatly stimulated by treatment with the permeating phosphatase inhibitor, pervanadate. When P-selectin immunoprecipitates were incubated with [gamma-32P]ATP (in vitro kinase assay), a fraction of P-selectin was phosphorylated on its tyrosine residues by a co-precipitated kinase. P-selectin phosphorylated in vitro co-migrated with 140 kDa surface-labelled 125I-P-selectin during SDS/PAGE under reducing conditions. Under non-reducing conditions, however, phosphorylated P-selectin was disulphide-linked to unknown protein(s) in a 205 kDa complex. In vitro kinase assays of the most abundant platelet tyrosine kinase, pp60c-src, demonstrated the presence of similar 140 and 205 kDa phosphorylated proteins in SDS/PAGE under reducing and non-reducing conditions respectively. Extraction and reprecipitation studies with proteins phosphorylated in vitro indicated that P-selectin and pp60c-src form a 205 kDa 1:1 disulphide-linked complex. In the complex, pp60c-src autophosphorylation is inhibited and P-selectin is phosphorylated on tyrosine residues. As protein disulphides in the cytoplasm of intact cells are extremely rare, our results suggest that P-selectin and pp60c-src, which co-localize in platelet dense granules, may be non-covalently associated and spontaneously form disulphide bridges during lysis. In addition, the observed tyrosine phosphorylation of P-selectin in intact platelets suggests that its function might be regulated by phosphorylation by pp60c-src.

Biochemical characterization of HIV-1 Rev as a potent activator of casein kinase II in vitro

FEBS Letters ◽  
1998 ◽  
Vol 428 (3) ◽  
pp. 235-240 ◽  
Author(s):  
Kenzo Ohtsuki ◽  
Toshiro Maekawa ◽  
Shigeyoshi Harada ◽  
Atsushi Karino ◽  
Yuko Morikawa ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Hiv 1

Regulation of eukaryotic initiation factor-2B activity in muscle of diabetic rats

1993 ◽  
Vol 264 (1) ◽  
pp. E101-E108 ◽  
Author(s):  
A. M. Karinch ◽  
S. R. Kimball ◽  
T. C. Vary ◽  
L. S. Jefferson
Keyword(s):  
Skeletal Muscle ◽  
Protein Synthesis ◽  
Cardiac Muscle ◽  
Casein Kinase Ii ◽  
Diabetic Rats ◽  
Casein Kinase ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Fast Twitch

Peptide-chain initiation is inhibited in fast-twitch skeletal muscle, but not heart, of diabetic rats. We have investigated mechanisms that might maintain eukaryotic initiation factor (eIF)-2B activity, preventing loss of efficiency of protein synthesis in heart of diabetic rats but not in fast-twitch skeletal muscle. There was no change in the amount or phosphorylation state of eIF-2 in skeletal or cardiac muscle during diabetes. In contrast, eIF-2B activity was decreased in fast-twitch but not slow-twitch muscle from diabetic animals. NADP+ inhibited partially purified eIF-2B in vitro, but addition of equimolar NADPH reversed the inhibition. The NADPH-to-NADP+ ratio was unchanged in fast-twitch muscle after induction of diabetes but was increased in heart of diabetic rats, suggesting that NADPH also prevents inhibition of eIF-2B in vivo. The activity of casein kinase II, which can phosphorylate and activate eIF-2B in vitro, was significantly lower in extracts of fast-twitch, but not cardiac muscle, of diabetic rats compared with controls. The results presented here demonstrate that changes in eIF-2 alpha phosphorylation are not responsible for the effect of diabetes on eIF-2B activity in fast-twitch skeletal muscle. Modulation of casein kinase II activity may be a factor in the regulation of protein synthesis in muscle during acute diabetes. The activity of eIF-2B in heart might be maintained by the increased NADPH/NADP+.

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