scholarly journals Casein Kinase II Phosphorylates the Fragile X Mental Retardation Protein and Modulates Its Biological Properties

2002 ◽  
Vol 22 (24) ◽  
pp. 8438-8447 ◽  
Author(s):  
Mikiko C. Siomi ◽  
Kyoko Higashijima ◽  
Akira Ishizuka ◽  
Haruhiko Siomi
Keyword(s):  
Gene Expression ◽  
Rna Binding ◽  
Fragile X ◽  
Regulation Of Gene Expression ◽  
Phosphorylation Site ◽  
Casein Kinase Ii ◽  
Biological Properties ◽  
Casein Kinase

ABSTRACT Fragile X syndrome is caused by loss of FMR1 protein expression. FMR1 binds RNA and associates with polysomes in the cytoplasm; thus, it has been proposed to function as a regulator of gene expression at the posttranscriptional level. Posttranslational modification of FMR1 had previously been suggested to regulate its activity, but no experimental support for this model has been reported to date. Here we report that FMR1 in Drosophila melanogaster (dFMR1) is phosphorylated in vivo and that the homomer formation and the RNA-binding activities of dFMR1 are modulated by phosphorylation in vitro. Identification of a protein phosphorylating dFMR1 showed it to be Drosophila casein kinase II (dCKII). dCKII directly interacts with and phosphorylates dFMR1 in vitro. The phosphorylation site in dFMR1 was identified as Ser406, which is highly conserved among FMR1 family members from several species. Using mass spectrometry, we established that Ser406 of dFMR1 is indeed phosphorylated in vivo. Furthermore, human FMR1 (hFMR1) is also phosphorylated in vivo, and alteration of the conserved Ser500 in hFMR1 abolishes phosphorylation by CKII in vitro. These studies support the model that the biological functions of FMR1, such as regulation of gene expression, are likely regulated by its phosphorylation.

scholarly journals Casein kinase II phosphorylation site mutations in c-Myb affect DNA binding and transcriptional cooperativity with NF-M.

1995 ◽  
Vol 15 (11) ◽  
pp. 5966-5974 ◽  
Author(s):  
M Oelgeschläger ◽  
J Krieg ◽  
J M Lüscher-Firzlaff ◽  
B Lüscher
Keyword(s):  
Dna Binding ◽  
Phosphorylation Site ◽  
Direct Interaction ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Wild Type ◽  
Type C ◽  
A Site

Phosphorylation of c-Myb has been implicated in the regulation of the binding of c-Myb to DNA. We show that murine c-Myb is phosphorylated at Ser-11 and -12 in vivo and that these sites can be phosphorylated in vitro by casein kinase II (CKII), analogous to chicken c-Myb. An efficient method to study DNA binding properties of full-length c-Myb and Myb mutants under nondenaturing conditions was developed. It was found that a Myb mutant in which Ser-11 and -12 were replaced with Ala (Myb Ala-11/12), wild-type c-Myb, and Myb Asp-11/12 bound to the A site of the mim-1 promoter with decreasing affinities. In agreement with this finding, Myb Ala-11/12 transactivated better than wild-type c-Myb and Myb Asp-11/12 on the mim-1 promoter or a synthetic Myb-responsive promoter. Similar observations were made for the myeloid-specific neutrophil elastase promoter. The presence of NF-M or an NF-M-like activity abolished partially the differences seen with the Ser-11/12 mutants, suggesting that the reduced DNA binding due to negative charge at positions 11 and 12 can be compensated for by NF-M. Since no direct interaction of c-Myb and NF-M was observed, we propose that the cooperativity is mediated by a third factor. Our data offer two possibilities for how casein kinase II phosphorylation can influence c-Myb function: first, by reducing c-Myb DNA binding and thereby influencing transactivation, and second, by enhancing the apparent cooperativity between c-Myb and NF-M or an NF-M-like activity.

scholarly journals Identification of the In Vivo Casein Kinase II Phosphorylation Site within the Homeodomain of the Cardiac Tisue-Specifying Homeobox Gene Product Csx/Nkx2.5

1999 ◽  
Vol 19 (1) ◽  
pp. 526-536 ◽  
Author(s):  
Hideko Kasahara ◽  
Seigo Izumo
Keyword(s):  
Mutational Analysis ◽  
Intracellular Localization ◽  
Phosphorylation Site ◽  
Homeobox Gene ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Site Directed Mutagenesis ◽  
Phosphopeptide Mapping

ABSTRACT Csx/Nkx2.5, a member of the homeodomain-containing transcription factors, serves critical developmental functions in heart formation in vertebrates and nonvertebrates. In this study the putative nuclear localization signal (NLS) of Csx/Nkx2.5 was identified by site-directed mutagenesis to the amino terminus of the homeodomain, which is conserved in almost all homeodomain proteins. When the putative NLS of Csx/Nkx2.5 was mutated a significant amount of the cytoplasmically localized Csx/Nkx2.5 was unphosphorylated, in contrast to the nuclearly localized Csx/Nkx2.5, which is serine- and threonine-phosphorylated, suggesting that Csx/Nkx2.5 phosphorylation is regulated, at least in part, by intracellular localization. Tryptic phosphopeptide mapping indicated that Csx/Nkx2.5 has at least five phosphorylation sites. Using in-gel kinase assays, we detected a Csx/Nkx2.5 kinase whose molecular mass is approximately 40 kDa in both cytoplasmic and nuclear extracts. Mutational analysis and in vitro kinase assays suggested that this 40-kDa Csx/Nkx2.5 kinase is a catalytic subunit of casein kinase II (CKII) that phosphorylates the serine residue between the first and second helix of the homeodomain. This CKII site is phosphorylated in vivo. CKII-dependent phosphorylation of the homeodomain increased Csx/Nkx2.5 DNA binding. Serine-to-alanine mutation at the CKII phosphorylation site reduced transcriptional activity when the carboxyl-terminal repressor domain was deleted. Although the precise biological function of Csx/Nkx2.5 phosphorylation by CKII remains to be determined, it may play an important role, as this CKII phosphorylation site within the homeodomain is fully conserved in all known members of the NK2 family of the homeobox genes.

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 Cytomegalovirus Replicon-Based Regulation of Gene Expression In Vitro and In Vivo

2012 ◽  
Vol 8 (6) ◽  
pp. e1002728 ◽  
Author(s):  
Hermine Mohr ◽  
Christian A. Mohr ◽  
Marlon R. Schneider ◽  
Laura Scrivano ◽  
Barbara Adler ◽  
...  
Keyword(s):  
Gene Expression ◽  
Regulation Of Gene Expression

Protein signaling and regulation of gene transcription in leukemia: role of the Casein Kinase II-Ikaros axis

2016 ◽  
Vol 64 (3) ◽  
pp. 735-739 ◽  
Author(s):  
Chandrika S Gowda ◽  
Chunhua Song ◽  
Yali Ding ◽  
Malika Kapadia ◽  
Sinisa Dovat
Keyword(s):  
Gene Transcription ◽  
Target Genes ◽  
Cellular Proliferation ◽  
Lymphoblastic Leukemia ◽  
Regulation Of Gene Expression ◽  
Regulatory Elements ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Protein Signaling

Protein signaling and regulation of gene expression are the two major mechanisms that regulate cellular proliferation in leukemia. Discerning the function of these processes is essential for understanding the pathogenesis of leukemia and for developing the targeted therapies. Here, we provide an overview of one of the mechanisms that regulates gene transcription in leukemia. This mechanism involves the direct interaction between Casein Kinase II (CK2) and the Ikaros transcription factor. Ikaros (IKZF1) functions as a master regulator of hematopoiesis and a tumor suppressor in acute lymphoblastic leukemia (ALL). Impaired Ikaros function results in the development of high-risk leukemia. Ikaros binds to the upstream regulatory elements of its target genes and regulates their transcription via chromatin remodeling. In vivo, Ikaros is a target for CK2, a pro-oncogenic kinase. CK2 directly phosphorylates Ikaros at multiple amino acids. Functional experiments showed that CK2-mediated phosphorylation of Ikaros, regulates Ikaros’ DNA binding affinity, subcellular localization and protein stability. Recent studies revealed that phosphorylation of Ikaros by CK2 regulates Ikaros binding and repression of the terminal deoxytransferase (TdT) gene in normal thymocytes and in T-cell ALL. Available data suggest that the oncogenic activity of CK2 in leukemia involves functional inactivation of Ikaros and provide a rationale for CK2 inhibitors as a potential treatment for ALL.

scholarly journals A Retinoid-Resistant Acute Promyelocytic Leukemia Subclone Expresses a Dominant Negative PML-RARα Mutation

Blood ◽  
1997 ◽  
Vol 89 (12) ◽  
pp. 4282-4289 ◽  
Author(s):  
Wenlin Shao ◽  
Laura Benedetti ◽  
William W. Lamph ◽  
Clara Nervi ◽  
Wilson H. Miller
Keyword(s):  
Gene Expression ◽  
Retinoic Acid ◽  
Ligand Binding ◽  
Cell Line ◽  
Acute Promyelocytic Leukemia ◽  
Regulation Of Gene Expression ◽  
Promyelocytic Leukemia ◽  
Dominant Negative

Abstract The unique t(15; 17) of acute promyelocytic leukemia (APL) fuses the PML gene with the retinoic acid receptor α (RARα) gene. Although retinoic acid (RA) inhibits cell growth and induces differentiation in human APL cells, resistance to RA develops both in vitro and in patients. We have developed RA-resistant subclones of the human APL cell line, NB4, whose nuclear extracts display altered RA binding. In the RA-resistant subclone, R4, we find an absence of ligand binding of PML-RARα associated with a point mutation changing a leucine to proline in the ligand-binding domain of the fusion PML-RARα protein. In contrast to mutations in RARα found in retinoid-resistant HL60 cells, in this NB4 subclone, the coexpressed RARα remains wild-type. In vitro expression of a cloned PML-RARα with the observed mutation in R4 confirms that this amino acid change causes the loss of ligand binding, but the mutant PML-RARα protein retains the ability to heterodimerize with RXRα and thus to bind to retinoid response elements (RAREs). This leads to a dominant negative block of transcription from RAREs that is dose-dependent and not relieved by RA. An unrearranged RARα engineered with this mutation also lost ligand binding and inhibited transcription in a dominant negative manner. We then found that the mutant PML-RARα selectively alters regulation of gene expression in the R4 cell line. R4 cells have lost retinoid-regulation of RXRα and RARβ and the RA-induced loss of PML-RARα protein seen in NB4 cells, but retain retinoid-induction of CD18 and CD38. Thus, the R4 cell line provides data supporting the presence of an RARα-mediated pathway that is independent from gene expression induced or repressed by PML-RARα. The high level of retinoid resistance in vitro and in vivo of cells from some relapsed APL patients suggests similar molecular changes may occur clinically.

scholarly journals Epigenetic regulation of gene expression by Ikaros, HDAC1 and Casein Kinase II in leukemia

Leukemia ◽  
2015 ◽  
Vol 30 (6) ◽  
pp. 1436-1440 ◽  
Author(s):  
C Song ◽  
X Pan ◽  
Z Ge ◽  
C Gowda ◽  
Y Ding ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epigenetic Regulation ◽  
Regulation Of Gene Expression ◽  
Casein Kinase Ii ◽  
Casein Kinase

Inhibition of Casein Kinase 2 Impairs Wnt Signaling and Cell Survival in Chronic Lymphocytic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2050-2050
Author(s):  
Christina Wu ◽  
Fitzgerald S Lao ◽  
Emily Nan ◽  
Hongying Li ◽  
Michael Y. Choi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Wnt Signaling ◽  
Conflicts Of Interest ◽  
Casein Kinase ◽  
Casein Kinase 2 ◽  
Lymphocytic Leukemia ◽  
Similar Reduction ◽  
Gene Assay

Abstract The oncogenic Wnt pathway is aberrantly activated in most CLL clones, and hence is an attractive target for therapy. The casein kinase 2 (CK2) enzyme is an established positive regulator of Wnt signaling. The inhibitor Silmitasertib, also known as CX-4945, is a nanomolar inhibitor of CK2. It has been reported that CK2 is overexpressed in CLL. Here we have investigated the effects of CX-4945 on WNT signaling in primary CLL cells. We confirmed that CX-4945 displayed in vitro cytotoxic activity toward CLL cells at very low µM concentration, as previously reported by others. However, at least 2-3 fold higher concentration of CX-4945 was required to achieve a similar toxicity against normal PBMC. Previously, our laboratory has successfully utilized a short-term CLL "parking" model in immunodeficient RAG/gamma chain knock out (RG-KO) mice to evaluate the in vivo efficacy and potential toxicity of anti-CLL agents. CX-4945 at dosages of 0.3-10 mg/kg was administered by oral gavage daily for 6 days to mice injected i.p. with 10 million CLL cells. These dosages of drug were well tolerated, and potently inhibited CLL persistence in the xenotransplanted mice. In a reporter gene assay, CX-4945 dose-dependently inhibited Wnt target gene expression. Furthermore, inhibition of dishevelled-2 (Dvl-2) protein expression was observed in primary CLL patient samples treated with 3-10 µM CX-4945 for 4-16 hours. Similar reduction in p-GSK3b(S9) protein was also observed. Quantitative RT-PCR also confirmed down regulation of b-catenin gene expression in primary CLL patient samples treated with 10 µM CX-4945 for 4h. Further molecular analyses of predictive or correlative biomarkers is ongoing using Nanostring PanCancer multipathway gene analysis. In a preliminary study, we found that CX-4945 perturbed the expression of multiple genes implicated in CLL development and survival. In summary, the CK2 inhibitor CX-4945 inhibited Wnt signaling and CLL survival, and displayed oral activity in mice. CK2 inhibitors are thus potential therapeutic agents for CLL. Disclosures No relevant conflicts of interest to declare.

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