Identity of the in vivo phosphorylation site in high mobility group 14 protein in HeLa cells with the site phosphorylated by casein kinase II in vitro.

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.

Download Full-text

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

Molecular and Cellular Biology ◽

10.1128/mcb.22.24.8438-8447.2002 ◽

2002 ◽

Vol 22 (24) ◽

pp. 8438-8447 ◽

Cited By ~ 57

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.

Download Full-text

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

Molecular and Cellular Biology ◽

10.1128/mcb.19.1.526 ◽

1999 ◽

Vol 19 (1) ◽

pp. 526-536 ◽

Cited By ~ 65

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.

Download Full-text

Phosphorylation of high mobility group protein 14 by casein kinase II.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)89134-7 ◽

1985 ◽

Vol 260 (8) ◽

pp. 4745-4750

Author(s):

G M Walton ◽

J Spiess ◽

G N Gill

Keyword(s):

High Mobility ◽

Casein Kinase Ii ◽

Casein Kinase ◽

High Mobility Group ◽

High Mobility Group Protein ◽

Group Protein

Download Full-text

Phosphorylation of serine 208 in the human vitamin D receptor. The predominant amino acid phosphorylated by casein kinase II, in vitro, and identification as a significant phosphorylation site in intact cells.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)53319-6 ◽

1993 ◽

Vol 268 (9) ◽

pp. 6791-6799

Author(s):

P.W. Jurutka ◽

J.C. Hsieh ◽

P.N. MacDonald ◽

C.M. Terpening ◽

C.A. Haussler ◽

...

Keyword(s):

Vitamin D ◽

Amino Acid ◽

Vitamin D Receptor ◽

Phosphorylation Site ◽

Casein Kinase Ii ◽

Casein Kinase ◽

Intact Cells ◽

Predominant Amino Acid

Download Full-text

Defective Calmodulin-Mediated Nuclear Transport of the Sex-Determining Region of the Y Chromosome (SRY) in XY Sex Reversal

Molecular Endocrinology ◽

10.1210/me.2004-0334 ◽

2005 ◽

Vol 19 (7) ◽

pp. 1884-1892 ◽

Cited By ~ 39

Author(s):

Helena Sim ◽

Kieran Rimmer ◽

Sabine Kelly ◽

Louisa M. Ludbrook ◽

Andrew H. A. Clayton ◽

...

Keyword(s):

Y Chromosome ◽

Nuclear Import ◽

High Mobility ◽

Sex Reversal ◽

High Mobility Group ◽

Missense Mutations ◽

Nuclear Localization Signals ◽

Xy Sex Reversal

Abstract The sex-determining region of the Y chromosome (SRY) plays a key role in human sex determination, as mutations in SRY can cause XY sex reversal. Although some SRY missense mutations affect DNA binding and bending activities, it is unclear how others contribute to disease. The high mobility group domain of SRY has two nuclear localization signals (NLS). Sex-reversing mutations in the NLSs affect nuclear import in some patients, associated with defective importin-β binding to the C-terminal NLS (c-NLS), whereas in others, importin-β recognition is normal, suggesting the existence of an importin-β-independent nuclear import pathway. The SRY N-terminal NLS (n-NLS) binds calmodulin (CaM) in vitro, and here we show that this protein interaction is reduced in vivo by calmidazolium, a CaM antagonist. In calmidazolium-treated cells, the dramatic reduction in nuclear entry of SRY and an SRY-c-NLS mutant was not observed for two SRY-n-NLS mutants. Fluorescence spectroscopy studies reveal an unusual conformation of SRY.CaM complexes formed by the two n-NLS mutants. Thus, CaM may be involved directly in SRY nuclear import during gonadal development, and disruption of SRY.CaM recognition could underlie XY sex reversal. Given that the CaM-binding region of SRY is well-conserved among high mobility group box proteins, CaM-dependent nuclear import may underlie additional disease states.

Download Full-text

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

Journal of Virology ◽

10.1128/jvi.73.2.1320-1330.1999 ◽

1999 ◽

Vol 73 (2) ◽

pp. 1320-1330 ◽

Cited By ~ 34

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.

Download Full-text