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 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 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 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 A casein kinase II-related activity is involved in phosphorylation of microtubule-associated protein MAP-1B during neuroblastoma cell differentiation.

1988 ◽  
Vol 106 (6) ◽  
pp. 2057-2065 ◽  
Author(s):  
J Díaz-Nido ◽  
L Serrano ◽  
E Méndez ◽  
J Avila
Keyword(s):  
Protein Kinase ◽  
Neuroblastoma Cells ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Related Activity ◽  
Dependent Protein Kinase ◽  
Protein Map ◽  
Dependent Protein

A neuroblastoma protein related to the brain microtubule-associated protein, MAP-1B, as determined by immunoprecipitation and coassembly with brain microtubules, becomes phosphorylated when N2A mouse neuroblastoma cells are induced to generate microtubule-containing neurites. To characterize the protein kinases that may be involved in this in vivo phosphorylation of MAP-1B, we have studied its in vitro phosphorylation. In brain microtubule protein, MAP-1B appears to be phosphorylated in vitro by an endogenous casein kinase II-like activity which also phosphorylates the related protein MAP-1A but scarcely phosphorylates MAP-2. A similar kinase activity has been detected in cell-free extracts of differentiating N2A cells. Using brain MAP preparations devoid of endogenous kinase activities and different purified protein kinases, we have found that MAP-1B is barely phosphorylated by cAMP-dependent protein kinase, Ca/calmodulin-dependent protein kinase, or Ca/phospholipid-dependent protein kinase whereas MAP-1B is one of the preferred substrates, together with MAP-1A, for casein kinase II. Brain MAP-1B phosphorylated in vitro by casein kinase II efficiently coassembles with microtubule proteins in the same way as in vivo phosphorylated MAP-1B from neuroblastoma cells. Furthermore, the phosphopeptide patterns of brain MAP-1B phosphorylated in vitro by either purified casein kinase II or an extract obtained from differentiating neuroblastoma cells are identical to each other and similar to that of in vivo phosphorylated neuroblastoma MAP-1B. Thus, we suggest that the observed phosphorylation of a protein identified as MAP-1B during neurite outgrowth is mainly due to the activation of a casein kinase II-related activity in differentiating neuroblastoma cells. This kinase activity, previously implicated in beta-tubulin phosphorylation (Serrano, L., J. Díaz-Nido, F. Wandosell, and J. Avila, 1987. J. Cell Biol. 105: 1731-1739), may consequently have an important role in posttranslational modifications of microtubule proteins required for neuronal differentiation.

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 Full-length cardiac Na+/Ca2+ exchanger 1 protein is not phosphorylated by protein kinase A

2011 ◽  
Vol 300 (5) ◽  
pp. C989-C997 ◽  
Author(s):  
Pimthanya Wanichawan ◽  
William E. Louch ◽  
Kristin H. Hortemo ◽  
Bjørg Austbø ◽  
Per Kristian Lunde ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Caspase 3 ◽  
Fluorescent Protein ◽  
Mutational Analysis ◽  
Phosphorylation Site ◽  
Full Length ◽  
Kinase A

The cardiac Na+/Ca2+ exchanger 1 (NCX1) is an important regulator of intracellular Ca2+ homeostasis and cardiac function. Several studies have indicated that NCX1 is phosphorylated by the cAMP-dependent protein kinase A (PKA) in vitro, which increases its activity. However, this finding is controversial and no phosphorylation site has so far been identified. Using bioinformatic analysis and peptide arrays, we screened NCX1 for putative PKA phosphorylation sites. Although several NCX1 synthetic peptides were phosphorylated by PKA in vitro, only one PKA site (threonine 731) was identified after mutational analysis. To further examine whether NCX1 protein could be PKA phosphorylated, wild-type and alanine-substituted NCX1-green fluorescent protein (GFP)-fusion proteins expressed in human embryonic kidney (HEK)293 cells were generated. No phosphorylation of full-length or calpain- or caspase-3 digested NCX1-GFP was observed with purified PKA-C and [γ-32P]ATP. Immunoblotting experiments with anti-PKA substrate and phosphothreonine-specific antibodies were further performed to investigate phosphorylation of endogenous NCX1. Phospho-NCX1 levels were also not increased after forskolin or isoproterenol treatment in vivo, in isolated neonatal cardiomyocytes, or in total heart homogenate. These data indicate that the novel in vitro PKA phosphorylation site is inaccessible in full-length as well as in calpain- or caspase-3 digested NCX1 protein, suggesting that NCX1 is not a direct target for PKA phosphorylation.

scholarly journals Tubulin phosphorylation by casein kinase II is similar to that found in vivo.

1987 ◽  
Vol 105 (4) ◽  
pp. 1731-1739 ◽  
Author(s):  
L Serrano ◽  
J Díaz-Nido ◽  
F Wandosell ◽  
J Avila
Keyword(s):  
Neuroblastoma Cells ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Carboxy Terminal Domain ◽  
Phosphatase Treatment ◽  
Microtubule Protein ◽  
Carboxy Terminal ◽  
Brain Tubulin

Purified brain tubulin subjected to an exhaustive phosphatase treatment can be rephosphorylated by casein kinase II. This phosphorylation takes place mainly on a serine residue, which has been located at the carboxy-terminal domain of the beta-subunit. Interestingly, tubulin phosphorylated by casein kinase II retains its ability to polymerize in accordance with descriptions by other authors of in vivo phosphorylated tubulin. Moreover, the V8 phosphopeptide patterns of both tubulin phosphorylated in vitro by casein kinase II and tubulin phosphorylated in vivo in N2A cells are quite similar, and different from that of tubulin phosphorylated in vitro by Ca/calmodulin-dependent kinase II. On the other hand, we have found an endogenous casein kinase II-like activity in purified brain microtubule protein that uses GTP and ATP as phosphate donors, is inhibited by heparin, and phosphorylates phosphatase-treated tubulin. Thus it appears that a casein kinase II-like activity should be considered a candidate for the observed phosphorylation of beta-tubulin in vivo in brain or neuroblastoma cells.

scholarly journals cdk1- and cdk2-Mediated Phosphorylation of MyoD Ser200 in Growing C2 Myoblasts: Role in Modulating MyoD Half-Life and Myogenic Activity

1999 ◽  
Vol 19 (4) ◽  
pp. 3167-3176 ◽  
Author(s):  
Magali Kitzmann ◽  
Marie Vandromme ◽  
Valerie Schaeffer ◽  
Gilles Carnac ◽  
Jean-Claude Labbé ◽  
...  
Keyword(s):  
Half Life ◽  
Site Directed Mutagenesis ◽  
Cyclin B ◽  
Wild Type ◽  
E Box ◽  
Integral Role ◽  
Phosphopeptide Mapping

ABSTRACT We have examined the role of protein phosphorylation in the modulation of the key muscle-specific transcription factor MyoD. We show that MyoD is highly phosphorylated in growing myoblasts and undergoes substantial dephosphorylation during differentiation. MyoD can be efficiently phosphorylated in vitro by either purified cdk1-cyclin B or cdk1 and cdk2 immunoprecipitated from proliferative myoblasts. Comparative two-dimensional tryptic phosphopeptide mapping combined with site-directed mutagenesis revealed that cdk1 and cdk2 phosphorylate MyoD on serine 200 in proliferative myoblasts. In addition, when the seven proline-directed sites in MyoD were individually mutated, only substitution of serine 200 to a nonphosphorylatable alanine (MyoD-Ala200) abolished the slower-migrating hyperphosphorylated form of MyoD, seen either in vitro after phosphorylation by cdk1-cyclin B or in vivo following overexpression in 10T1/2 cells. The MyoD-Ala200 mutant displayed activity threefold higher than that of wild-type MyoD in transactivation of an E-box-dependent reporter gene and promoted markedly enhanced myogenic conversion and fusion of 10T1/2 fibroblasts into muscle cells. In addition, the half-life of MyoD-Ala200 protein was longer than that of wild-type MyoD, substantiating a role of Ser200 phosphorylation in regulating MyoD turnover in proliferative myoblasts. Taken together, our data show that direct phosphorylation of MyoD Ser200 by cdk1 and cdk2 plays an integral role in compromising MyoD activity during myoblast proliferation.

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