scholarly journals Protein Kinase CK2 Phosphorylates a Conserved Motif in the Notch Effector E(spl)-Mγ

2021 ◽  
Author(s):  
Lucas Jozwick ◽  
Ashok Bidwai
Keyword(s):  
Protein Kinase Ck2 ◽  
Large Family ◽  
Conserved Motif ◽  
Conserved Sequence ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
Biochemical Analyses ◽  
Kinase Ck2 ◽  
Enhancer Of Split ◽  
Drosophila Embryos

Abstract Across metazoans, the effects of Notch signaling are mediated via the Enhancer of Split (E(spl)/HES) basic Helix-Loop-Helix-Orange (bHLH-O) repressors. Although conserved, sequence diversity is, in large part, restricted to the C-terminal domain (CtD), which separates the O-domain from the penultimate WRPW motif that binds the co-repressor Groucho. While the kinases CK2 and MAPK target the CtD and regulate Drosophila E(spl)-M8 and mammalian HES6, the generality of this regulation to other E(spl)/HES repressors has remained unknown. To determine the broader impact of phosphorylation on this large family of repressors, we conducted bioinformatics, evolutionary and biochemical analyses. Our studies identify E(spl)-Mγ as a new target of native CK2 purified from Drosophila embryos, reveal that phosphorylation is specific to CK2 and independent of the regulatory CK2-β subunit, and identify that the site of phosphorylation is juxtaposed to the WRPW motif, a feature unique to and conserved in the Mγ homologues over 50x106 years of Drosophila evolution. Thus, a preponderance of E(spl) homologues in Drosophila are targets for CK2, and the distinct positioning of the CK2 and MAPK sites, raises the prospect that phosphorylation underlies functional diversity of bHLH-O proteins.

scholarly journals Hes6 Promotes Cortical Neurogenesis and Inhibits Hes1 Transcription Repression Activity by Multiple Mechanisms

2003 ◽  
Vol 23 (19) ◽  
pp. 6922-6935 ◽  
Author(s):  
Michel-Olivier Gratton ◽  
Elena Torban ◽  
Stephanie Belanger Jasmin ◽  
Francesca M. Theriault ◽  
Michael S. German ◽  
...  
Keyword(s):  
Neuronal Differentiation ◽  
Cortical Neurons ◽  
Transcriptional Repression ◽  
Protein Kinase Ck2 ◽  
Molecular Mechanisms ◽  
Neural Progenitor Cells ◽  
Helix Loop Helix ◽  
Molecular Events ◽  
Kinase Ck2 ◽  
Enhancer Of Split

ABSTRACT Hes1 is a mammalian basic helix-loop-helix transcriptional repressor that inhibits neuronal differentiation together with corepressors of the Groucho (Gro)/Transducin-like Enhancer of split (TLE) family. The interaction of Hes1 with Gro/TLE is mediated by a WRPW tetrapeptide present in all Hairy/Enhancer of split (Hes) family members. In contrast to Hes1, the related protein Hes6 promotes neuronal differentiation. Little is known about the molecular mechanisms that underlie the neurogenic activity of Hes6. It is shown here that Hes6 antagonizes Hes1 function by two mechanisms. Hes6 inhibits the interaction of Hes1 with its transcriptional corepressor Gro/TLE. Moreover, it promotes proteolytic degradation of Hes1. This effect is maximal when both Hes1 and Hes6 contain the WRPW motif and is reduced when Hes6 is mutated to eliminate a conserved site (Ser183) that can be phosphorylated by protein kinase CK2. Consistent with these findings, Hes6 inhibits Hes1-mediated transcriptional repression in cortical neural progenitor cells and promotes the differentiation of cortical neurons, a process that is normally inhibited by Hes1. Mutation of Ser183 impairs the neurogenic ability of Hes6. Taken together, these findings clarify the molecular events underlying the neurogenic function of Hes6 and suggest that this factor can antagonize Hes1 activity by multiple mechanisms.

The bHLH gene Hes6, an inhibitor of Hes1, promotes neuronal differentiation

Development ◽  
2000 ◽  
Vol 127 (13) ◽  
pp. 2933-2943 ◽  
Author(s):  
S. Bae ◽  
Y. Bessho ◽  
M. Hojo ◽  
R. Kageyama
Keyword(s):  
Cell Differentiation ◽  
Neuronal Differentiation ◽  
Rod Photoreceptor ◽  
Bhlh Gene ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
Differentiated Cells ◽  
Loop Region ◽  
The Family ◽  
Enhancer Of Split

We have isolated the basic helix-loop-helix (bHLH) gene Hes6, a novel member of the family of mammalian homologues of Drosophila hairy and Enhancer of split. Hes6 is expressed by both undifferentiated and differentiated cells, unlike Hes1, which is expressed only by the former cells. Hes6 alone does not bind to the DNA but suppresses Hes1 from repressing transcription. In addition, Hes6 suppresses Hes1 from inhibiting Mash1-E47 heterodimer and thereby enables Mash1 and E47 to upregulate transcription in the presence of Hes1. Furthermore, misexpression of Hes6 with retrovirus in the developing retina promotes rod photoreceptor differentiation, like Mash1, in sharp contrast to Hes1, which inhibits cell differentiation. These results suggest that Hes6 is an inhibitor of Hes1, supports Mash1 activity and promotes cell differentiation. Mutation analysis revealed that Hes1- and Hes6-specific functions are, at least in part, interchangeable by alteration of the loop region, suggesting that the loop is not simply a nonfunctional spacer but plays an important role in the specific functions.

scholarly journals The Notch signalling pathway is required for Enhancer of split bHLH protein expression during neurogenesis in the Drosophila embryo

Development ◽  
1994 ◽  
Vol 120 (12) ◽  
pp. 3537-3548 ◽  
Author(s):  
B. Jennings ◽  
A. Preiss ◽  
C. Delidakis ◽  
S. Bray
Keyword(s):  
Protein Expression ◽  
Notch Signalling ◽  
Signalling Pathway ◽  
Drosophila Embryo ◽  
Protein Accumulation ◽  
Notch Signalling Pathway ◽  
Cell Fate Decisions ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
Enhancer Of Split

The Enhancer of split locus is required during many cell-fate decisions in Drosophila, including the segregation of neural precursors in the embryo. We have generated monoclonal antibodies that recognise some of the basic helix-loop-helix proteins encoded by the Enhancer of split locus and have used them to examine expression of Enhancer of split proteins during neurogenesis. The proteins are expressed in a dynamic pattern in the ventral neurogenic region and are confined to those ectodermal cells that surround a neuroblast in the process of delaminating. There is no staining in the neuroblasts themselves. We have also examined the relationship between Enhancer of split protein accumulation and the Notch signalling pathway. Protein expression is abolished in a number of neurogenic mutant backgrounds, including Notch, but is increased as a result of expressing a constitutively active Notch product. We conclude that Notch signalling activity is directly responsible for the accumulation of basic helix-loop-helix proteins encoded by the Enhancer of split locus.

scholarly journals Role for Hes1-Induced Phosphorylation in Groucho-Mediated Transcriptional Repression

2002 ◽  
Vol 22 (2) ◽  
pp. 389-399 ◽  
Author(s):  
Hugh N. Nuthall ◽  
Junaid Husain ◽  
Keith W. McLarren ◽  
Stefano Stifani
Keyword(s):  
Protein Kinase ◽  
Dna Binding ◽  
Transcriptional Repression ◽  
Protein Kinase Ck2 ◽  
Protein Complexes ◽  
Transcription Repression ◽  
Tight Association ◽  
Kinase Ck2 ◽  
Enhancer Of Split

ABSTRACT Transcriptional corepressors of the Groucho/transducin-like Enhancer of split (Gro/TLE) family regulate a number of developmental pathways in both invertebrates and vertebrates. They form transcription repression complexes with members of several DNA-binding protein families and participate in the regulation of the expression of numerous genes. Despite their pleiotropic roles, little is known about the mechanisms that regulate the functions of Gro/TLE proteins. It is shown here that Gro/TLEs become hyperphosphorylated in response to neural cell differentiation and interaction with the DNA-binding cofactor Hairy/Enhancer of split 1 (Hes1). Hyperphosphorylation of Gro/TLEs is correlated with a tight association with the nuclear compartment through interaction with chromatin, suggesting that hyperphosphorylated Gro/TLEs may mediate transcriptional repression via chromatin remodeling mechanisms. Pharmacological inhibition of protein kinase CK2 reduces the Hes1-induced hyperphosphorylation of Gro/TLEs and causes a decrease in the chromatin association of the latter. Moreover, the transcription repression activity of Gro/TLEs is reduced by protein kinase CK2 inhibition. Consistent with these observations, Gro/TLEs are phosphorylated in vitro by purified protein kinase CK2. Taken together, these results implicate protein kinase CK2 in Gro/TLE functions. They suggest further that this kinase is involved in a hyperphosphorylation mechanism activated by Hes1 that promotes the transcription repression functions of Hes1-Gro/TLE protein complexes.

The basic helix-loop-helix region of the transcriptional repressor hairy and enhancer of split 1 is preorganized to bind DNA

2014 ◽  
Vol 82 (4) ◽  
pp. 537-545 ◽  
Author(s):  
Matija Popovic ◽  
Hans Wienk ◽  
Maristella Coglievina ◽  
Rolf Boelens ◽  
Sándor Pongor ◽  
...  
Keyword(s):  
Transcriptional Repressor ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
Enhancer Of Split

scholarly journals Specificity for the hairy/enhancer of split basic helix-loop-helix (bHLH) proteins maps outside the bHLH domain and suggests two separable modes of transcriptional repression.

1995 ◽  
Vol 15 (12) ◽  
pp. 6923-6931 ◽  
Author(s):  
S R Dawson ◽  
D L Turner ◽  
H Weintraub ◽  
S M Parkhurst
Keyword(s):  
Transcriptional Repression ◽  
Domain Swapping ◽  
Functional Domain ◽  
Transcriptional Repressors ◽  
Wild Type ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
Evolutionarily Conserved ◽  
Bhlh Proteins ◽  
Enhancer Of Split

The Hairy/Enhancer of split/Deadpan family of basic helix-loop-helix (bHLH) proteins function as transcriptional repressors. We have examined the mechanisms of repression used by the Hairy and E(SPL) proteins by assaying the antagonism between wild-type or altered Hairy/E(SPL) and Scute bHLH proteins during sex determination in Drosophila melanogaster. Domain swapping and mutagenesis of the Hairy and E(SPL) proteins show that three evolutionarily conserved domains are required for their function: the bHLH, Orange, and WRPW domains. However, the suppression of Scute activity by Hairy does not require the WRPW domain. We show that the Orange domain is an important functional domain that confers specificity among members of the Hairy/E(SPL) family. In addition, we show that a Xenopus Hairy homology conserves not only Hairy's structure but also its biological activity in our assays. We propose that transcriptional repression by the Hairy/E(SPL) family of bHLH proteins involves two separable mechanisms: repression of specific transcriptional activators, such as Scute, through the bHLH and Orange domains and repression of other activators via interaction of the C-terminal WRPW motif with corepressors, such as the Groucho protein.

scholarly journals Identification of a Domain in Human Immunodeficiency Virus Type 1 Rev That Is Required for Functional Activity and Modulates Association with Subnuclear Compartments Containing Splicing Factor SC35

2000 ◽  
Vol 74 (24) ◽  
pp. 11899-11910 ◽  
Author(s):  
Donna M. D'Agostino ◽  
Tiziana Ferro ◽  
Lorenza Zotti ◽  
Flavio Meggio ◽  
Lorenzo A. Pinna ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Protein Kinase ◽  
Nuclear Export ◽  
Protein Kinase Ck2 ◽  
Rna Binding ◽  
Nuclear Bodies ◽  
Helix Loop Helix ◽  
Loop Region ◽  
Immunodeficiency Virus ◽  
Kinase Ck2

ABSTRACT The activity of human immunodeficiency virus Rev as a regulator of viral mRNA expression is tightly linked to its ability to shuttle between the nucleus and cytoplasm; these properties are conferred by a leucine-rich nuclear export signal (NES) and by an arginine-rich nuclear localization signal/RNA binding domain (NLS/RBD) required for binding to the Rev-responsive element (RRE) located on viral unspliced and singly spliced mRNAs. Structure predictions and biophysical measurements indicate that Rev consists of an unstructured region followed by a helix-loop-helix motif containing the NLS/RBD and sequences directing multimerization and by a carboxy-terminal tail containing the NES. We present evidence that the loop portion of the helix-loop-helix region is an essential functional determinant that is required for binding to the RRE and for correct intracellular routing. Data obtained using a protein kinase CK2 phosphorylation assay indicated that the loop region is essential for juxtaposition of helices 1 and 2 and phosphorylation by protein kinase CK2. Deletion of the loop resulted in partial accumulation of Rev in SC35-positive nuclear bodies that resembled nuclear bodies that form in response to inhibition of transcription. Accumulation of the ΔLoop mutant in nuclear bodies depended on the presence of an intact NES, suggesting that both the loop and the NES play a role in controlling intranuclear compartmentalization of Rev and its association with splicing factors.

scholarly journals Target Specificities of DrosophilaEnhancer of split Basic Helix-Loop-Helix Proteins

1999 ◽  
Vol 19 (7) ◽  
pp. 4600-4610 ◽  
Author(s):  
Barbara H. Jennings ◽  
David M. Tyler ◽  
Sarah J. Bray
Keyword(s):  
Dna Sequences ◽  
Target Genes ◽  
Notch Signalling Pathway ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
Dna Binding Site ◽  
E Box ◽  
The Individual ◽  
Enhancer Of Split

ABSTRACT Seven Enhancer of split genes in Drosophila melanogaster encode basic-helix-loop-helix transcription factors which are components of the Notch signalling pathway. They are expressed in response to Notch activation and mediate some effects of the pathway by regulating the expression of target genes. Here we have determined that the optimal DNA binding site for the Enhancer of split proteins is a palindromic 12-bp sequence, 5′-TGGCACGTG(C/T)(C/T)A-3′, which contains an E-box core (CACGTG). This site is recognized by all of the individual Enhancer of split basic helix-loop-helix proteins, consistent with their ability to regulate similar target genes in vivo. We demonstrate that the 3 bp flanking the E-box core are intrinsic to DNA recognition by these proteins and that the Enhancer of split and proneural proteins can compete for binding on specific DNA sequences. Furthermore, the regulation conferred on a reporter gene in Drosophila by three closely related sequences demonstrates that even subtle sequence changes within an E box or flanking bases have dramatic consequences on the overall repertoire of proteins that can bind in vivo.

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