Mutation of serum response factor phosphorylation sites and the mechanism by which its DNA-binding activity is increased by casein kinase II

1991 ◽  
Vol 11 (7) ◽  
pp. 3652-3659
Author(s):  
J R Manak ◽  
R Prywes
Keyword(s):  
Dna Binding ◽  
Serum Response Factor ◽  
Casein Kinase Ii ◽  
Binding Activity ◽  
Casein Kinase ◽  
Binding Domain ◽  
Response Factor ◽  
Dna Binding Domain ◽  
Dna Binding Activity ◽  
Serum Response

Casein kinase II (CKII) phosphorylates the mammalian transcription factor serum response factor (SRF) on a serine residue(s) located within a region of the protein spanning amino acids 70 to 92, thereby enhancing its DNA-binding activity in vitro. We report here that serine 83 appears to be the residue phosphorylated by CKII but that three other serines in this region can also be involved in phosphorylation and the enhancement of DNA-binding activity. A mutant that contained glutamate residues in place of these serines had only low-level binding activity; however, when the serines were replaced with glutamates and further mutations were made that increased the negative charge of the region, the resulting mutant showed a constitutively high level of binding equal to that achieved by phosphorylation of wild-type SRF. We have investigated the mechanism by which phosphorylation of SRF increases its DNA-binding activity. We have ruled out the possibilities that phosphorylation affects SRF dimerization or relieves inhibition due to masking of the DNA-binding domain by an amino-terminal region of the protein. Rather, using partial proteolysis to probe SRF's structure, we find that the conformation of SRF's DNA-binding domain is altered by phosphorylation.

scholarly journals Mutation of serum response factor phosphorylation sites and the mechanism by which its DNA-binding activity is increased by casein kinase II.

1991 ◽  
Vol 11 (7) ◽  
pp. 3652-3659 ◽  
Author(s):  
J R Manak ◽  
R Prywes
Keyword(s):  
Dna Binding ◽  
Serum Response Factor ◽  
Casein Kinase Ii ◽  
Binding Activity ◽  
Casein Kinase ◽  
Binding Domain ◽  
Response Factor ◽  
Dna Binding Domain ◽  
Dna Binding Activity ◽  
Serum Response

Casein kinase II (CKII) phosphorylates the mammalian transcription factor serum response factor (SRF) on a serine residue(s) located within a region of the protein spanning amino acids 70 to 92, thereby enhancing its DNA-binding activity in vitro. We report here that serine 83 appears to be the residue phosphorylated by CKII but that three other serines in this region can also be involved in phosphorylation and the enhancement of DNA-binding activity. A mutant that contained glutamate residues in place of these serines had only low-level binding activity; however, when the serines were replaced with glutamates and further mutations were made that increased the negative charge of the region, the resulting mutant showed a constitutively high level of binding equal to that achieved by phosphorylation of wild-type SRF. We have investigated the mechanism by which phosphorylation of SRF increases its DNA-binding activity. We have ruled out the possibilities that phosphorylation affects SRF dimerization or relieves inhibition due to masking of the DNA-binding domain by an amino-terminal region of the protein. Rather, using partial proteolysis to probe SRF's structure, we find that the conformation of SRF's DNA-binding domain is altered by phosphorylation.

scholarly journals Recruitment of the tinman homolog Nkx-2.5 by serum response factor activates cardiac alpha-actin gene transcription.

1996 ◽  
Vol 16 (11) ◽  
pp. 6372-6384 ◽  
Author(s):  
C Y Chen ◽  
R J Schwartz
Keyword(s):  
Dna Binding ◽  
Serum Response Factor ◽  
Binding Activity ◽  
Dominant Negative ◽  
Actin Gene ◽  
Response Factor ◽  
Dna Binding Activity ◽  
Actin Promoter ◽  
Alpha Actin ◽  
Serum Response

We recently showed that the cardiogenic homeodomain factor Nkx-2.5 served as a positive acting accessory factor for serum response factor (SRF) and that together they provided strong transcriptional activation of the cardiac alpha-actin promoter, depending upon intact serum response elements (SREs) (C. Y. Chen, J. Croissant, M. Majesky, S. Topouz, T. McQuinn, M. J. Frankovsky, and R. J. Schwartz, Dev. Genet. 19:119-130, 1996). As shown here, Nkx-2.5 and SRF collaborated to activate the endogenous murine cardiac alpha-actin gene in 10T1/2 fibroblasts by a mechanism in which SRF recruited Nkx-2.5 to the alpha-actin promoter. Activation of a truncated promoter consisting of the proximal alpha-actin SRE1 occurred even when Nkx-2.5 DNA-binding activity was blocked by a point mutation in the third helix of its homeodomain. Investigation of protein-protein interactions showed that Nkx-2.5 was bound to SRF in the absence of DNA in soluble protein complexes retrieved from cardiac myocyte nuclei but could also be detected in coassociated binding complexes on the proximal SRE1. Recruitment of Nkx-2.5 to an SRE depended upon SRF DNA-binding activity and was blocked by the dominant negative SRFpm1 mutant, which allowed for dimerization of SRF monomers but prevented DNA binding. Interactive regions shared by Nkx-2.5 and SRF were mapped to N-terminal/helix I and helix II/helix III regions of the Nkx-2.5 homeodomain and to the N-terminal extension of the MADS box. Our study suggests that physical association between Nkx-2.5 and SRF is one way that cardiac specified genes are activated in cardiac cell lineages.

Human and Drosophila Homeodomain Proteins That Enhance the DNA-Binding Activity of Serum Response Factor

Science ◽  
1992 ◽  
Vol 257 (5073) ◽  
pp. 1089-1095 ◽  
Author(s):  
D. A. Grueneberg ◽  
S. Natesan ◽  
C. Alexandre ◽  
M. Z. Gilman
Keyword(s):  
Dna Binding ◽  
Serum Response Factor ◽  
Binding Activity ◽  
Response Factor ◽  
Homeodomain Proteins ◽  
Dna Binding Activity ◽  
Serum Response

scholarly journals Identification of transcriptional activation and inhibitory domains in serum response factor (SRF) by using GAL4-SRF constructs

1993 ◽  
Vol 13 (8) ◽  
pp. 4640-4647
Author(s):  
F E Johansen ◽  
R Prywes
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Serum Response Factor ◽  
Binding Domain ◽  
Response Factor ◽  
Dna Binding Domain ◽  
Activation Domain ◽  
Transcriptional Activation Domain ◽  
Serum Response

The binding of serum response factor (SRF) to the c-fos serum response element has been shown to be essential for serum and growth factor activation of c-Fos. Since SRF is ubiquitously expressed, it has been difficult to measure the activity of SRF introduced into cells. To assay for functions of SRF in cells, we have changed its DNA binding specificity by fusing it to the DNA binding domain of GAL4. Transfection of GAL4-SRF constructs into cells has allowed us to identify SRF's transcriptional activation domain as well as domains which inhibit this activity. First, we found that the transcriptional activation domain maps to between amino acids 339 and 508 in HeLa cells and to between amino acids 414 and 508 in NIH 3T3 cells. Second, we show that in the context of GAL4-SRF constructs, there are two separate domains of SRF that can inhibit its activation domain. Although these domains overlap the DNA binding and dimerization domains of SRF, these functions were not required for inhibition. Finally, we show that one of the inhibitory domains is modular in that it can also inhibit activation when it is moved amino terminal to GAL4's DNA binding domain in an SRF-GAL4-SRF construct. The implications of these inhibitory domains for SRF regulation are discussed.

scholarly journals MAL and Ternary Complex Factor Use Different Mechanisms To Contact a Common Surface on the Serum Response Factor DNA-Binding Domain

2006 ◽  
Vol 26 (11) ◽  
pp. 4134-4148 ◽  
Author(s):  
Alexia-Ileana Zaromytidou ◽  
Francesc Miralles ◽  
Richard Treisman
Keyword(s):  
Dna Binding ◽  
Complex Formation ◽  
Ternary Complex ◽  
Serum Response Factor ◽  
Dna Bending ◽  
Binding Domain ◽  
Response Factor ◽  
Dna Binding Domain ◽  
Serum Response ◽  
Ternary Complex Factor

ABSTRACT The transcription factor serum response factor (SRF) interacts with its cofactor, MAL/MKL1, a member of the myocardin-related transcription factor (MRTF) family, through its DNA-binding domain. We define a seven-residue sequence within the conserved MAL B1 region essential and sufficient for complex formation. The neighboring Q-box sequence facilitates this interaction. The B1 and Q-box regions also have antagonistic effects on MAL nuclear import, but the residues involved are largely distinct. Both MAL and the ternary complex factor (TCF) family of SRF cofactors interact with a hydrophobic groove and pocket on the SRF DNA-binding domain. Unlike the TCFs, however, interaction of MAL with SRF is impaired by SRF αI-helix mutations that reduce DNA bending in the SRF-DNA complex. A clustered SRF αI-helix mutation strongly impairs MAL-SRF complex formation but does not affect DNA distortion in the MAL-SRF complex. MAL-SRF complex formation is facilitated by DNA binding. DNase I footprinting indicates that in the SRF-MAL complex MAL directly contacts DNA. These contacts, which flank the DNA sequences protected from DNase I by SRF, are required for effective MAL-SRF complex formation in gel mobility shift assays. We propose a model of MAL-SRF complex formation in which MAL interacts with SRF by the addition of a β-strand to the SRF DNA-binding domain β-sheet region, while SRF-induced DNA bending facilitates MAL-DNA contact.

scholarly journals Physical Interaction between the MADS Box of Serum Response Factor and the TEA/ATTS DNA-binding Domain of Transcription Enhancer Factor-1

2001 ◽  
Vol 276 (13) ◽  
pp. 10413-10422 ◽  
Author(s):  
Madhu Gupta ◽  
Paul Kogut ◽  
Francesca J. Davis ◽  
Narasimhaswamy S. Belaguli ◽  
Robert J. Schwartz ◽  
...  
Keyword(s):  
Dna Binding ◽  
Serum Response Factor ◽  
Binding Domain ◽  
Physical Interaction ◽  
Response Factor ◽  
Dna Binding Domain ◽  
Mads Box ◽  
Serum Response ◽  
Transcription Enhancer ◽  
Enhancer Factor

scholarly journals Identification of transcriptional activation and inhibitory domains in serum response factor (SRF) by using GAL4-SRF constructs.

1993 ◽  
Vol 13 (8) ◽  
pp. 4640-4647 ◽  
Author(s):  
F E Johansen ◽  
R Prywes
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Serum Response Factor ◽  
Binding Domain ◽  
Response Factor ◽  
Dna Binding Domain ◽  
Activation Domain ◽  
Transcriptional Activation Domain ◽  
Serum Response

The binding of serum response factor (SRF) to the c-fos serum response element has been shown to be essential for serum and growth factor activation of c-Fos. Since SRF is ubiquitously expressed, it has been difficult to measure the activity of SRF introduced into cells. To assay for functions of SRF in cells, we have changed its DNA binding specificity by fusing it to the DNA binding domain of GAL4. Transfection of GAL4-SRF constructs into cells has allowed us to identify SRF's transcriptional activation domain as well as domains which inhibit this activity. First, we found that the transcriptional activation domain maps to between amino acids 339 and 508 in HeLa cells and to between amino acids 414 and 508 in NIH 3T3 cells. Second, we show that in the context of GAL4-SRF constructs, there are two separate domains of SRF that can inhibit its activation domain. Although these domains overlap the DNA binding and dimerization domains of SRF, these functions were not required for inhibition. Finally, we show that one of the inhibitory domains is modular in that it can also inhibit activation when it is moved amino terminal to GAL4's DNA binding domain in an SRF-GAL4-SRF construct. The implications of these inhibitory domains for SRF regulation are discussed.

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