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

J Robert Manak; Ron Prywes

doi:10.1128/mcb.11.7.3652-3659.1991

Back

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

Journal article

Peer reviewed

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

J Robert Manak and Ron Prywes

Molecular and cellular biology, Vol.11(7), pp.3652-3659

07/1991

DOI: 10.1128/mcb.11.7.3652-3659.1991

PMCID: PMC361119

PMID: 2046671

View Online

Abstract

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.

Phosphorylation

Plasmids

Protein Kinases - metabolism

Recombinant Proteins - metabolism

Amino Acid Sequence

Oligonucleotide Probes

Mutagenesis, Site-Directed

Casein Kinases

Nuclear Proteins - isolation & purification

Molecular Sequence Data

Nuclear Proteins - metabolism

DNA-Binding Proteins - genetics

DNA-Binding Proteins - isolation & purification

Macromolecular Substances

DNA-Binding Proteins - metabolism

Recombinant Proteins - isolation & purification

Serum Response Factor

Transcription Factors - metabolism

Animals

Base Sequence

Protein Conformation

Nuclear Proteins - genetics

Details

Title: Subtitle: Mutation of serum response factor phosphorylation sites and the mechanism by which its DNA-binding activity is increased by casein kinase II
Creators: J Robert Manak - Department of Biological Sciences, Columbia University, New York, New York 10027
Ron Prywes
Resource Type: Journal article
Publication Details: Molecular and cellular biology, Vol.11(7), pp.3652-3659
DOI: 10.1128/mcb.11.7.3652-3659.1991
PMID: 2046671
PMCID: PMC361119
NLM abbreviation: Mol Cell Biol
ISSN: 0270-7306
eISSN: 1098-5549
Publisher: United States
Grant note: CA 50329-01 / NCI NIH HHS
Language: English
Date published: 07/1991
Academic Unit: Stead Family Department of Pediatrics; Biology; Craniofacial Anomalies Research Center
Record Identifier: 9983991946002771

Metrics

13 Record Views

94 Times Cited - Web of Science