Related Experiment Video

Updated: Aug 14, 2026

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
12:26

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Published on: May 3, 2018

Phosphorylation of CCAAT-enhancer binding protein by protein kinase C attenuates site-selective DNA binding

C W Mahoney1, J Shuman, S L McKnight

  • 1Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.

The Journal of Biological Chemistry
|September 25, 1992
PubMed

Insights

Protein kinase C (PKC) phosphorylation of CCAAT/enhancer binding protein (C/EBP) DNA-binding domains attenuates DNA binding. Specifically, phosphorylation at Ser299 significantly reduces C/EBP

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Protein Kinase Signaling

Background:

  • CCAAT/enhancer binding protein (C/EBP) is a transcription factor crucial for gene regulation.
  • Protein kinase C (PKC) is a family of enzymes involved in various cellular signaling pathways.
  • Post-translational modifications, such as phosphorylation, can modulate protein function and DNA binding activity.

Purpose of the Study:

  • To investigate the effects of protein kinase C (PKC) phosphorylation on the DNA-binding activity of CCAAT/enhancer binding protein (C/EBP).
  • To identify specific phosphorylation sites on C/EBP that influence its interaction with DNA.
  • To determine if DNA binding affects the phosphorylation status of C/EBP.

Main Methods:

  • In vitro phosphorylation of C/EBP DNA-binding domain proteins using PKC.
  • High-performance liquid chromatography (HPLC)-peptide mapping to identify phosphorylated peptides.
  • Analysis of DNA-binding affinity using truncated C/EBP variants.
  • Immunoprecipitation and peptide mapping of phosphorylated intact C/EBP from nuclear extracts.

Main Results:

  • PKC phosphorylation of C/EBP DNA-binding domains resulted in attenuated binding to CCAAT oligodeoxynucleotides.
  • Three major phosphorylated peptides were identified, with phosphorylation occurring at Ser299, Ser277, and Ser248.
  • Phosphorylation at Ser299 was critical for the observed attenuation of DNA binding; mutating Ser299 to Cys abolished this effect.
  • PKC could not phosphorylate Ser299 if the C/EBP protein was already bound to DNA.
  • Intact C/EBP from liver nuclear extract showed phosphorylation at Ser299, Ser277, and an additional site.

Conclusions:

  • Phosphorylation of C/EBP by PKC significantly impairs its DNA-binding capability.
  • Serine 299 is a key site for inhibitory phosphorylation by PKC, affecting C/EBP's interaction with CCAAT sequences.
  • The ability of C/EBP to bind DNA may regulate its phosphorylation status, suggesting a feedback mechanism.

More Related Videos

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers10:28

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Published on: September 20, 2018

CD Spectroscopy to Study DNA-Protein Interactions06:48

CD Spectroscopy to Study DNA-Protein Interactions

Published on: February 10, 2022

Related Concept Videos

Phosphorylation01:02

Phosphorylation

The addition or removal of phosphate groups from proteins is the most common chemical modification that regulates cellular processes. These modifications can affect the structure, activity, stability, and localization of proteins within cells as well as their interactions with other proteins.
During phosphorylation, protein kinases transfer the terminal phosphate group of ATP to specific amino acid side chains of substrate proteins. Serine, threonine, and tyrosine are the most commonly...
Protein Kinases and Phosphatases02:54

Protein Kinases and Phosphatases

Proteins undergo chemical modifications that trigger changes in the charge, structure, and conformation of the proteins. Phosphorylation, acetylation, glycosylation, nitrosylation, ubiquitination, lipidation, methylation, and proteolysis are various protein modifications that regulate protein activity. Such modifications are usually enzyme-driven.
Protein kinases
Many proteins in the cell are regulated by phosphorylation, the addition of a phosphate group. A family of enzymes called kinases...
Covalently Linked Protein Regulators02:04

Covalently Linked Protein Regulators

Proteins can undergo many types of post-translational modifications, often in response to changes in their environment. These modifications play an important role in the function and stability of these proteins. Covalently linked molecules include functional groups, such as methyl, acetyl, and phosphate groups, and also small proteins, such as ubiquitin. There are around 200 different types of covalent regulators that have been identified.
These groups modify specific amino acids in a protein.
Cell Specific Gene Expression01:58

Cell Specific Gene Expression

Multicellular organisms contain a variety of structurally and functionally distinct cell types, but the DNA in all the cells originated from the same parent cells. The differences in the cells can be attributed to the differential gene expression. Liver cells, whose functions include detoxification of blood, production of bile to metabolize fats, and synthesis of proteins essential for metabolism, must express a specific set of genes to perform their functions. Gene expression also varies with...
Allosteric Proteins-ATCase01:19

Allosteric Proteins-ATCase

Binding sites linkages can regulate a protein's function.  For example, enzyme activity is often regulated through a feedback mechanism where the end product of the biochemical process serves as an inhibitor.
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to  N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis pathway,...
Calmodulin-dependent Signaling01:16

Calmodulin-dependent Signaling

Calmodulin (CaM) is a calcium-binding protein in eukaryotes that controls various calcium-regulated cellular processes. It has four calcium-binding sites that bind calcium to form the calcium-calmodulin ( Ca2+-CaM) complex. GPCR stimulation increases the calcium levels in the cells that bind to CaM and induces a conformational change.
The Ca2+-CaM complex does not have enzymatic activity by itself. Instead, the complex binds downstream target proteins, including membrane proteins or enzymes,...