Related Experiment Video

Updated: Jul 16, 2026

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation
09:40

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

Published on: June 27, 2017

Protein kinase D induces transcription through direct phosphorylation of the cAMP-response element-binding protein

Mona Johannessen1, Marit Pedersen Delghandi, An Rykx

  • 1Department of Microbiology and Virology, Institute of Medical Biology, University of Tromsø, N-9037 Tromsø, Norway.

The Journal of Biological Chemistry
|March 29, 2007
PubMed

Insights

Protein kinase D (PKD) directly phosphorylates the transcription factor cAMP-response element-binding protein (CREB) at Ser-133. This novel PKD signaling pathway enhances CREB-mediated gene expression, impacting cellular processes.

Area of Science:

  • Molecular Biology
  • Cell Signaling
  • Biochemistry

Background:

  • Protein kinase D (PKD) is a serine/threonine kinase family involved in diverse cellular functions like proliferation, apoptosis, and protein trafficking.
  • Despite its broad roles, the specific in vivo substrates for PKD remain largely unidentified.
  • Understanding PKD substrates is crucial for elucidating its signaling pathways.

Purpose of the Study:

  • To identify direct in vivo substrates of Protein kinase D (PKD).
  • To investigate the interaction and functional consequences of PKD on the transcription factor cAMP-response element-binding protein (CREB).
  • To elucidate a novel signaling mechanism involving PKD and CREB.

Main Methods:

  • Co-immunoprecipitation assays to demonstrate interaction between PKD1 and CREB.
  • In vitro and in vivo kinase assays to assess CREB phosphorylation at Ser-133 by activated PKD1.
  • Reporter gene assays using GAL4-CREB constructs and CRE-responsive promoters to measure transcriptional activity.
  • Analysis of CREB target gene expression.

Main Results:

  • The transcription factor cAMP-response element-binding protein (CREB) was identified as a direct substrate for PKD.
  • PKD1 and CREB were shown to interact in cells, with activated PKD1 phosphorylating CREB at Ser-133 both in vitro and in vivo.
  • A constitutively active PKD1 mutant stimulated CREB-mediated transcription in a Ser-133-dependent manner, activating CRE-responsive promoters and increasing target gene expression.
  • PKD1 also enhanced transcription mediated by ATF-1 and CREM, other members of the CREB family.

Conclusions:

  • PKD directly phosphorylates CREB at Ser-133, establishing a novel mechanism for PKD-mediated signaling.
  • This pathway regulates CREB transcriptional activity and the expression of CREB target genes.
  • The findings suggest that Protein kinase C-mediated CREB phosphorylation may involve downstream activation of PKD, offering new insights into cellular signaling networks.

More Related Videos

Oligopeptide Competition Assay for Phosphorylation Site Determination09:16

Oligopeptide Competition Assay for Phosphorylation Site Determination

Published on: May 18, 2017

Assaying Protein Kinase Activity with Radiolabeled ATP08:05

Assaying Protein Kinase Activity with Radiolabeled ATP

Published on: May 26, 2017

Related Concept Videos

cAMP-dependent Protein Kinase Pathways01:25

cAMP-dependent Protein Kinase Pathways

Cyclic Adenosine Monophosphate (cAMP) is an essential second messenger that activates protein kinase A (PKA) and regulates various biological processes. A single epinephrine molecule binds to GPCR and activates several heterotrimeric G proteins, each stimulating multiple adenylyl cyclase, amplifying the signal, and synthesizing large numbers of cAMP molecules. Small changes in cAMP concentration affect PKA activity. The binding of four cAMP molecules induces a conformational change in PKA,...
MAPK Signaling Cascades01:07

MAPK Signaling Cascades

Mitogen-activated protein kinase, or MAPK pathway, activates three sequential kinases to regulate cellular responses such as proliferation, differentiation, survival, and apoptosis. The canonical MAPK pathway starts with a mitogen or growth factor binding to an RTK. The activated RTKs stimulate Ras, which recruits Raf or MAP3 Kinase (MAPKKK), the first kinase of the MAPK signaling cascade. Raf further phosphorylates and activates MEK or MAP2 Kinases (MAPKK), which in turn phosphorylates MAP...
PI3K/mTOR/AKT Signaling Pathway01:22

PI3K/mTOR/AKT Signaling Pathway

The mammalian target of rapamycin  (mTOR) is a serine/threonine kinase that regulates growth, proliferation, and cell survival in response to hormones, growth factors, or nutrient availability. This kinase exists in two structurally and functionally distinct forms: mTOR complex 1  (mTORC1) and mTOR complex 2  (mTORC2). The first form (mTORC1) is composed of a rapamycin-sensitive Raptor and proline-rich Akt substrate, PRAS40. In contrast,  mTORC2 consists of a rapamycin-insensitive companion...
RNA Polymerase II Accessory Proteins02:36

RNA Polymerase II Accessory Proteins

Proteins that regulate transcription can do so either via direct contact with RNA Polymerase or through indirect interactions facilitated by adaptors, mediators, histone-modifying proteins, and nucleosome remodelers. Direct interactions to activate transcription is seen in bacteria as well as in some eukaryotic genes. In these cases, upstream activation sequences are adjacent to the promoters, and the activator proteins interact directly with the transcriptional machinery. For example, in...
Amplifying Signals via Enzymatic Cascade01:22

Amplifying Signals via Enzymatic Cascade

When a ligand binds to a cell-surface receptor, the receptor's intracellular domain changes shape, which may either activate its enzyme function or allow its binding to other molecules. The initial signal is amplified by most signal transduction pathways. This means that a single ligand molecule can activate multiple molecules of a downstream target. Proteins that relay a signal are most commonly phosphorylated at one or more sites, activating or inactivating the protein. Kinases catalyze the...
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,...