Related Experiment Video

Updated: Jul 19, 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

Identification of a novel phosphorylation site in protein phosphatase inhibitor-1 as a negative regulator of cardiac

Patricia Rodriguez1, Bryan Mitton, Jason R Waggoner

  • 1Department of Pharmacology and Cell Biophysics, College of Medicine, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, OH 45267, USA.

The Journal of Biological Chemistry
|October 19, 2006
PubMed

Insights

A new phosphorylation site on protein phosphatase inhibitor-1 (threonine 75) was discovered. This finding reveals a novel mechanism regulating cardiac contractility and heart failure progression.

Area of Science:

  • Cardiovascular Biology
  • Molecular Cardiology
  • Protein Phosphorylation

Background:

  • Heart failure is linked to increased type-1 protein phosphatase activity.
  • Protein phosphatase inhibitor-1 regulates this activity and integrates cAMP and calcium signaling.
  • Known phosphorylation sites on inhibitor-1 include threonine 35 and serine 67.

Purpose of the Study:

  • To identify novel regulatory mechanisms of protein phosphatase inhibitor-1.
  • To investigate the role of inhibitor-1 phosphorylation in cardiac function.

Main Methods:

  • Utilized recombinant wild-type and mutated inhibitor-1 proteins.
  • Generated specific antibodies for phosphorylated threonine 75.
  • Employed adenoviral-mediated gene transfer in isolated myocytes.

Main Results:

  • Identified threonine 75 as a novel phosphorylation site on inhibitor-1, phosphorylated by protein kinase Calpha.
  • Confirmed threonine 75 phosphorylation in rat and dog hearts.
  • Constitutive phosphorylation of threonine 75 in myocytes stimulated type-1 protein phosphatase activity and impaired calcium pump function, reducing contractility.

Conclusions:

  • Phosphorylation of inhibitor-1 at threonine 75 is a newly identified mechanism in cardiac contractility regulation.
  • This phosphorylation impacts sarcoplasmic reticulum calcium transport.
  • Alterations in inhibitor-1 phosphorylation may contribute to the pathophysiology of heart failure.

More Related Videos

Development and Application of Rapamycin-regulated Tyrosine Phosphatases06:56

Development and Application of Rapamycin-regulated Tyrosine Phosphatases

Published on: September 6, 2024

Oligopeptide Competition Assay for Phosphorylation Site Determination09:16

Oligopeptide Competition Assay for Phosphorylation Site Determination

Published on: May 18, 2017

Related Concept Videos

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...
Transducer Mechanism: Enzyme-Linked Receptors01:27

Transducer Mechanism: Enzyme-Linked Receptors

Enzyme-linked receptors are cell-surface receptors acting as an enzyme or associating with an enzyme intracellularly. They make excellent drug targets. Drugs can bind to the extracellular ligand-binding domain or directly affect their enzymatic domain and alter their activity.
Major types that are helpful drug targets include:
The JAK-STAT Signaling Pathway01:20

The JAK-STAT Signaling Pathway

Several cytokine receptors have tightly bound Janus kinase or JAK proteins attached at their cytosolic tail. Small signaling molecules such as cytokines, growth hormones, or prolactins bind to the cytokine receptors and initiate their dimerization. The dimerization brings the cytosolic JAKs together that trans-phosphorylate and activates each other. The activated JAKs now phosphorylate cytosolic tails of the cytokine receptors, which serve as binding sites for adaptor proteins such as  SH2...
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...
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...
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,...