Related Experiment Video

Updated: Jul 14, 2026

Purification of Ubiquitinated p53 Proteins from Mammalian Cells
10:55

Purification of Ubiquitinated p53 Proteins from Mammalian Cells

Published on: March 21, 2022

Phosphorylation of Pirh2 by calmodulin-dependent kinase II impairs its ability to ubiquitinate p53

Shanshan Duan1, Zhan Yao, Dezhi Hou

  • 1Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, People's Republic of China.

The EMBO Journal
|June 15, 2007
PubMed

Insights

Pirh2 protein phosphorylation by CaMK II regulates its E3 ligase activity, impacting p53 stability. This fine-tuning mechanism is crucial for maintaining the p53-Pirh2 feedback loop and tumor suppression.

Area of Science:

  • Molecular Biology
  • Cancer Research
  • Protein Biochemistry

Background:

  • Pirh2 is a p53-induced ubiquitin-protein E3 ligase that negatively regulates p53.
  • The precise regulatory mechanisms controlling Pirh2 activity are not fully understood.
  • Understanding Pirh2 regulation is critical for comprehending p53 stability and tumor suppression.

Purpose of the Study:

  • To elucidate the regulatory mechanism of Pirh2.
  • To investigate the role of Pirh2 phosphorylation in its function.
  • To determine how Pirh2 phosphorylation affects its interaction with p53.

Main Methods:

  • Analysis of Pirh2 phosphorylation status in normal and tumor tissues/cell lines.
  • Identification of the kinase responsible for Pirh2 phosphorylation (CaMK II).
  • Site-directed mutagenesis to pinpoint phosphorylation sites (Thr-154, Ser-155).
  • Assessment of Pirh2 E3 ligase activity following phosphorylation.

Main Results:

  • Pirh2 is predominantly unphosphorylated in tumor cells/tissues, unlike normal tissues.
  • Calmodulin-dependent kinase II (CaMK II) phosphorylates Pirh2 at Thr-154 and Ser-155.
  • Phosphorylated Pirh2 exhibits reduced E3 ligase activity towards p53.
  • Pirh2 phosphorylation is cell cycle-dependent and affects protein stability.

Conclusions:

  • CaMK II-mediated phosphorylation of Pirh2 abrogates its E3 ligase activity.
  • Pirh2 phosphorylation acts as a fine-tuning mechanism in the p53-Pirh2 feedback loop.
  • This regulation is essential for maintaining p53 stability and tumor suppression.

More Related Videos

A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF311:44

A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3

Published on: January 24, 2016

Related Concept Videos

Abnormal Proliferation02:23

Abnormal Proliferation

Under normal conditions, most adult cells remain in a non-proliferative state unless stimulated by internal or external factors to replace lost cells. Abnormal cell proliferation is a condition in which the cell's growth exceeds and is uncoordinated with normal cells. In such situations, cell division persists in the same excessive manner even after cessation of the stimuli, leading to persistent tumors. The tumor arises from the damaged cells that replicate to pass the damage to the daughter...
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,...
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...
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.
Phosphoinositides and PIPs01:42

Phosphoinositides and PIPs

Phosphoinositides are a group of phospholipids containing a glycerol backbone with two fatty acid chains and a phosphate attached to a myoinositol sugar ring. The inositol head group extends into the cytoplasm, where it is modified by adding phosphate groups to form phosphatidylinositol phosphates or PIPs.
Different phosphoinositides are synthesized and recruited on the cytosolic face of the plasma membrane. The localization of specific phosphoinositides concentrated in separate membrane...
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...