Related Experiment Video

Updated: Jul 17, 2026

A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors
10:17

A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors

Published on: April 29, 2022

Phosphatase type 2A-dependent and -independent pathways for ATR phosphorylation of Chk1

Ge Li1, Robert T Elder, Kefeng Qin

  • 1Department of Pathology, Department of Microbiology-Immunology, Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.

The Journal of Biological Chemistry
|January 11, 2007
PubMed

Insights

Human immunodeficiency virus-1 Vpr protein uniquely utilizes protein phosphatase type 2A (PP2A) to activate ATR kinase for cell cycle arrest. This contrasts with hydroxyurea and UV, which activate ATR independently of PP2A.

Area of Science:

  • Cellular Biology
  • Virology
  • Molecular Biology

Background:

  • ATM and Rad3-related (ATR) kinase regulates cell cycle arrest via Chk1 phosphorylation.
  • ATR activation can be induced by hydroxyurea, UV radiation, or human immunodeficiency virus-1 Vpr protein.

Purpose of the Study:

  • To investigate the specific mechanisms by which Vpr activates ATR compared to other known activators.
  • To determine the role of protein phosphatase type 2A (PP2A) in Vpr-mediated ATR activation.

Main Methods:

  • Utilized PP2A-specific inhibitor okadaic acid.
  • Employed small interference RNA (siRNA) to down-regulate specific PP2A subunits (PP2A(Cbeta) and PP2A(Aalpha)).
  • Assessed phosphorylation of Chk1-Ser(345), Cdk1-Tyr(15), and gammaH2AX-Ser(139), as well as cell cycle arrest and Claspin expression.

Main Results:

  • Vpr-induced G(2) arrest and Chk1-Ser(345) phosphorylation are dependent on PP2A, unlike hydroxyurea or UV.
  • Down-regulation of PP2A(Cbeta) and PP2A(Aalpha) specifically impaired Vpr-induced ATR signaling.
  • PP2A subunits did not affect ATR-mediated gammaH2AX phosphorylation by any agent.
  • Vpr expression increased Claspin amount and phosphorylation, with PP2A down-regulation reducing Claspin levels.

Conclusions:

  • Vpr uniquely employs PP2A for ATR activation, distinct from hydroxyurea and UV.
  • PP2A subunits PP2A(Cbeta) and PP2A(Aalpha) are crucial for Vpr-mediated G(2) arrest and Chk1 phosphorylation.
  • Claspin may be a key downstream target linking PP2A activity to Vpr-induced Chk1 phosphorylation by ATR.

More Related Videos

Assaying Protein Kinase Activity with Radiolabeled ATP08:05

Assaying Protein Kinase Activity with Radiolabeled ATP

Published on: May 26, 2017

Oligopeptide Competition Assay for Phosphorylation Site Determination09:16

Oligopeptide Competition Assay for Phosphorylation Site Determination

Published on: May 18, 2017

Related Concept Videos

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...
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...
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...
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...
Amplifying Signals via Second Messengers01:15

Amplifying Signals via Second Messengers

Many receptor binding ligands are hydrophilic; they do not cross the cell membrane but bind to cell-surface receptors. Thus, their message must be relayed by second messengers present in the cell cytoplasm. There are several second messenger pathways, each with its own way of relaying information. For example, the G protein-coupled receptors can activate both phosphoinositol and cyclic AMP (cAMP) second messenger pathways. The phosphoinositol pathway is active when the receptor induces...
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,...