Related Experiment Video

Updated: Jun 16, 2026

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors
12:40

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

Published on: December 7, 2014

Function of activation loop tyrosine phosphorylation in the mechanism of c-Kit auto-activation and its implication in

Jonathan P DiNitto1, Gayatri D Deshmukh, Yan Zhang

  • 1Pfizer Research Technology Center, 620 Memorial Drive, Cambridge, MA 02139, USA. jdinitto@msn.com

Journal of Biochemistry
|February 12, 2010
PubMed

Insights

Receptor tyrosine kinase (RTK) activation, specifically c-Kit, relies on JM domain phosphorylation, not the activation loop (A-loop). The Y823F mutation highlights A-loop flexibility in drug binding post-activation.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Pharmacology

Background:

  • Receptor tyrosine kinases (RTKs) play crucial roles in cell signaling and are tightly regulated.
  • c-Kit RTK activation involves complex autophosphorylation mechanisms.
  • Understanding RTK activation is vital for targeted cancer therapies.

Purpose of the Study:

  • To investigate the role of specific phosphorylation sites in c-Kit RTK activation.
  • To determine the contribution of the activation loop (A-loop) tyrosine (Y823) to c-Kit kinase activity and drug binding.
  • To elucidate the impact of mutations on c-Kit auto-activation and inhibitor sensitivity.

Main Methods:

  • Kinetic studies of c-Kit RTK activation.
  • Phosphopeptide mapping to identify phosphorylation sites.
  • Site-directed mutagenesis to create loss-of-phosphorylation mutants (Y823F) and drug-resistant mutants (D816H).
  • Binding assays to assess drug affinity (sunitinib, imatinib) to wild-type and mutant c-Kit enzymes in activated and unactivated states.

Main Results:

  • c-Kit activation primarily depends on phosphorylation sites within the JM domain, not the A-loop.
  • Phosphorylation of A-loop tyrosine Y823 occurs late in activation and is not required for kinase auto-activation.
  • The Y823F mutant exhibits normal auto-activation and retains high-affinity binding to sunitinib and imatinib even after activation, unlike wild-type c-Kit.
  • A sunitinib-resistant mutant (D816H) also shows no A-loop phosphorylation upon full activation.

Conclusions:

  • A-loop phosphorylation is dispensable for c-Kit auto-activation.
  • The A-loop tyrosine Y823 does not appear to be a prerequisite for kinase activation but influences drug binding.
  • The Y823F mutation confers sustained drug binding capacity by maintaining an unactivated conformation, suggesting therapeutic implications for inhibitor design.

More Related Videos

Deciphering the Structural Effects of Activating EGFR Somatic Mutations with Molecular Dynamics Simulation15:05

Deciphering the Structural Effects of Activating EGFR Somatic Mutations with Molecular Dynamics Simulation

Published on: May 20, 2020

Related Concept Videos

Receptor Tyrosine Kinases01:26

Receptor Tyrosine Kinases

Receptor tyrosine kinases or RTKs are membrane-bound receptors that phosphorylate specific tyrosine on protein substrates. RTKs regulate cellular growth, differentiation, survival, and migration. They contain an extracellular ligand binding domain, a transmembrane domain, and a cytosolic tail with intrinsic kinase activity. Several extracellular signaling molecules activate RTKs in one or more ways and relay the signal downstream. Ligands such as platelet-derived growth factor (PDGF) or...
mTOR Signaling and Cancer Progression03:03

mTOR Signaling and Cancer Progression

The mammalian target of rapamycin or mTOR protein was discovered in 1994 due to its direct interaction with rapamycin. The protein gets its name from a yeast homolog called TOR. The mTOR protein complex in mammalian cells plays a major role in balancing anabolic processes such as the synthesis of proteins, lipids, and nucleotides and catabolic processes, such as autophagy in response to environmental cues, such as availability of nutrients and growth factors.
The mTOR pathway or the...
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...
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...
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:
Inhibition of Cdk Activity02:34

Inhibition of Cdk Activity

The orderly progression of the cell cycle depends on the activation of Cdk protein by binding to its cyclin partner. However, the cell cycle must be restricted when undergoing abnormal changes. Most cancers correlate to the deregulated cell cycle, and since Cdks are a central component of the cell cycle, Cdk inhibitors are extensively studied to develop anticancer agents. For instance, cyclin D associates with several Cdks, such as Cdk 4/6, to form an active complex. The cyclin D-Cdk4/6 complex...