Related Experiment Video

Updated: May 28, 2026

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays
11:31

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Published on: July 4, 2018

Sequestration of phosphoinositides by mutated MARCKS effector domain inhibits stimulated Ca(2+) mobilization and

Deepti Gadi1, Alice Wagenknecht-Wiesner, David Holowka

  • 1Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA.

Molecular Biology of the Cell
|October 21, 2011
PubMed

Insights

Myristoylated alanine-rich protein kinase C substrate (MARCKS) effector domain regulates mast cell degranulation by sequestering phosphoinositides. PKCβ-dependent phosphorylation controls MARCKS dissociation, impacting calcium mobilization and granule exocytosis.

Area of Science:

  • Immunology
  • Cell Biology
  • Biochemistry

Background:

  • Protein kinase C beta (PKCβ) is involved in mast cell degranulation via FcεRI signaling.
  • The molecular mechanisms linking PKCβ to degranulation are not fully understood.
  • Myristoylated alanine-rich protein kinase C substrate (MARCKS) is a key intracellular substrate of PKC.

Purpose of the Study:

  • To investigate the role of the MARCKS effector domain (ED) in regulating phosphoinositide sequestration at the plasma membrane.
  • To determine if PKC-mediated phosphorylation of MARCKS controls its dissociation and subsequent mast cell degranulation.
  • To elucidate the molecular basis of PKCβ's participation in FcεRI-stimulated mast cell degranulation.

Main Methods:

  • Real-time fluorescence imaging to monitor intracellular Ca(2+) oscillations and plasma membrane association of PKCβ and MARCKS-ED.
  • Utilizing mutant MARCKS-ED (SA4) with non-phosphorylatable serine residues.
  • Assessing antigen-stimulated Ca(2+) mobilization and granule exocytosis.
  • Investigating degranulation triggered by thapsigargin and store-operated calcium entry.

Main Results:

  • PKCβ association with the plasma membrane is synchronized with intracellular Ca(2+) oscillations.
  • MARCKS-ED dissociation from the plasma membrane is synchronized with PKCβ association and is dependent on PKC phosphorylation sites.
  • Mutant MARCKS-ED SA4 expression delays Ca(2+) mobilization and inhibits antigen-stimulated and thapsigargin-induced degranulation.
  • Store-operated calcium entry remains unaffected by MARCKS-ED SA4.

Conclusions:

  • The MARCKS effector domain regulates granule exocytosis in a PKC-dependent manner.
  • MARCKS-ED acts by sequestering phosphoinositides, which are crucial for granule fusion at the plasma membrane.
  • This study clarifies a key molecular mechanism in FcεRI-mediated mast cell degranulation.

More Related Videos

Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis16:01

Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis

Published on: January 26, 2015

Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice09:07

Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice

Published on: May 27, 2015

Related Concept Videos

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,...
IP3/DAG Signaling Pathway01:11

IP3/DAG Signaling Pathway

Membrane lipids such as phosphatidylinositol (PI) are precursors for several membrane-bound and soluble second messengers. Specific kinases phosphorylate PI and produce phosphorylated inositol phospholipids. One such inositol phospholipids are the  phosphatidylinositol-4,5 bisphosphate [PI(4,5)P2], present in the inner half of the lipid bilayer. Upon ligand binding, GPCR stimulates Gq proteins to turn on phospholipase Cꞵ. Activated phospholipase Cꞵ cleaves PI(4,5)P2 and produces two-second...
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...
GPCR Desensitization01:12

GPCR Desensitization

G protein-coupled receptor (GPCR) signaling plays a crucial role in cell functioning. GPCR desensitization is an equally essential process. It allows cells to respond to changing environments and regain sensitivity to new stimuli while preventing unnecessary stimulation when no longer needed. Prolonged exposure to stimuli leads to GPCR desensitization. It involves blocking the receptors from binding and activating additional G proteins. This inhibits activation of downstream effectors, thereby...
Overview of Secretory Vesicles01:33

Overview of Secretory Vesicles

Secretory vesicles, also known as dense core vesicles (DCVs), are membrane-bound vesicles that transport secretory proteins, such as hormones or neurotransmitters. Regulated secretory vesicles transport proteins from the trans-Golgi network to the exterior of the cell. Proteins present in regulated secretory vesicles are required to be rapidly exocytosed in large amounts upon a specific stimulus.
Various proteins regulate the aggregation of molecules inside the secretory vesicles. Chromogranins...
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...