Sequestration of phosphoinositides by mutated MARCKS effector domain inhibits stimulated Ca(2+) mobilization and degranulation in mast cells.
Protein kinase C β (PKCβ) participates in antigen-stimulated mast cell degranulation mediated by the high-affinity receptor for immunoglobulin E, FcεRI, but the molecular basis is unclear. We investigated the hypothesis that the polybasic effector domain (ED) of the abundant intracellular substrate for protein kinase C known as myristoylated alanine-rich protein kinase C substrate (MARCKS) sequesters phosphoinositides at the inner leaflet of the plasma membrane until MARCKS dissociates after phosphorylation by activated PKC. Real-time fluorescence imaging confirms synchronization between stimulated oscillations of intracellular Ca(2+) concentrations and oscillatory association of PKCβ-enhanced green fluorescent protein with the plasma membrane. Similarly, MARCKS-ED tagged with monomeric red fluorescent protein undergoes antigen-stimulated oscillatory dissociation and rebinding to the plasma membrane with a time course that is synchronized with reversible plasma membrane association of PKCβ. We find that MARCKS-ED dissociation is prevented by mutation of four serine residues that are potential sites of phosphorylation by PKC. Cells expressing this mutated MARCKS-ED SA4 show delayed onset of antigen-stimulated Ca(2+) mobilization and substantial inhibition of granule exocytosis. Stimulation of degranulation by thapsigargin, which bypasses inositol 1,4,5-trisphosphate production, is also substantially reduced in the presence of MARCKS-ED SA4, but store-operated Ca(2+) entry is not inhibited. These results show the capacity of MARCKS-ED to regulate granule exocytosis in a PKC-dependent manner, consistent with regulated sequestration of phosphoinositides that mediate granule fusion at the plasma membrane.
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA.
Deepti
D
Gadi
Alice
A
Wagenknecht-Wiesner
David
D
Holowka
Barbara
B
Baird
D000818:Animals; D000941:Antigens / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D015550:Cell Degranulation / Q000502:physiology*; D002460:Cell Line; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D054878:Lipoylation / Q000502:physiology; D008407:Mast Cells / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009154:Mutation; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010716:Phosphatidylinositols / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism; D064546:Protein Kinase C beta; D017434:Protein Structure, Tertiary; D022142:Secretory Vesicles / Q000235:genetics / Q000378:metabolism*
D000941:Antigens; D047908:Intracellular Signaling Peptides and Proteins; C000619711:Marcks protein, mouse; D008565:Membrane Proteins; D010716:Phosphatidylinositols; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D002118:Calcium