Chromaffin Cell F-actin Disassembly and Potentiation of Catecholamine Release in Response to Protein Kinase C Activation by Phorbol Esters Is Mediated through Myristoylated Alanine-rich C Kinase Substrate Phosphorylation

Abstract

MARCKS binds and crosslinks F-actin, the latter is inhibited by protein kinase C-induced MARCKS phosphorylation. MARCKS was found in chromaffin cells by immunoblotting. MARCKS was also detected by immunoprecipitation. In intact or permeabilized cells MARCKS phosphorylation increased upon stimulation with 10؊7 M phorbol 12-myristate 13-acetate. This was accompanied by cortical F-actin disassembly and potentiation of secretion. MARCKS phosphorylation, cortical F-actin disassembly, and potentiation of Ca 2؉ -evoked secretion were inhibited by a peptide (MARCKS phosphorylation site domain sequence (MPSD)) with amino acid sequence corresponding to MARCKS phosphorylation site. MPSD was phosphorylated in the process. A similar peptide (alanine-substituted phosphorylated site domain) with four serine residues of MPSD substituted by alanines was ineffective. These results provide the first evidence for MARCKS involvement in chromaffin cell secretion and suggest that regulation of cortical F-actin crosslinking might be involved in this process.

Involvement of Protein Kinase C-ε in Activity-Dependent Potentiation of Large Dense-Core Vesicle Exocytosis in Chromaffin Cells

“…Staining of F-actin was performed in chromaffin cells as described previously (Rose et al, 2001;Park et al, 2006). After washout with PBS, cells were stained for F-actin with 0.5 U/ml rhodamine-phalloidin for 20 min at room temperature.…”

“…Stimulation of nAChR induces cortical F-actin disassembly (Rose et al, 2001), which is regulated by phosphorylation of (Fig. 1 A), cells were subjected to SDS-PAGE.…”

“…Phosphorylation of MARCKS by PKC disrupts the electrostatic interactions between MARCKS and the plasma membrane, thereby inducing translocation of MARCKS to the cytosolic fraction (Arbuzova et al, 2002). In addition, phosphorylation of MARCKS also results in F-actin disassembly, which is associated with vesicle recruitment to the plasma membrane (Rose et al, 2001).…”

Myristoylated alanine rich C kinase substrate (MARCKS) heterozygous mutant mice exhibit deficits in hippocampal mossy fiber-CA3 long-term potentiation

“…F-actin cytoskeletal plasticity is required for vesicular trafficking (Doussau and Augustine, 2000;Morales et al, 2000;Trifaro et al, 2002), dendritic motility (Matus, 2000), and the maintenance phase of LTP (Kim and Lisman, 1999;Krucker et al, 2000;Fukazawa et al, 2003). MARCKS binds and crosslinks F-actin in a PKC phosphorylation-dependent manner (Hartwig et al, 1992;Bubb et al, 1999;Yarmola et al, 2001), and PKC-mediated phosphorylation of MARCKS results in the movement of synaptic vesicles from reserve pools to active release sites in cultured cells and synaptosomes (Vitale et al, 1995;Stevens and Sullivan, 1998;Walaas and Sefland, 2000;Rose et al, 2001;Yang et al, 2002). Furthermore, anti-MARCKS antibodies, or F-actin destabilizing agents, increase spontaneous neurotransmitter release in PC12 cells (Shoji-Kasai et al, 2002), whereas MARCKS overexpression (two-fold) impaired PKCinduced enhancement of neurotransmitter release in human neuroblastoma SH-SY5Y cells (Hartness et al, 2001).…”

Phosphorylation of neurofibromin by PKC is a possible molecular switch in EGF receptor signaling in neural cells

“…F-actin networks are important in immature neurons for neuritic elongation, neuronal migration, and regulated secretion (Cheng et al, 2000a). Disruption of the cortical F-actin cytoskeleton affects the translocation of exocytotic vesicles to the plasma membrane, a process that requires PKC activation and phosphorylation of F-actin-binding proteins (Rose et al, 2001). Analysis of the primary structure of neurofibromin reveals several PKC phosphorylation consensus sites.…”