Membrane Targeting and Cytoplasmic Sequestration in the Spatiotemporal Localization of Human Protein Kinase C α

Arachidonic acid (AA) directly activates protein kinases C (PKC) and may thereby serve as a regulatory signal during cell stimulation. The effect, however, requires a >20 M concentration of the fatty acid. We find that human polymorphonuclear neutrophils ( or PKC␦ fused to the reporter enhanced green fluorescent protein (EGFP) were studied. AA caused EGFP-PKC␤ translocation from cytosol to plasma membrane at >0.5 M, and EGFP-PKC␦ translocation from cytosol to nuclear and, to a lesser extent, plasma membrane at as little as 30 nM. We conclude that AA induces PKC translocations to specific membrane targets at concentrations 2-4 orders of magnitude below those activating the enzymes. These responses, at least as they occur in PMN, do not require changes in cell Ca 2؉ or oxygenation of the fatty acid. AA seems more suited for signaling the movement than activation of PKC.

Section: Pdb Binding-[mentioning

confidence: 93%

Protein Kinases C Translocation Responses to Low Concentrations of Arachidonic Acid

O’Flaherty¹,

Chadwell²,

Kearns

et al. 2001

“…In this respect, we tested the kinase domain including the substrate-binding site and the PKC␣ V3 region. This region affects the intracellular localization of PKC␣ (46), and a disruption of its interaction with ␤ 1 -integrins blocks the PKC-dependent chemotaxis of carcinoma cells (9). Neither PKC␣ V3 nor the kinase domain, however, strengthened substantially the interaction of the PKC regulatory domain with filamin.…”

Section: Discussionmentioning

confidence: 99%

“…To identify the main characteristics of the interaction with filamin, domain constructs of the lipid and Ca 2ϩ -binding PKC regulatory domain (rd), the variable hinge region (V3), that is involved in intercellular targeting (46), and ligand binding (9), and the kinase domain (kd) were employed in overlay assays. The domain structure of PKC is shown in Fig.…”

Section: The Interaction Of Flna Constructs [N1-4] and [22-24c] Witmentioning

confidence: 99%

The F-actin Cross-linking and Focal Adhesion Protein Filamin A Is a Ligand and in Vivo Substrate for Protein Kinase Cα

Tigges

Koch

Wissing

et al. 2003

Filamin A is an established structural component of cell-matrix adhesion sites. In addition, it serves as a scaffold for the subcellular targeting of different signaling molecules. Protein kinase C (PKC) has been found associated with filamin; however, details about this interaction and its significance for cell-matrix adhesiondependent signaling have remained elusive. We performed a yeast two-hybrid analysis using protein kinase C␣ as a bait and identified filamin as a direct binding partner. The interaction was confirmed in transfected HeLa cells, and serial truncation fragments of filamin A were employed to identify two binding sites on filamin. In vitro ligand binding assays revealed a Ca 2؉ and phospholipid-dependent association of the regulatory domain of protein kinase C with these sites. Phosphorylation of filamin was found to be isoform-restricted, leading to phosphate incorporation in the C termini of filamin A and C, but not B. PKC-dependent phosphorylation of filamin was also detected in cells. Our data suggest an intimate interaction between filamin and PKC in cell signaling.

“…22). As prototype C2 proteins with a Ca 2ϩ -dependent plasma membrane binding activity (18,19), we also tested the localization of PKC␣ and -␥ in our assay. Remarkably, under the conditions used (1 M ionophore), the DOC2B-EGFP translocation was clearly more pronounced than that of the PKC isoforms, which require more stringent concentrations for their activation (e.g.…”

Section: Discussionmentioning

confidence: 99%

“…Expression vectors encoding Myc-tagged DOC2B were derived from this vector by subcloning the NheI-BamHI fragment into the corresponding sites of pCDNA3.1-Myc-HisC (Invitrogen). Expression vectors encoding Munc13-1, PKC-␣, and PKC-␥ were described previously (17)(18)(19).…”

Section: Methodsmentioning

confidence: 99%

Ca2+-induced Recruitment of the Secretory Vesicle Protein DOC2B to the Target Membrane

Groffen

Brian

Dudok

et al. 2004

Ca 2؉ -dependent fusion of transport vesicles at their target can be enhanced by intracellular Ca 2؉ and diacylglycerol. Diacylglycerol induces translocation of the vesicle priming factor Munc13 and association of the secretory vesicle protein DOC2B to the membrane. Here we demonstrate that a rise in intracellular Ca 2؉ is sufficient for a Munc13-independent recruitment of DOC2B to the target membrane. This novel mechanism occurred readily in the absence of Munc13 and was not influenced by DOC2B mutations that abolish Munc13 binding. Purified DOC2B (expressed as a bacterial fusion protein) bound phospholipids in a Ca 2؉ -dependent way, suggesting that the translocation is the result of a C2 domain activation mechanism. Ca 2؉ -induced translocation was also observed in cultured neurons expressing DOC2B-enhanced green fluorescent protein. In this case, however, various degrees of membrane association occurred under resting conditions, suggesting that physiological Ca 2؉ concentrations modulate DOC2B localization. Depolarization of the neurons induced a complete translocation of DOC2B-enhanced green fluorescent protein to the target membrane within 5 s. We hypothesize that this novel Ca 2؉ -induced activity of DOC2B functions synergistically with diacylglycerol-induced Munc13 binding to enhance exocytosis during episodes of high secretory activity.Ca 2ϩ -induced exocytosis is a widely conserved mechanism of importance for a broad range of secretory systems, such as the release of neurotransmitters and neuropeptides in the central and peripheral nervous system. To attain fusion competence, secretory vesicles must first dock at the target membrane and undergo a process called priming. The release of fusion-ready vesicles is coupled to the opening of Ca 2ϩ channels in the plasma membrane, producing a transient high Ca 2ϩ concentration (10 -100 M for ϳ1 ms) in close proximity to the channels. The inward Ca 2ϩ current then diffuses into the cytoplasm, resulting in a residual concentration in the range of 1 M during tens of seconds. It is widely accepted that this residual Ca 2ϩ concentration induces short term changes in exocytotic strength (e.g. see Refs. 1 and 2), although the mechanisms that contribute to this phenomenon remain largely unclear. As a result, the secretory strength of neuronal and endocrine release sites is modulated in an activity-dependent way. This phenomenon is important to avoid vesicle depletion during repetitive stimulation and is furthermore considered in neurons to contribute to memory and learning.Although Ca 2ϩ is the most important messenger after high frequency activation of the release site, exocytotic potentiation can also be induced by diacylglycerol (synthesized by members of the phospholipase family) or by its phorbolester analogues. Diacylglycerol has multiple intracellular targets (e.g. protein kinase C isoforms), but the potentiating effect in regulated exocytosis is mediated by the priming factor Munc13-1. Munc13-1-deficient mice die shortly after birth and show a defect in synapt...