Suppression of Synaptotagmin II restrains phorbolester-induced downregulation of protein kinase Cα by diverting the kinase from a degradative pathway to the recycling endocytic compartment

2002 ◽  
Vol 115 (15) ◽  
pp. 3083-3092 ◽  
Author(s):  
Ze Peng ◽  
Elena Grimberg ◽  
Ronit Sagi-Eisenberg
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Phorbol Esters ◽  
Secretory Granules ◽  
Critical Factor ◽  
Cellular Level ◽  
Endocytic Compartment ◽  
Early Endosomes ◽  
Protein Kinase Cα ◽  
Rbl Cells

Downregulation of protein kinase Cα (PKCα) following long-term exposure to phorbol esters such as TPA is traffic dependent and involves delivery of the active, membrane-associated PKCα to endosomes. In this study, we show that synaptotagmin II (Syt II), a member of the Syt family of proteins, is required for TPA-induced degradation of PKCα. Thus, whereas the kinase half-life in TPA-treated cultured mast cells (the mast cell line rat basophilic leukemia RBL-2H3) is 2 hours, it is doubled in RBL-Syt II- cells, in which the cellular level of Syt II is reduced by>95% by transfection with Syt II antisense cDNA. We demonstrate that in TPA-treated RBL cells, PKCα travels from the cytosol to the plasma membrane, where it is delivered to early endosomes on its route to degradation. By contrast, in TPA-treated RBL-Syt II- cells,PKCα is diverted to recycling endosomes and remains distributed between the plasma membrane and the perinuclear recycling endocytic compartment. Notably, in both RBL and RBL-Syt II- cells, a fraction of PKCα is delivered and maintained in the secretory granules (SG). These results implicate Syt II as a critical factor for the delivery of internalized cargo for degradation. As shown here, one consequence of Syt II suppression is a delay in PKCα downregulation, resulting in its prolonged signaling.

Tamoxifen inhibits phorbol ester stimulated osteoclastic bone resorption: An effect mediated by calmodulin

2000 ◽  
Vol 78 (6) ◽  
pp. 715-723 ◽  
Author(s):  
John P Williams ◽  
Margaret A McKenna ◽  
Allyn M Thames III ◽  
Jay M McDonald
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Bone Resorption ◽  
Phorbol Ester ◽  
Phorbol Esters ◽  
Fold Increase ◽  
Dependent Manner ◽  
Osteoclast Activity ◽  
Kinase C ◽  
Protein Kinase Cα

Tamoxifen inhibits bone resorption by disrupting calmodulin-dependent processes. Since tamoxifen inhibits protein kinase C in other cells, we compared the effects of tamoxifen and the phorbol ester, phorbol myristate acetate, on osteoclast activity. Phorbol esters stimulate bone resorption and calmodulin levels four-fold (k0.5 = 0.1–0.3 µM). In contrast, tamoxifen inhibited osteoclast activity ~60% with an IC50 of 1.5 µM, had no apparent effect on protein kinase C activity in whole-cell lysates, and reduced protein kinase Cα recovered by immunoprecipitation 75%. Phorbol esters stimulated resorption in a time-dependent manner that was closely correlated with a similar-fold increase in calmodulin. Protein kinase Cα, β, δ, ε, and ζ were all down-regulated in response to phorbol ester treatment. Tamoxifen and trifluoperazine inhibited PMA-dependent increases in bone resorption and calmodulin by 85 ± 10%. Down-regulation of protein kinase C isoforms by phorbol esters suggests that the observed increases in bone resorption and calmodulin levels are most likely due to a mechanism independent of protein kinase C and dependent on calmodulin. In conclusion, the data suggest that protein kinase C negatively regulates calmodulin expression and support the hypothesis that the effects of both phorbol esters and tamoxifen on osteoclast activity is mediated by calmodulin.Key words: osteoclast, calmodulin, tamoxifen, osteoporosis, protein kinase C.

scholarly journals Specific Translocation of Protein Kinase Cα to the Plasma Membrane Requires Both Ca2+ and PIP2 Recognition by Its C2 Domain

2006 ◽  
Vol 17 (1) ◽  
pp. 56-66 ◽  
Author(s):  
John H. Evans ◽  
Diana Murray ◽  
Christina C. Leslie ◽  
Joseph J. Falke
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Membrane Binding ◽  
Membrane Lipid ◽  
C2 Domain ◽  
Plasma Membrane Lipid ◽  
Protein Kinase Cα ◽  
Golgi Network

The C2 domain of protein kinase Cα (PKCα) controls the translocation of this kinase from the cytoplasm to the plasma membrane during cytoplasmic Ca2+ signals. The present study uses intracellular coimaging of fluorescent fusion proteins and an in vitro FRET membrane-binding assay to further investigate the nature of this translocation. We find that Ca2+-activated PKCα and its isolated C2 domain localize exclusively to the plasma membrane in vivo and that a plasma membrane lipid, phosphatidylinositol-4,5-bisphosphate (PIP2), dramatically enhances the Ca2+-triggered binding of the C2 domain to membranes in vitro. Similarly, a hybrid construct substituting the PKCα Ca2+-binding loops (CBLs) and PIP2 binding site (β-strands 3–4) into a different C2 domain exhibits native Ca2+-triggered targeting to plasma membrane and recognizes PIP2. Conversely, a hybrid containing the CBLs but lacking the PIP2 site translocates primarily to trans-Golgi network (TGN) and fails to recognize PIP2. Similarly, PKCα C2 domains possessing mutations in the PIP2 site target primarily to TGN and fail to recognize PIP2. Overall, these findings demonstrate that the CBLs are essential for Ca2+-triggered membrane binding but are not sufficient for specific plasma membrane targeting. Instead, targeting specificity is provided by basic residues on β-strands 3–4, which bind to plasma membrane PIP2.

scholarly journals Role of the Ca2+/Phosphatidylserine Binding Region of the C2 Domain in the Translocation of Protein Kinase Cα to the Plasma Membrane

2003 ◽  
Vol 278 (12) ◽  
pp. 10282-10290 ◽  
Author(s):  
Stephen R. Bolsover ◽  
Juan C. Gomez-Fernandez ◽  
Senena Corbalan-Garcia
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
C2 Domain ◽  
Binding Region ◽  
Protein Kinase Cα

scholarly journals GLUT8 Subcellular Localization and Absence of Translocation to the Plasma Membrane in PC12 Cells and Hippocampal Neurons

Endocrinology ◽  
2005 ◽  
Vol 146 (11) ◽  
pp. 4727-4736 ◽  
Author(s):  
Mathieu Widmer ◽  
Marc Uldry ◽  
Bernard Thorens
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Cell Surface ◽  
Pc12 Cells ◽  
Hippocampal Neurons ◽  
Osmotic Shock ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Intracellular Compartment ◽  
Early Endosomes

GLUT8 is a high-affinity glucose transporter present mostly in testes and a subset of brain neurons. At the cellular level, it is found in a poorly defined intracellular compartment in which it is retained by an N-terminal dileucine motif. Here we assessed GLUT8 colocalization with markers for different cellular compartments and searched for signals, which could trigger its cell surface expression. We showed that when expressed in PC12 cells, GLUT8 was located in a perinuclear compartment in which it showed partial colocalization with markers for the endoplasmic reticulum but not with markers for the trans-Golgi network, early endosomes, lysosomes, and synaptic-like vesicles. To evaluate its presence at the plasma membrane, we generated a recombinant adenovirus for the expression of GLUT8 containing an extracellular myc epitope. Cell surface expression was evaluated by immunofluorescence microscopy of transduced PC12 cells or primary hippocampal neurons exposed to different stimuli. Those included substances inducing depolarization, activation of protein kinase A and C, activation or inhibition of tyrosine kinase-linked signaling pathways, glucose deprivation, AMP-activated protein kinase stimulation, and osmotic shock. None of these stimuli-induced GLUT8 cell surface translocation. Furthermore, when GLUT8myc was cotransduced with a dominant-negative form of dynamin or GLUT8myc-expressing PC-12 cells or neurons were incubated with an anti-myc antibody, no evidence for constitutive recycling of the transporter through the cell surface could be obtained. Thus, in cells normally expressing it, GLUT8 was associated with a specific intracellular compartment in which it may play an as-yet-uncharacterized role.

scholarly journals The ATP-dependent Membrane Localization of Protein Kinase Cα Is Regulated by Ca2+ Influx and Phosphatidylinositol 4,5-Bisphosphate in Differentiated PC12 Cells

2005 ◽  
Vol 16 (6) ◽  
pp. 2848-2861 ◽  
Author(s):  
Consuelo Marín-Vicente ◽  
Juan C. Gómez-Fernández ◽  
Senena Corbalán-García
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Pc12 Cells ◽  
Specific Inhibitor ◽  
Enzyme Activation ◽  
C2 Domain ◽  
Differentiation Process ◽  
Rich Cluster ◽  
Differentiated Pc12 Cells ◽  
Protein Kinase Cα

Signal transduction through protein kinase Cs (PKCs) strongly depends on their subcellular localization. Here, we investigate the molecular determinants of PKCα localization by using a model system of neural growth factor (NGF)-differentiated pheochromocytoma (PC12) cells and extracellular stimulation with ATP. Strikingly, the Ca2+ influx, initiated by the ATP stimulation of P2X receptors, rather than the Ca2+ released from the intracellular stores, was the driving force behind the translocation of PKCα to the plasma membrane. Furthermore, the localization process depended on two regions of the C2 domain: the Ca2+-binding region and the lysine-rich cluster, which bind Ca2+ and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], respectively. It was demonstrated that diacylglycerol was not involved in the localization of PKCα through its C1 domain, and in lieu, the presence of PtdIns(4,5)P2 increased the permanence of PKCα in the plasma membrane. Finally, it also was shown that ATP cooperated with NGF during the differentiation process of PC12 cells by increasing the length of the neurites, an effect that was inhibited when the cells were incubated in the presence of a specific inhibitor of PKCα, suggesting a possible role for this isoenzyme in the neural differentiation process. Overall, these results show a novel mechanism of PKCα activation in differentiated PC12 cells, where Ca2+ influx, together with the endogenous PtdIns(4,5)P2, anchor PKCα to the plasma membrane through two distinct motifs of its C2 domain, leading to enzyme activation.

scholarly journals Inhibition of Membrane Lipid-independent Protein Kinase Cα Activity by Phorbol Esters, Diacylglycerols, and Bryostatin-1

1998 ◽  
Vol 273 (36) ◽  
pp. 23160-23168 ◽  
Author(s):  
Simon J. Slater ◽  
Frank J. Taddeo ◽  
Anthony Mazurek ◽  
Brigid A. Stagliano ◽  
Shawn K. Milano ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Phorbol Esters ◽  
Membrane Lipid ◽  
Bryostatin 1 ◽  
Protein Kinase Cα ◽  
Independent Protein

Stimulation of protein kinase C pathway mediates endocytosis of human nongastric H+-K+-ATPase, ATP1AL1

2002 ◽  
Vol 283 (2) ◽  
pp. F335-F343 ◽  
Author(s):  
J. Reinhardt ◽  
M. Kosch ◽  
M. Lerner ◽  
H. Bertram ◽  
D. Lemke ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Molecular Mechanisms ◽  
Phorbol Esters ◽  
Ion Pump ◽  
Membrane Expression ◽  
Kinase C ◽  
Plasma Membrane Expression ◽  
Functional Inactivation

The human nongastric H+-K+-ATPase, ATP1AL1, shown to reabsorb K+ in exchange for H+ or Na+, is localized in the luminal plasma membrane of renal epithelial cells. It is presumed that renal H+-K+-ATPases can be regulated by endocytosis. However, little is known about the molecular mechanisms that control plasma membrane expression of renal H+-K+-ATPases. In our study, activation of protein kinase C (PKC) using phorbol esters (phorbol 12-myristate 13-acetate) leads to clathrin-dependent internalization and intracellular accumulation of the ion pump in stably transfected Madin-Darby canine kidney cells. Functional inactivation of the H+-K+-ATPase by PKC activation is shown by intracellular pH measurements. Proton extrusion capacity of ATP1AL1-transfected cells is drastically reduced after phorbol 12-myristate 13-acetate incubation and can be prevented with the PKC blocker bisindolylmaleimide. Ion pump internalization and inactivation are specifically mediated by the PKC pathway, whereas activation of the protein kinase A pathway has no influence. Our results show that the nongastric H+-K+-ATPase is a specific target for the PKC pathway. Therefore, PKC-mediated phosphorylation is a potential regulatory mechanism for apical nongastric H+-K+-ATPase plasma membrane expression.

Protein Kinase C-Dependent Supply of Secretory Granules to the Plasma Membrane

2001 ◽  
Vol 282 (2) ◽  
pp. 621-628 ◽  
Author(s):  
Takashi Tsuboi ◽  
Toshiteru Kikuta ◽  
Akira Warashina ◽  
Susumu Terakawa
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Secretory Granules ◽  
Kinase C
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