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 Determination of the calcium-binding sites of the C2 domain of protein kinase Cα that are critical for its translocation to the plasma membrane

1999 ◽  
Vol 337 (3) ◽  
pp. 513-521 ◽  
Author(s):  
Senena CORBALÁN-GARCÍA ◽  
José A. RODRÍGUEZ-ALFARO ◽  
Juan C. GÓMEZ-FERNÁNDEZ
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Hela Cells ◽  
Calcium Binding ◽  
Lipid Vesicles ◽  
Site Directed Mutagenesis ◽  
C2 Domain ◽  
Protein Kinase Cα

The C2 domain is a conserved protein module present in various signal-transducing proteins. To investigate the function of the C2 domain of protein kinase Cα (PKCα), we have generated a recombinant glutathione S-transferase-fused C2 domain from rat PKCα, PKC-C2. We found that PKC-C2 binds with high affinity (half-maximal binding at 0.6 µM) to lipid vesicles containing the negatively charged phospholipid phosphatidylserine. When expressed into COS and HeLa cells, most of the PKC-C2 was found at the plasma membrane, whereas when the cells were depleted of Ca2+ by incubation with EGTA and ionophore, the C2 domain was localized preferentially in the cytosol. Ca2+ titration was performed in vivo and the critical Ca2+ concentration ranged from 0.1 to 0.32 µM. We also identified, by site-directed mutagenesis, three aspartic residues critical for that Ca2+ interaction, namely Asp-187, Asp-246 and Asp-248. Mutation of these residues to asparagine, to abolish their negative charge, resulted in a domain expressed as the same extension as wild-type protein that could interact in vitro with neither Ca2+ nor phosphatidylserine. Overexpression of these mutants into COS and HeLa cells also showed that they cannot localize at the plasma membrane, as demonstrated by immunofluorescence staining and subcellular fractionation. These results suggest that the Ca2+-binding site might be involved in promoting the interaction of the C2 domain of PKCα with the plasma membrane in vivo.

Effects of membrane lipid and fluidity modifications on HIV-1 infectibility of primate lymphocytes in vitro

1990 ◽  
Vol 10 (3) ◽  
pp. 263-270 ◽  
Author(s):  
J. Pascal Zimmer ◽  
Hans A. Lehr ◽  
Christoph Hübner ◽  
Stephan G. Lindner ◽  
Ralf Ramsperger ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Fluidity ◽  
Lipid Composition ◽  
Membrane Lipid ◽  
Serum Factors ◽  
Membrane Lipid Composition ◽  
Plasma Membrane Lipid ◽  
Hiv 1

Although most non-human primates, except the chimpanzee and the gibbon in vivo are not infectible by HIV-1, lymphocytes of several of these species can be infected by HIV-1 in vitro.In order to investigate whether the in vitro infectibility of primate lymphocytes might be attributed to plasma membrane adaptation processes or to serum factors, we compared HIV-1 infectibility of cultivated peripheral blood lymphocytes of macaques and of baboons on day one and on day ten of cultivation. These data were correlated to plasma membrane lipid composition and membrane fluidity.We found a correlation between increased HIV-1 in vitro infectibility and changes in plasma membrane lipid composition resulting in decreased membrane fluidity of cultured primate lymphocytes.

scholarly journals Regulation of Oxysterol-binding Protein Golgi Localization through Protein Kinase D–mediated Phosphorylation

2010 ◽  
Vol 21 (13) ◽  
pp. 2327-2337 ◽  
Author(s):  
Sokha Nhek ◽  
Mike Ngo ◽  
Xuemei Yang ◽  
Michelle M. Ng ◽  
Seth J. Field ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Binding Protein ◽  
Critical Role ◽  
Protein Kinase D ◽  
Cholesterol Depletion ◽  
Oxysterol Binding Protein ◽  
Golgi Localization ◽  
Golgi Network

Protein kinase D (PKD) plays a critical role at the trans-Golgi network by regulating the fission of transport carriers destined for the plasma membrane. Two known Golgi-localized PKD substrates, PI4-kinase IIIβ and the ceramide transfer protein CERT, mediate PKD signaling to influence vesicle trafficking to the plasma membrane and sphingomyelin synthesis, respectively. PKD is recruited and activated at the Golgi through interaction with diacylglycerol, a pool of which is generated as a by-product of sphingomyelin synthesis from ceramide. Here we identify a novel substrate of PKD at the Golgi, the oxysterol-binding protein OSBP. Using a substrate-directed phospho-specific antibody that recognizes the optimal PKD consensus motif, we show that PKD phosphorylates OSBP at Ser240 in vitro and in cells. We further show that OSBP phosphorylation occurs at the Golgi. Phosphorylation of OSBP by PKD does not modulate dimerization, sterol binding, or affinity for PI(4)P. Instead, phosphorylation attenuates OSBP Golgi localization in response to 25-hydroxycholesterol and cholesterol depletion, impairs CERT Golgi localization, and promotes Golgi fragmentation.

scholarly journals Protein kinase D inhibits plasma membrane Na+/H+exchanger activity

1999 ◽  
Vol 277 (6) ◽  
pp. C1202-C1209 ◽  
Author(s):  
Robert S. Haworth ◽  
James Sinnett-Smith ◽  
Enrique Rozengurt ◽  
Metin Avkiran
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Fluorescent Protein ◽  
Dominant Negative ◽  
Protein Kinase D ◽  
Wild Type ◽  
Ph Indicator

The regulation of plasma membrane Na+/H+exchanger (NHE) activity by protein kinase D (PKD), a novel protein kinase C- and phorbol ester-regulated kinase, was investigated. To determine the effect of PKD on NHE activity in vivo, intracellular pH (pHi) measurements were made in COS-7 cells by microepifluorescence using the pH indicator cSNARF-1. Cells were transfected with empty vector (control), wild-type PKD, or its kinase-deficient mutant PKD-K618M, together with green fluorescent protein (GFP). NHE activity, as reflected by the rate of acid efflux ( J H), was determined in single GFP-positive cells following intracellular acidification. Overexpression of wild-type PKD had no significant effect on J H(3.48 ± 0.25 vs. 3.78 ± 0.24 mM/min in control at pHi 7.0). In contrast, overexpression of PKD-K618M increased J H (5.31 ± 0.57 mM/min at pHi 7.0; P < 0.05 vs. control). Transfection with these constructs produced similar effects also in A-10 cells, indicating that native PKD may have an inhibitory effect on NHE in both cell types, which is relieved by a dominant-negative action of PKD-K618M. Exposure of COS-7 cells to phorbol ester significantly increased J H in control cells but failed to do so in cells overexpressing either wild-type PKD (due to inhibition by the overexpressed PKD) or PKD-K618M (because basal J Hwas already near maximal). A fusion protein containing the cytosolic regulatory domain (amino acids 637–815) of NHE1 (the ubiquitous NHE isoform) was phosphorylated in vitro by wild-type PKD, but with low stoichiometry. These data suggest that PKD inhibits NHE activity, probably through an indirect mechanism, and represents a novel pathway in the regulation of the exchanger.

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 H3 Histamine Receptor–Mediated Activation of Protein Kinase Cα Inhibits the Growth of Cholangiocarcinoma In vitro and In vivo

2009 ◽  
Vol 7 (10) ◽  
pp. 1704-1713 ◽  
Author(s):  
Heather Francis ◽  
Paolo Onori ◽  
Eugenio Gaudio ◽  
Antonio Franchitto ◽  
Sharon DeMorrow ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Histamine Receptor ◽  
Protein Kinase Cα

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.

An in vitro1H-MRS Model of Oncogene Transfection

1997 ◽  
Vol 38 (6) ◽  
pp. 1083-1086
Author(s):  
T. Nakai ◽  
R. Ishima ◽  
H. Sakahara ◽  
K. Endo ◽  
J. Konishi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Lipid ◽  
Ras Gene ◽  
Membrane Viscosity ◽  
Nih3t3 Cells ◽  
Transfected Cells ◽  
H Nmr ◽  
Plasma Membrane Lipid ◽  
Reduced Mobility

Purpose: Malignancy is an abnormality of cell division and differentiation based on abnormal expression of oncogenes. This note describes the in vitro1H-NMR spectral features of oncogene-transfected NIH3T3 fibroblast cells compared to non-transfected cells Material and Methods: 1H-NMR spectra of cultured NIH3T3 cells and c-erbB-2 or c-Ha-ras gene-transfected cells were obtained by 400 MHz high resolution NMR. the peaks were assigned by 2D HOHAHA spectra of the cell suspension and the spectral changes were evaluated in 1D and 1D differential spectra Results: the 1H spectra obtained from both transfected cell lines were broadened over all peaks, suggesting reduced mobility in plasma membrane lipid molecules. No other differential spectra for characterizing metabolic change was detected Conclusion: Broadened 1H spectra observed after c-erbB-2 or c-Ha-ras transfection suggest changes of plasma membrane viscosity, which may be related to the oncogene expression

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