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

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 = 0.10.3 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.

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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

Journal of Cell Science ◽

10.1242/jcs.115.15.3083 ◽

2002 ◽

Vol 115 (15) ◽

pp. 3083-3092 ◽

Cited By ~ 1

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.

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

Molecular Biology of the Cell ◽

10.1091/mbc.e05-06-0499 ◽

2006 ◽

Vol 17 (1) ◽

pp. 56-66 ◽

Cited By ~ 77

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.

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Bryostatin 1 activates protein kinase C and induces monocytic differentiation of HL-60 cells

Blood ◽

10.1182/blood.v72.1.208.bloodjournal721208 ◽

1988 ◽

Vol 72 (1) ◽

pp. 208-213 ◽

Cited By ~ 2

Author(s):

RM Stone ◽

E Sariban ◽

GR Pettit ◽

DW Kufe

Keyword(s):

Gene Expression ◽

Protein Kinase C ◽

Protein Kinase ◽

Growth Inhibition ◽

Phorbol Ester ◽

Phorbol Esters ◽

Cell Protein ◽

Monocytic Differentiation ◽

Bryostatin 1 ◽

Kinase C

Phorbol esters induce the human HL-60 promyelocytic cell line to differentiate along a monocytic pathway. This induction of differentiation may involve phorbol ester-induced activation of the phospholipid- and calcium-dependent protein kinase C. Bryostatin 1, a macrocyclic lactone, has been shown to compete with phorbol esters for binding to protein kinase C. We have confirmed that bryostatin 1 translocates activity of protein kinase C from the cytosolic to membrane fractions of HL-60 cells. The present results also demonstrate that bryostatin 1 (10 nmol/L) induces monocytic differentiation of HL- 60 cells as determined by adherence, growth inhibition, appearance of monocyte cell surface antigens, and alpha-naphthyl acetate esterase staining. Furthermore, bryostatin 1 (10 nmol/L) downregulated c-myc expression and induced c-fos, c-fms, and tumor necrosis factor transcripts. These changes in gene expression induced by bryostatin 1 are similar to those associated with phorbol ester-induced monocytic differentiation of HL-60 cells. In contrast, exposure to a higher concentration of bryostatin 1 (100 nmol/L) had less of an effect on growth inhibition of HL-60 cells and changes in gene expression. Moreover, 100 nmol/L bryostatin 1 antagonized the cytostatic effects and adherence induced by phorbol esters. Our results thus suggest that bryostatin 1 activates HL-60 cell protein kinase C and that this effect is associated with induction of monocytic differentiation.

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Receptor-independent Protein Kinase Cα (PKCα) Signaling by Calpain-generated Free Catalytic Domains Induces HDAC5 Nuclear Export and Regulates Cardiac Transcription

Journal of Biological Chemistry ◽

10.1074/jbc.m111.234757 ◽

2011 ◽

Vol 286 (30) ◽

pp. 26943-26951 ◽

Cited By ~ 30

Author(s):

Yan Zhang ◽

Scot J. Matkovich ◽

Xiujun Duan ◽

Abhinav Diwan ◽

Min-Young Kang ◽

...

Keyword(s):

Protein Kinase ◽

Nuclear Export ◽

Catalytic Domains ◽

Protein Kinase Cα ◽

Independent Protein

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Bryostatin 1 activates protein kinase C and induces monocytic differentiation of HL-60 cells

Blood ◽

10.1182/blood.v72.1.208.208 ◽

1988 ◽

Vol 72 (1) ◽

pp. 208-213 ◽

Cited By ~ 85

Author(s):

RM Stone ◽

E Sariban ◽

GR Pettit ◽

DW Kufe

Keyword(s):

Gene Expression ◽

Protein Kinase C ◽

Protein Kinase ◽

Growth Inhibition ◽

Phorbol Ester ◽

Phorbol Esters ◽

Cell Protein ◽

Monocytic Differentiation ◽

Bryostatin 1 ◽

Kinase C

Abstract Phorbol esters induce the human HL-60 promyelocytic cell line to differentiate along a monocytic pathway. This induction of differentiation may involve phorbol ester-induced activation of the phospholipid- and calcium-dependent protein kinase C. Bryostatin 1, a macrocyclic lactone, has been shown to compete with phorbol esters for binding to protein kinase C. We have confirmed that bryostatin 1 translocates activity of protein kinase C from the cytosolic to membrane fractions of HL-60 cells. The present results also demonstrate that bryostatin 1 (10 nmol/L) induces monocytic differentiation of HL- 60 cells as determined by adherence, growth inhibition, appearance of monocyte cell surface antigens, and alpha-naphthyl acetate esterase staining. Furthermore, bryostatin 1 (10 nmol/L) downregulated c-myc expression and induced c-fos, c-fms, and tumor necrosis factor transcripts. These changes in gene expression induced by bryostatin 1 are similar to those associated with phorbol ester-induced monocytic differentiation of HL-60 cells. In contrast, exposure to a higher concentration of bryostatin 1 (100 nmol/L) had less of an effect on growth inhibition of HL-60 cells and changes in gene expression. Moreover, 100 nmol/L bryostatin 1 antagonized the cytostatic effects and adherence induced by phorbol esters. Our results thus suggest that bryostatin 1 activates HL-60 cell protein kinase C and that this effect is associated with induction of monocytic differentiation.

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Differing modulation of protein kinase C by bryostatin 1 and phorbol esters in JB6 mouse epidermal cells.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)68496-0 ◽

1988 ◽

Vol 263 (17) ◽

pp. 8437-8442

Author(s):

A S Kraft ◽

J A Reeves ◽

C L Ashendel

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Phorbol Esters ◽

Epidermal Cells ◽

Bryostatin 1 ◽

Kinase C

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Protein Kinase Cα Contains Two Activator Binding Sites That Bind Phorbol Esters and Diacylglycerols with Opposite Affinities

Journal of Biological Chemistry ◽

10.1074/jbc.271.9.4627 ◽

1996 ◽

Vol 271 (9) ◽

pp. 4627-4631 ◽

Cited By ~ 71

Author(s):

Simon J. Slater ◽

Cojen Ho ◽

Mary Beth Kelly ◽

Jonathan D. Larkin ◽

Frank J. Taddeo ◽

...

Keyword(s):

Protein Kinase ◽

Binding Sites ◽

Phorbol Esters ◽

Protein Kinase Cα

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Tumor promotion by depleting cells of protein kinase C delta.

Molecular and Cellular Biology ◽

10.1128/mcb.17.6.3418 ◽

1997 ◽

Vol 17 (6) ◽

pp. 3418-3428 ◽

Cited By ~ 150

Author(s):

Z Lu ◽

A Hornia ◽

Y W Jiang ◽

Q Zang ◽

S Ohno ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Phorbol Ester ◽

Phorbol Esters ◽

Kinase Inhibitor ◽

Tumor Promotion ◽

Dominant Negative ◽

Prolonged Treatment ◽

Bryostatin 1 ◽

Kinase C

Tumor-promoting phorbol esters activate, but then deplete cells of, protein kinase C (PKC) with prolonged treatment. It is not known whether phorbol ester-induced tumor promotion is due to activation or depletion of PKC. In rat fibroblasts overexpressing the c-Src proto-oncogene, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induced anchorage-independent growth and other transformation-related phenotypes. The appearance of transformed phenotypes induced by TPA in these cells correlated not with activation but rather with depletion of expressed PKC isoforms. Consistent with this observation, PKC inhibitors also induced transformed phenotypes in c-Src-overexpressing cells. Bryostatin 1, which inhibited the TPA-induced down-regulation of the PKCdelta isoform specifically, blocked the tumor-promoting effects of TPA, implicating PKCdelta as the target of the tumor-promoting phorbol esters. Consistent with this hypothesis, expression of a dominant negative PKCdelta mutant in cells expressing c-Src caused transformation of these cells, and rottlerin, a protein kinase inhibitor with specificity for PKCdelta, like TPA, caused transformation of c-Src-overexpressing cells. These data suggest that the tumor-promoting effect of phorbol esters is due to depletion of PKCdelta, which has an apparent tumor suppressor function.

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