bryostatin-1 and lactacystin

Cotreatment with a minimally toxic concentration of the protein kinase C (PKC) activator (and down-regulator) bryostatin 1 (BRY) induced a marked increase in mitochondrial dysfunction and apoptosis in U937 monocytic leukemia cells exposed to the proteasome inhibitor lactacystin (LC). This effect was blocked by cycloheximide, but not by alpha-amanitin or actinomycin D. Qualitatively similar interactions were observed with other PKC activators (eg, phorbol 12-myristate 13-acetate and mezerein), but not phospholipase C, which does not down-regulate the enzyme. These events were examined in relationship to functional alterations in stress (eg, SAPK, JNK) and survival (eg, MAPK, ERK) signaling pathways. The observations that LC/BRY treatment failed to trigger JNK activation and that cell death was unaffected by a dominant-interfering form of c-JUN (TAM67) or by pretreatment with either curcumin or the p38/RK inhibitor, SB203580, suggested that the SAPK pathway was not involved in potentiation of apoptosis. In marked contrast, perturbations in the PKC/Raf/MAPK pathway played an integral role in LC/BRY-mediated cell death based on evidence that pretreatment of cells with bisindolylmaleimide I, a selective PKC inhibitor, or geldanamycin, a benzoquinone ansamycin, which destabilizes and depletes Raf-1, markedly suppressed apoptosis. Furthermore, ERK phosphorylation was substantially prolonged in LC/BRY-treated cells compared to those exposed to BRY alone, and pretreatment with the highly specific MEK inhibitors, PD98059, U0126, and SL327, opposed ERK activation while protecting cells from LC/BRY-induced lethality. Together, these findings suggest a role for activation and/or dysregulation of the PKC/MAPK cascade in modulation of leukemic cell apoptosis following exposure to the proteasome inhibitor LC. (Blood. 2001;97:2105-2114)

Topics: Acetylcysteine; Amanitins; Aminoacetonitrile; Apoptosis; Benzoquinones; Bryostatins; Butadienes; Curcumin; Cysteine Endopeptidases; Dactinomycin; Diterpenes; Drug Synergism; Enzyme Activation; Flavonoids; Humans; Imidazoles; Indoles; JNK Mitogen-Activated Protein Kinases; Lactams, Macrocyclic; Lactones; Macrolides; Maleimides; MAP Kinase Kinase 4; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Multienzyme Complexes; Neoplasm Proteins; Nitriles; Nucleic Acid Synthesis Inhibitors; p38 Mitogen-Activated Protein Kinases; Protease Inhibitors; Proteasome Endopeptidase Complex; Protein Kinase C; Protein Synthesis Inhibitors; Proto-Oncogene Proteins c-raf; Pyridines; Quinones; Terpenes; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Type C Phospholipases; U937 Cells; Ubiquitins

The ubiquitin-mediated proteolytic system has been implicated in the turnover of a number of intracellular proteins. In the present study, we investigated the novelty and potential role of bryostatin 1, a macrocyclic lactone isolated from the marine bryozoan, Bugula neritina, in inducing the ubiquitin-mediated proteolysis of the oncoprotein Bcl-2. Immunoprecipitation and immunoblotting analyses revealed that Bcl-2 is ubiquitinated following exposure of the acute lymphoblastic leukemia (ALL) cell line Reh to 1 nM bryostatin 1. Bcl-2 protein rapidly decreases to 50% of that recorded in the control after 24 h of bryostatin 1 treatment. In the subsequent 24 h, Bcl-2 protein again rapidly decreases to 6% of its pre-bryostatin 1 level at which time a plateau is reached and maintained for another 72 h. Furthermore, ubiquitin-Bcl-2 conjugates are detected in untreated as well as bryostatin 1 treated cells, indicating that ubiquitin-dependent proteolysis plays a role in the normal turnover of Bcl-2. However, ubiquitin-Bcl-2 conjugates increase in a time-dependent manner following bryostatin 1 treatment. Lactacystin, which inhibits the proteinase activities of the proteasome, inhibited the bryostatin 1-induced decrease of Bcl-2 protein. The effect of bryostatin 1 on the proteolytic efficiency of the 26S proteasome in Reh cell extracts was also investigated and shown to increase following 1 h of bryostatin 1 treatment. Proteolytic activity reached its highest point by 3 h, and subsequently returned to control levels by 12 h, post-bryostatin 1 treatment. In addition, bryostatin 1 treatment of the Reh cell line decreased expression of bcl-2 mRNA within 3 h. However, bcl-2 mRNA expression returned after 24 h. We speculate that this decrease in mRNA together with increased 26S proteolytic activity accounts for the initial rapid decrease recorded in Bcl-2 protein. These findings indicate that bryostatin 1 treatment of Reh ALL cells decreases Bcl-2 expression through two processes: a) enhanced Bcl-2 protein degradation through the activation of the ubiquitin-proteasome pathway and b) decreased bcl-2 mRNA expression.

Topics: Acetylcysteine; Antineoplastic Agents; Blotting, Northern; Blotting, Western; Bryostatins; Cell Extracts; Cysteine Endopeptidases; Humans; Lactones; Macrolides; Multienzyme Complexes; Precipitin Tests; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Proteasome Endopeptidase Complex; Protein Binding; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured; Ubiquitins

We evaluated the possibility that distinct proteolytic pathways contribute to the down-regulation of a novel (epsilon) or conventional (alpha) isoform of protein kinase C (PKC) in nonimmortalized human fibroblasts. Inhibitors of calpains and other cysteine proteinases, vesicle trafficking, or lysosomal proteolysis did not affect the down-regulation of PKC-alpha or -epsilon produced by bryostatin 1 (Bryo). Lactacystin (Lacta) and certain terminal aldehyde tripeptides or tetrapeptides, which selectively inhibit the proteasome, preserved substantial PKC-alpha and -epsilon protein from down-regulation by Bryo or phorbol-12-myristate-13-acetate. Lacta preserved active kinase in vivo, as shown by the retention of Bryo-induced autophosphorylated PKC-alpha. Concomitant with down-regulation, Bryo produced PKC-alpha and -epsilon species that were larger than the native proteins (80 and 90 kDa, respectively). Western blot analysis showed that the larger PKC-alpha species were ubiquitinylated. Treatment with Bryo plus Lacta synergistically increased multiubiquitinylated PKC-alpha, as expected if Bryo induces ubiquitinylation of PKC-alpha and Lacta blocks its degradation. Bryo also produced a 76-kDa, nonphosphorylated form of PKC-alpha and an 86-kDa form of PKC-epsilon. Phosphatase inhibitors decreased production of 76- and 86-kDa PKC-alpha and -epsilon by Bryo and preserved 80- and 90-kDa PKC-alpha and -epsilon, respectively. Our results suggest that the down-modulation of PKC-alpha and -epsilon occurs principally via the ubiquitin/ proteasome pathway. Dephosphorylation seems to predispose PKC to ubiquitinylation.

Topics: Acetylcysteine; Bryostatins; Cells, Cultured; Cysteine Proteinase Inhibitors; Down-Regulation; Fibroblasts; Glycoproteins; Humans; Isoenzymes; Lactones; Macrolides; Protein Kinase C; Protein Kinase C-alpha; Protein Kinase C-epsilon; Tetradecanoylphorbol Acetate; Ubiquitins

Bryostatins and phorbol esters acutely activate and subsequently down-regulate protein kinase C (PKC) by inducing its proteolysis via an unknown pathway. Here we show that treatment of renal epithelial cells with bryostatin 1 (Bryo) produced novel PKC-alpha species, which were larger than the native protein (80 kDa). The >80 kDa PKC-alpha species contained Ubi as indicated by immunostaining and accumulated in the presence of lactacystin, a selective inhibitor of proteolysis by the proteasome. In vitro experiments with 125I-ubiquitin and membranes from Bryo-treated cells showed that PKC-alpha became ubiquitinated by a reaction that depended on ATP and a cytosolic fraction. Lactacystin or a peptidyl aldehyde, Bz-Gly-Leu-Ala-leucinal, which inhibits certain proteinase activities of the proteasome, inhibited Bryo-evoked disappearance of PKC-alpha protein from the cells. Lacta preserved Bryo-induced 32P-labeled PKC-alpha indicating that the proteasome inhibitor spared activated enzyme from down-regulation in vivo. These findings show that Bryo induces the degradation of PKC-alpha by the ubiquitin-proteasome complex.

Topics: Acetylcysteine; Adenosine Triphosphate; Amino Acid Sequence; Animals; Bryostatins; Cells, Cultured; Cysteine Endopeptidases; Cytosol; Down-Regulation; Hydrolysis; Isoenzymes; Lactones; Leucine; Macaca mulatta; Macrolides; Molecular Sequence Data; Multienzyme Complexes; Proteasome Endopeptidase Complex; Protein Kinase C; Protein Kinase C-alpha; Ubiquitins