Bryostatin 1 and Phorbol Ester Down-Modulate Protein Kinase C-α and -ε via the Ubiquitin/Proteasome Pathway in Human Fibroblasts
- 1Department of Pharmacology and Toxicology, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, Alabama 35294 (H.-W.L., L.S., J.B.S.), and the 2Cancer Research Institute and Department of Chemistry, Arizona State University, Tempe, Arizona 85287 (G.R.P.)
Abstract
We evaluated the possibility that distinct proteolytic pathways contribute to the down-regulation of a novel (ε) or conventional (α) 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-α or -ε produced by bryostatin 1 (Bryo). Lactacystin (Lacta) and certain terminal aldehyde tripeptides or tetrapeptides, which selectively inhibit the proteasome, preserved substantial PKC-α and -ε protein from down-regulation by Bryo or phorbol-12-myristate-13-acetate. Lacta preserved active kinasein vivo, as shown by the retention of Bryo-induced autophosphorylated PKC-α. Concomitant with down-regulation, Bryo produced PKC-α and -ε species that were larger than the native proteins (80 and 90 kDa, respectively). Western blot analysis showed that the larger PKC-α species were ubiquitinylated. Treatment with Bryo plus Lacta synergistically increased multiubiquitinylated PKC-α, as expected if Bryo induces ubiquitinylation of PKC-α and Lacta blocks its degradation. Bryo also produced a 76-kDa, nonphosphorylated form of PKC-α and an 86-kDa form of PKC-ε. Phosphatase inhibitors decreased production of 76- and 86-kDa PKC-α and -ε by Bryo and preserved 80- and 90-kDa PKC-α and -ε, respectively. Our results suggest that the down-modulation of PKC-α and -ε occurs principally via the ubiquitin/proteasome pathway. Dephosphorylation seems to predispose PKC to ubiquitinylation.
Footnotes
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Send reprint requests to: Dr. Jeffrey B. Smith, Department of Pharmacology and Toxicology, Volker Hall, G133E, 1670 University Boulevard, Birmingham, AL 35294-0019. E-mail:jeff.smith{at}ccc.uab.edu
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↵1 Lee, H.-W., and Smith, J. B., unpublished observations.
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This work was supported by National Institutes of Health Grant HL44408 (G.B.S.); by Outstanding Investigator Grant CA44344–01-08 (G.R.P.) from the United States National Cancer Institute, Divison of Cancer Treatment, Diagnosis Centers; and by the Department of Health and Human Services, and The Arizona Disease Control Research Commission.
- Abbreviations:
- PKC
- protein kinase C
- AcLLMal
- N-acetyl-Leu-Leu-methional
- AcLLNal
- N-acetyl-Leu-Leu-norleucinal
- BFA
- brefeldin A
- Bryo
- bryostatin 1
- DAG
- diacylglycerol
- DMEM
- Dulbecco’s modified Eagle’s medium
- Lacta
- lactacystin
- LB
- lysis buffer
- PAGE
- polyacrylamide gel electrophoresis
- PMA
- phorbol-12-myristate-13-acetate
- SDS
- sodium dodecyl sulfate
- TBS
- Tris-buffered salt solution
- TRH
- thyrotropin-releasing hormone
- Ub
- ubiquitin
- ZGLALal
- benzyloxycarbonyl-Gly-Leu-Ala-leucinal
- ZGLALol
- benzyloxycarbonyl-Gly-Leu-Ala-leucinol
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- Received October 16, 1996.
- Accepted December 4, 1996.
- The American Society for Pharmacology and Experimental Therapeutics
