calyculin-a and lactacystin

Recently, we have shown that two proteasome inhibitors, MG132 and lactacystin, induce hyperphosphorylation and trimerization of HSF1, and transactivate heat shock genes at 37 degrees C. Here, we examined the effects of these proteasome inhibitors and, in addition, a phosphatase inhibitor calyculin A (CCA) on the activation of HSF1 upon heat shock and during post-heat-shock recovery, with emphasis on HSF1 hyperphosphorylation and the ability of HSF1 to transactivate heat shock genes. When lactacystin, MG132, or CCA was present after heat shock, HSF1 remained hyperphosphorylated during post-heat-shock recovery at 37 degrees C. Failure of HSF1 to recover to its preheated dephosphorylated state correlated well with the suppression of the heat-induced hsp70 expression. In vitro, HSF1 from heat-shocked cells, when dephosphorylated, showed an increase in HSE-binding affinity. Taken together, these data suggest that phosphorylation of HSF1 plays an important role in the negative regulation of heat-shock response. Specifically, during post-heat-shock recovery phase, prolonged hyperphosphorylation of HSF1 suppresses heat-induced expression of heat shock genes.

Topics: Acetylcysteine; Animals; Cell Line; Cysteine Proteinase Inhibitors; DNA; DNA-Binding Proteins; Down-Regulation; Enzyme Activation; Enzyme Inhibitors; Gene Expression Regulation; Heat Shock Transcription Factors; Heat-Shock Proteins; Hot Temperature; HSP70 Heat-Shock Proteins; JNK Mitogen-Activated Protein Kinases; Leupeptins; Marine Toxins; Mice; Mitogen-Activated Protein Kinases; Oxazoles; Phosphorylation; Transcription Factors

Lactacystin (1.3 microM), a metabolite from an actinomycete, induced the formation of bipolar projections at both sides of the cell body of Neuro 2a cells 1 day after treatment and networks at and after 3 days and enhanced acetylcholinesterase activity (a marker of neuronal differentiation). Thus, the neuronal differentiation was characterized both morphologically and functionally. The experiments with various inhibitors of protein kinases and phosphatases revealed that the protein phosphatase inhibitors calyculin A (0.5 nM) and okadaic acid (0.6 nM) inhibit the formation of bipolar projections at 1 day, but does not inhibit the network formation at and after 3 days.

Topics: Acetylcholinesterase; Acetylcysteine; Animals; Antifungal Agents; Biomarkers; Bucladesine; Cell Differentiation; Cell Line; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Ethers, Cyclic; Hydroquinones; Kinetics; Marine Toxins; Neurites; Neurons; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Protein Kinase Inhibitors; Pyrans; Spiro Compounds; Time Factors