okadaic-acid and lactacystin

The chromosomal passenger complex (CPC) senses tension defects at the kinetochore to activate the spindle assembly checkpoint, and helps to position the cleavage furrow. The CPC, consisting of INCENP, Survivin, Borealin and Aurora B localizes to the inner centromere at metaphase and re-localizes to the spindle midzone at anaphase; several CPC functions are regulated by post-translational modification. Borealin is phosphorylated at multiple sites and phosphorylation at S219 causes Borealin to migrate more slowly upon electrophoresis. Here we find that Cdk1 can induce a mobility shift of Borealin, suggesting that S219 phosphorylation is under Cdk1 control. However, Cdk1 is inefficient at phosphorylating purified Borealin in vitro. A yeast orthologue of Borealin, Npl1, is dephosphorylated by the phosphatase Cdc14. We find no difference in the mobility shift of Borealin in human cells lacking either Cdc14A or Cdc14B. In contrast, the phosphatase inhibitor okadaic acid does delay the dephosphorylation of Borealin as cells exit mitosis. The proteasome inhibitor MG132 reduces Borealin phosphorylation in mitosis and increases the steady-state level of Borealin, especially in mutants lacking the C-terminus. However, a second, structurally unrelated proteasome inhibitor, lactacystin did not up-regulate Borealin. These results suggest that the effect of MG132 on Borealin is due to the inhibition of an intracellular protease other than the proteasome.

Topics: Acetylcysteine; Amino Acid Substitution; CDC2 Protein Kinase; Cell Cycle Proteins; Dual-Specificity Phosphatases; Gene Expression; HeLa Cells; Humans; Leupeptins; Okadaic Acid; Peptide Fragments; Phosphoric Monoester Hydrolases; Phosphorylation; Proteasome Inhibitors; Protein Processing, Post-Translational; Protein Transport; Proteolysis; Ubiquitin

α-Synuclein (a-Syn) is a major component of fibrillar aggregates in Lewy bodies (LBs), a characteristic hallmark of Parkinson disease. Almost 90% of a-Syn deposited in LBs is phosphorylated at Ser-129. However, the role of Ser-129-phosphorylated a-Syn in the biogenesis of LBs remains unclear. Here, we investigated the metabolism of Ser-129-phosphorylated a-Syn. In SH-SY5Y cells, inhibition of protein phosphatase 2A/1 by okadaic acid, and inhibition of the proteasome pathway by MG132 or lactacystin accumulated Ser-129-phosphorylated a-Syn. However, these inhibitions did not alter the amounts of total a-Syn within the observation time. Inhibition of the autophagy-lysosome pathway by 3-methyladenine or chloroquine accumulated Ser-129-phosphorylated a-Syn in parallel to total a-Syn during longer incubations. Experiments using cycloheximide showed that Ser-129-phosphorylated a-Syn diminished rapidly (t(½) = 54.9 ± 6.4 min), in contrast to the stably expressed total a-Syn. The short half-life of Ser-129-phosphorylated a-Syn was blocked by MG132 to a greater extent than okadaic acid. In rat primary cortical neurons, either MG132, lactacystin, or okadaic acid accumulated Ser-129-phosphorylated a-Syn. Additionally, we did not find that phosphorylated a-Syn was ubiquitinated in the presence of proteasome inhibitors. These data show that Ser-129-phosphorylated a-Syn is targeted to the proteasome pathway in a ubiquitin-independent manner, in addition to undergoing dephosphorylation. The proteasome pathway may play a role in the biogenesis of Ser-129-phosphorylated a-Syn-rich LBs.

Topics: Acetylcysteine; alpha-Synuclein; Animals; Cell Line, Tumor; Cerebral Cortex; Cycloheximide; Cysteine Proteinase Inhibitors; Humans; Leupeptins; Lewy Bodies; Neurons; Okadaic Acid; Parkinson Disease; Phosphorylation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Phosphatase 1; Protein Phosphatase 2; Protein Synthesis Inhibitors; Rabbits; Ubiquitin

Dysfunction of proteasome contributes to the accumulation of the abnormally hyperphosphorylated tau in Alzheimer's disease. However, whether tau hyperphosphorylation and accumulation affect the activity of proteasome is elusive. Here we found that a moderate tau phosphorylation activated the trypsin-like activity of proteasome, whereas further phosphorylation of tau inhibited the activity of the protease in HEK293 cells stably expressing tau441. Furthermore, tau hyperphosphorylation could partially reverse lactacystin-induced inhibition of proteasome. These results suggest that phosphorylation of tau plays a dual role in modulating the activity of proteasome.

Topics: Acetylcysteine; Cells, Cultured; Colforsin; Cysteine Proteinase Inhibitors; Humans; Okadaic Acid; Phosphorylation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; tau Proteins

Cyclin-dependent kinase 5 (cdk5) is involved in the development of the nervous system and neuronal process outgrowth, and it regulates several intracellular processes including cytoskeletal dynamics. Dysregulation of cdk5 has been implicated in many disorders of the nervous system. The activity of the kinase is regulated by binding of cdk5 activators (p35, p39, p67). We examined the phosphorylation of p35, and the role of phosphorylation in regulating the proteolysis of the p35 protein. By detecting changes in electrophoretic mobility, we observed that a significant proportion of p35 is phosphorylated in rat brain tissue. In cultured neurons, the phosphorylation was prevented by roscovitine, an inhibitor of cdk5 and some other cdks. The phosphatase inhibitor okadaic acid induced p35 degradation in neuronal cultures which was sensitive to the proteasome inhibitor lactacystin. These latter results agree with some previous studies showing that phosphorylation regulates proteasomal degradation of p35. Treatment of brain homogenate with okadaic acid in the presence of ATP led to accumulation of p35 phosphorylated also by a kinase that was not inhibited by roscovitine. This implies that the effect of okadaic acid on p35 degradation could also be contributed by a non-cdk kinase. The calpain protease has been shown to cleave p35. Our results suggest that this process may also be modulated by p35 phosphorylation under some conditions. We conclude that p35 phosphorylation influences the proteasome-mediated degradation of p35 and calpain-mediated cleavage of p35 to p25.

Topics: Acetylcysteine; Adenosine Triphosphate; Animals; Animals, Newborn; Brain Chemistry; Calpain; Cells, Cultured; Cyclin-Dependent Kinase 5; Cyclin-Dependent Kinases; Cysteine Endopeptidases; Embryo, Mammalian; Enzyme Inhibitors; Hippocampus; Multienzyme Complexes; Nerve Tissue Proteins; Neurons; Okadaic Acid; Phosphorylation; Proteasome Endopeptidase Complex; Purines; Rats; Rats, Wistar; Roscovitine

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