Analysis of mitotic phosphorylation of Borealin

Background  

The main role of the chromosomal passenger complex is to ensure that Aurora B kinase is properly localized and activated before and during mitosis. Borealin, a member of the chromosomal passenger complex, shows increased expression during G2/M phases and is involved in targeting the complex to the centromere and the spindle midzone, where it ensures proper chromosome segregation and cytokinesis. Borealin has a consensus CDK1 phosphorylation site, threonine 106 and can be phosphorylated by Aurora B Kinase at serine 165 in vitro.     

Effects of tau phosphorylation on proteasome activity

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.     

Ubiquitin-independent degradation of cell-cycle inhibitors by the REGgamma proteasome

The cell-cycle regulator p21(Cip1) is degraded by proteasomes independently of ubiquitination. We now show that degradation of p21 in vivo does not require the 19S proteasome lid, which contains the ubiquitin-binding subunit. Instead, the major proteasomal pathway for p21 degradation involves an alternative proteasome lid, the REGgamma complex. REGgamma binds to p21 in vivo, and deletion of p21's REGgamma-binding site greatly extends its half-life. Knockdown of REGgamma by RNA interference stabilizes p21, p21 has a significantly extended half-life in REGgamma(-/-) murine embryonic fibroblasts, and the p21 abundance is elevated in REGgamma(-/-) mice. The role of REGgamma in cell-cycle regulation may extend beyond p21 regulation, because p16(INK4A) and p19(Arf) also bind to REGgamma and are stabilized in REGgamma-deficient cells.     

Effects of phosphatase inhibitors on nuclease activity

Nucleases such as DNase I, which selectively digest chromatin, are inhibited by several commonly used phosphatase inhibitors including sodium bisulfite. Two inhibitors, sodium arsenate and fructose-1,6-diphosphate, did not significantly inhibit nuclease action. Two other effective phosphatase inhibitors, p-chloromercuriphenyl sulfonate and 5,5'-dithiobis(2-nitrobenzoate), can be used during nuclei isolation and then washed out of nuclei before nuclease digestion. Using this procedure, 1mM p-chloromercuriphenyl sulfonate is as effective as 50mM bisulfite in retaining the phosphatase-sensitive mitotic phosphorylations of histones H1 and H3.     

Involvement of histone phosphorylation in thymocyte apoptosis by protein phosphatase inhibitors

Incubation of rat thymocytes with the inhibitors of protein phosphatase such as calyculin A and okadaic acid resulted in an increase in DNA fragmentation. These effects were dependent on the concentration of the inhibitors and the incubation time. Analyses of the fragmented DNA revealed the production of approximately 50 kbp of DNA and a 180 bp DNA ladder. In addition, a laser scanning-microscopic analysis showed that these compounds caused nuclear condensation. Thus, these results demonstrated that protein phosphatase inhibitors induced thymocyte apoptosis. The inhibitors of protein phosphatase increased the phosphorylation of proteins of approximately 15 kDa. The phosphorylation of proteins preceded the DNA fragmentation induced by these inhibitors. Judging from acetic acid-urea-Triton X-100 gel electrophoresis, the phosphorylated proteins were histone H1 and H2A/H3. Therefore, these results suggest that phosphorylation of histones triggers the DNA fragmentation of thymocytes undergoing apoptosis.     

细菌蛋白酶体及蛋白酶体抑制剂研究进展

蛋白酶体在真核生物、古菌和部分细菌(主要是放线菌)的胞内蛋白质降解中起着至关重要的作用。虽然三域生物蛋白酶体的结构相似,但细菌蛋白酶体在组装、调节、生理功能等方面与真核生物和古菌都截然不同。研究细菌蛋白酶体不仅有助于认识其起源和进化历程,也将为发掘蛋白酶体抑制剂(proteasome inhibitor, PI)这类具有广阔药用前景的化合物提供指导。本文综述了细菌蛋白酶体的结构、功能和进化假说,并概括了细菌蛋白酶体抑制剂的最新研究进展,期望为相关研究提供参考。     

Protein degradation by the proteasome and dissection of its in vivo importance with synthetic inhibitors

Molecular Biology Reports -     

Effects of phosphatase inhibitors and a protein phosphatase on norepinephrine secretion by permeabilized bovine chromaffin cells

A protein phosphatase and phosphatase inhibitors were used to examine the role of protein phosphorylation in the regulation of norepinephrine secretion in digitonin-permeabilized bovine chromaffin cells. Addition of okadaic acid, a potent inhibitor of type 1 and type 2A protein phosphatases, or 1-naphthylphosphate, a more general phosphatase inhibitor, to digitonin-permeabilized chromaffin cells caused about a 100% increase in the amount of norepinephrine secreted in the absence of Ca²⁺ (in 5 mM EGTA) without affecting the amount of norepinephrine secreted in the presence of 10 μM free Ca²⁺. This stimulation of norepinephrine secretion by protein phosphatase inhibitors suggests that in the absence of Ca²⁺ there is a slow rate phosphorylation and that this phosphorylation triggers secretion. Addition of an exogenous type 2A protein phosphatase caused almost a 50% decrease in Ca²⁺-dependent norepinephrine secretion. Thus, the amounts of norepinephrine released both in the absence of Ca²⁺ and in the presence of Ca²⁺ appear to depend upon the level of protein phosphorylation.     

Cathepsin D-mediated yolk protein degradation is blocked by acid phosphatase inhibitors

Vitellin (VT) is a lipoglycophosphoprotein stored inside the eggs of every oviparous organism during oogenesis. In the blood-sucking bug Rhodnius prolixus, VT is deposited inside growing oocytes together with two acid hydrolases: acid phosphatase (AP) and cathepsin D (CD). Egg fertilization triggers AP activity and VT proteolysis in vivo [Insect Biochem. Mol. Biol. 2002 (32) 847]. Here, we show that CD is the main protease targeting VT proteolysis during egg development. CD activity in total egg homogenates is blocked by the classical aspartyl protease inhibitor, pepstatin A. Surprisingly, AP inhibitors such as NaF, Na+/K+ tartrate, and inorganic phosphate also block VT proteolysis, whereas this effect is not observed when tyrosine phosphatase inhibitors such as vanadate and phenylarsine oxide or an inhibitor of alkaline phosphatases such as levamisole are used in a VT proteolysis assay. NaF concentrations that block isolated AP activity do not affect the activity of partially purified CD. Therefore, a specific repressor of VT proteolysis must be dephosphorylated by AP in vivo. In conclusion, these results demonstrate for the first time that acid hydrolases act cooperatively to promote yolk degradation during egg development in arthropods.     

Effects of nucleotides on assembly of the 26S proteasome and degradation of ubiquitin conjugates

We have investigated three aspects of nucleotide usage by the 26S proteasome and its regulatory complex (RC). Both particles hydrolyze the four major ribonucleotides, but ATP and CTP have substantially lower K _s for hydrolysis than do GTP and UTP. The K _ for ATP hydrolysis is 15 m for the 26S proteasome and 30 m for the regulatory complex. Formation of the 26S proteasome from the RC and the 20S proteasome requires about 5 m ATP. Although measurable degradation of Ubiquitin(Ub)-lysozyme conjugates occurs in the presence of CTP, GTP, and UTP, the best nucleotide for Ub-conjugate degradation by the 26S proteasome is ATP, with an estimated K _ of 12 m. In summary, our studies show that micromolar concentrations of ATP are sufficient for several 26S proteasome activities.     

Effects of phosphatase inhibitors and a protein phosphatase on norepinephrine secretion by permeabilized bovine chromaffin cells.

A protein phosphatase and phosphatase inhibitors were used to examine the role of protein phosphorylation in the regulation of norepinephrine secretion in digitonin-permeabilized bovine chromaffin cells. Addition of okadaic acid, a potent inhibitor of type 1 and type 2A protein phosphatases, or 1-naphthylphosphate, a more general phosphatase inhibitor, to digitonin-permeabilized chromaffin cells caused about a 100% increase in the amount of norepinephrine secreted in the absence of Ca2+ (in 5 mM EGTA) without affecting the amount of norepinephrine secreted in the presence of 10 microM free Ca2+. This stimulation of norepinephrine secretion by protein phosphatase inhibitors suggests that in the absence of Ca2+ there is a slow rate phosphorylation and that this phosphorylation triggers secretion. Addition of an exogenous type 2A protein phosphatase caused almost a 50% decrease in Ca(2+)-dependent norepinephrine secretion. Thus, the amounts of norepinephrine released both in the absence of Ca2+ and in the presence of Ca2+ appear to depend upon the level of protein phosphorylation.     

Regulation of proteasome complexes by gamma-interferon and phosphorylation

Proteasomes play a major role in non-lysosomal proteolysis and also in the processing of proteins for presentation by the MHC class I pathway. In animal cells they exist in several distinct molecular forms which contribute to the different functions. 26S proteasomes contain the core 20S proteasome together with two 19S regulatory complexes. Alternatively, PA28 complexes can bind to the ends of the 20S proteasome to form PA28-proteasome complexes and PA28-proteasome-19S hybrid complexes have also been described. Immunoproteasome subunits occur in 26S proteasomes as well as in PA28-proteasome complexes. We have found differences in the subcellular distribution of the different forms of proteasomes. The gamma-interferon inducible PA28 alpha and beta subunits are predominantly located in the cytoplasm, while 19S regulatory complexes (present at significant levels only in 26S complexes) are present in the nucleus as well as in the cytoplasm. Immunoproteasomes are greatly enriched at the endoplasmic reticulum (ER) where they may facilitate the generation of peptides for transport into the lumen of the ER. We have also investigated the effects of gamma-interferon on the levels and subcellular distribution of inducible subunits and regulator subunits. In each case gamma-interferon was found to increase the level but not to alter the distribution. Several subunits of proteasomes are phosphorylated including alpha subunits C8 (alpha7) and C9 (alpha3), and ATPase subunit S4 (rpt2). Our studies have shown that gamma-interferon treatment decreases the level of phosphorylation of proteasomes. We have investigated the role of phosphorylation of C8 by casein kinase II by site directed mutagenesis. The results demonstrate that phosphorylation at either one of the two sites is essential for the association of 19S regulatory complexes and that the ability to undergo phosphorylation at both sites gives the most efficient incorporation of C8 into the 26S proteasome.     

Advances in and applications of proteasome inhibitors

With the recent US Food and Drug Administration approval of bortezomib (Velcade) for the treatment of relapsed multiple myeloma, the proteasome has emerged as a new therapeutic target with diverse pathology. Drug discovery programs in academia and the pharmaceutical industry have developed a range of low nanomolar synthetic and natural inhibitors of the 20S proteasome core particle that have entered human clinical trials as significant anti-cancer and anti-inflammatory leads. Moreover, proteasome inhibitors continue to serve as valuable research tools in cellular biology through the elucidation of important biological processes associated with the ubiquitin-proteasome pathway of protein degradation. This review will highlight recent advances in the development and application of proteasome inhibitors.     

Effects of tyrosine kinase and phosphatase inhibitors on microtubules in Arabidopsis root cells

To investigate the role of tyrosine phosphorylation/dephosphorylation processes in plant cells the morphology of Arabidopsis thaliana primary roots and the organization of cortical microtubules (MTs) were studied after inhibition of protein tyrosine kinases (PTKs) and tyrosine phosphatases (PTPs). It was found that all tested types of PTKs inhibitors (herbimycin A, genistein and tyrphostin AG 18) altered root hair growth and development, probably as a result of their significant influences on MTs organization in root hairs. The treatment also led to MTs reorientation and disruption in epidermis and cortex cells of both elongation and differentiation zones of primary roots. Enhanced tyrosine phosphorylation after treatment with a PTPs inhibitor (sodium orthovanadate) resulted in intense induction of root hair development and growth and caused a significant shortening of the elongation zone. It also led to changes of MTs orientation from transverse to longitudinal in epidermis and cortex cells of the elongation and differentiation zones of the root. From the data obtained we can suppose that tyrosine phosphorylation can be involved in the dynamics and organization of MTs in different types of plant cells.     

Effects of phosphoprotein phosphatase inhibitors (phenylarsine oxide and cantharidin) on Tetrahymena

The effects of phenylarsine oxide (PAO) (phosphotyrosine phosphatase inhibitor) and cantharidin (serine/threonine phosphatase [PP2A] inhibitor) treatments were analysed on the synthesis of phospholipids and glycolipids, and on the cytoskeletal elements (F-actin and tubulin containing structures) of Tetrahymena pyriformis. Both phosphatase inhibitors reduced the amount of incorporated 32P of the whole phospholipid content, but the ratio of phosphatidylserine (PS) and phosphatidylcholine (PC) to the total phospholipid content increased. Both treatments influenced the phosphatidylinositol (PI) system. These inhibitors also influenced the incorporation of palmitic acid into the phospholipids: in general PAO decreased, whereas cantharidin increased the amount of incorporated palmitic acid; 1 microM cantharidin significantly increased the labelling of PE and PA. The incorporation of mannose and glucosamine was influenced differently by PAO and cantharidin treatments: the latter elevated, while PAO decreased the labelling of glycolipids with these sugars. The effects of these treatments were visible also in the case of confocal scanning laser microscopic (CSLM) images: after treatments with both inhibitors, the F-actin containing cortical elements were destroyed, but the tubulin containing ones (longitudinal and transversal microtubules, oral apparatus and deep fibres) did not display significant alterations. The different effects of phosphatase inhibitors were visible also on the scanning electron microscopic (sEM) images: cantharidin treatments (1 microM) decreased the amount of dissolved membrane lipids after chemical dehydration of the cells with 2, 2-dimethoxy propane (DMP), but in the case of treatments with 10 microM, the surface pattern of cells was similar to the controls. On the other hand, after PAO treatments the surface pattern of Tetrahymena showed significant alterations. Both phosphatase inhibitors inhibited the phagocytotic activity of the cells. On the basis of present experiments we suppose that these treatments are able to influence signalling systems (e.g. PI) of Tetrahymena, and also the structure of the cytoskeleton and the functions (e.g. phagocytosis) which are connected with skeletal elements.     

Stable acid phosphatase: II. Effects of pH and inhibitors

Summary Microdensitometry demonstrated that stable acid phosphatase (SAPhase) in rat and hamster osteoclasts, chondroclasts, and chondrocytes has very similar properties. The differences that were observed suggest that conformational alterations in the enzymes may be responsible for inhibition by some agents such as tartrate. These differences in response to inhibitors depend on the method of embedding as well as on species differences. SAPhase appears to correspond to acid nitrophenyl phosphatase, as shown by its pH dependent re-activation, resistance to fluoride inhibition at nearneutral pH, and the inverse effect of pH on inhibition by zinc versus aluminium ions. That proportion of SAPhase resistant to fluoride is an acid phosphatase with activity at near-neutral pH rather than a strict neutral phosphatase. The difference between fluoride sensitive and fluoride resistant SAPhase may relate to the varying association of a single enzyme with cell or lysosomal membrances. The close similarity of acid and neutral SAPhase suggests that both may represent a single enzyme in two forms rather than two distinct enzymes.Supported by the Medical Research Council of Great Britain     

Stable acid phosphatase: II. Effects of pH and inhibitors

Microdensitometry demonstrated that stable acid phosphatase (SAPhase) in rat and hamster osteoclasts, chondroclasts, and chondrocytes has very similar properties. The differences that were observed suggest that conformational alterations in the enzymes may be responsible for inhibition by some agents such as tartrate. These differences in response to inhibitors depend on the method of embedding as well as on species differences. SAPhase appears to correspond to acid nitrophenyl posphatase, as shown by its pH dependent re-activation, resistance to fluoride inhibition at near-neutral pH, and the inverse effect of pH on inhibition by zinc versus aluminium ions. That proportion of SAPhase resistant to fluoride is an acid phosphatase with activity at near-neutral pH rather than a strict neutral phosphatase. The difference between fluoride sensitive and fluoride resistant SAPhase may relate to the varying association of a single enzyme with cell or lysosomal membranes. The close similarity of acid and neutral SAPhase suggests that both may represent a single enzyme in two forms rather than two distinct enzymes.     

P'-extended alpha-ketoamide inhibitors of proteasome.

A series of potent P'-extended alpha-ketoamide inhibitors of chymotrypsin-like activity of proteasome is described.