Stathmin 1 in normal and malignant hematopoiesis

Stathmin 1 is a microtubule destabilizer that plays an important role in cell cycle progression, segregation of chromosomes, clonogenicity, cell motility and survival. Stathmin 1 overexpression has been reported in malignant hematopoietic cells and Stathmin 1 inhibition reduces the highly proliferative potential of leukemia cell lines. However, during the differentiation of primary hematopoietic cells, Stathmin 1 expression decreases in parallel to decreases in the proliferative potential of early hematopoietic progenitors. The scope of the present review is to survey the current knowledge and highlight future perspectives for Stathmin 1 in normal and malignant hematopoiesis, with regard to the expression, function and clinical implications of this protein. [BMB Reports 2014; 47(12): 660-665]     

Drosophila stathmin is required to maintain tubulin pools

Stathmin, or Oncoprotein 18 (Op18), is the founding member of a phosphoprotein family that can regulate the microtubule cytoskeleton by sequestering tubulin and promoting microtubule catastrophe. Stathmin is subject to spatially and temporally controlled regulatory phosphorylation, which inhibits its interaction with tubulin. Drosophila Stathmin has similar properties to the mammalian proteins. We find that Drosophila Stathmin is required for specific microtubule-dependent processes: maintenance of oocyte identity within a germline cyst and localization of polarity determinants. Unexpectedly, microtubules are less abundant in stathmin mutant cells compared to normal cells, showing that a key function of Stathmin in vivo is the long-term maintenance of the microtubule cytoskeleton. The microtubule network re-forms more slowly after coldshock in stathmin mutant follicle cells. Surprisingly, stathmin mutant animals and tissues show a marked decrease in total tubulin-protein levels, and this might explain the effect on the microtubule cytoskeleton. Stathmin overexpression also increases tubulin protein. Free alpha- and beta-tubulin have been shown to negatively autoregulate their own synthesis. We suggest that Stathmin serves to maintain a noninhibitory, soluble, and releasable tubulin pool.     

An oncogenic role for the phosphorylated h-subunit of human translation initiation factor eIF3

Dysregulation of protein synthesis has been implicated in oncogenesis through a mechanism whereby "weak" mRNAs encoding proteins involved in cell proliferation are strongly translated when the protein synthesis apparatus is activated. Previous work has determined that many cancer cells contain high levels of eIF3h, a protein subunit of translation initiation factor eIF3, and overexpression of eIF3h malignantly transforms immortal NIH-3T3 cells. This is a general feature of eIF3h, as high levels also affect translation, proliferation, and a number of malignant phenotypes of CHO-K1 and HeLa cells and, most significantly, of a primary prostate cell line. Furthermore, overexpressed eIF3h inhibits Myc-dependent induction of apoptosis of primary prostate cells. eIF3h appears to function through translation, as the initial appearance of overexpressed eIF3h in rapidly induced NIH-3T3 cells correlates tightly with the stimulation of protein synthesis and the generation of malignant phenotypes. This oncogenic potential of eIF3h is enhanced by phosphorylation at Ser(183). Finally, reduction of eIF3h levels in breast and prostate cancer cell lines by short interfering RNA methods reduces their rates of proliferation and anchorage-independent growth in soft agar. The results provide compelling evidence that high eIF3h levels directly stimulate protein synthesis, resulting in the establishment and maintenance of the malignant state in cells.     

POSH, a scaffold protein for JNK signaling, binds to ALG-2 and ALIX in Drosophila

Plenty of SH3s (POSH) functions as a scaffold protein for the Jun N-terminal kinase (JNK) signal transduction pathway, which leads to cell death in mammalian cultured cells and Drosophila. Here, we show that POSH forms a complex with Apoptosis-linked gene-2 (ALG-2) and ALG-2-interacting protein (ALIX/AIP1) in a calcium-dependent manner. Overexpression of ALG-2 or ALIX in developing imaginal eye discs resulted in roughened or melanized eyes, respectively. These phenotypes were enhanced by co-overexpression of POSH. We found that overexpression of either gene could induce ectopic JNK activation, suggesting that POSH/ALG-2/ALIX may function together in the regulation of the JNK pathway.     

Suppression of v-Src transformation by andrographolide via degradation of the v-Src protein and attenuation of the Erk signaling pathway

Elevated expression and aberrant activation of the src oncogene are strongly associated with cancer initiation and progression, thereby making Src a promising molecular target for anti-cancer therapy. Through drug screening using a temperature-inducible v-Src-transformed epithelial cell line, we found that andrographolide could suppress v-Src-induced transformation and down-regulate v-Src protein expression. In addition, actin cable dissolution and E-cadherin down-regulation, features of transformed phenotype, are perturbed by andrographolide. Moreover, andrographolide promoted v-Src degradation via a ubiquitin-dependent manner. Although andrographolide treatment altered the tyrosine phosphorylation pattern in v-Src-expressing cells, it did not directly affect the kinase activity of v-Src. Both the Erk and phosphatidylinositol 3-kinase signaling pathways were strongly inhibited in andrographolide-treated v-Src cells. However, only MKK inhibitors (PD98059 and U0126) were able to cause a non-transformed morphology similar to that of andrographolide-treated v-Src cells. Moreover, overexpression of constitutively active MKK1 in v-Src cells blocked andrographolide-mediated morphological inhibition. Interestingly, andrographolide treatment could also reduce the protein level of the c-Src truncation mutant (Src531), an Src mutant originally identified from human colon cancer cells. In summary, we demonstrated that andrographolide antagonized v-Src action through promotion of v-Src protein degradation. Furthermore, attenuation of the Erk1/2 signaling pathway is essential for andrographolide-mediated inhibition of v-Src transformation. Our results demonstrate that andrographolide can act as a v-Src inhibitor and reveal a novel action mechanism of andrographolide.     

p300-mediated acetylation of TRF2 is required for maintaining functional telomeres

The human telomeric protein TRF2 is required to protect chromosome ends by facilitating their organization into the protective capping structure. Post-translational modifications of TRF2 such as phosphorylation, ubiquitination, SUMOylation, methylation and poly(ADP-ribosyl)ation have been shown to play important roles in telomere function. Here we show that TRF2 specifically interacts with the histone acetyltransferase p300, and that p300 acetylates the lysine residue at position 293 of TRF2. We also report that p300-mediated acetylation stabilizes the TRF2 protein by inhibiting its ubiquitin-dependent proteolysis and is required for efficient telomere binding of TRF2. Furthermore, overexpression of the acetylation-deficient mutant, K293R, induces DNA-damage response foci at telomeres, thereby leading to induction of impaired cell growth, cellular senescence and altered cell cycle distribution. A small but significant number of metaphase chromosomes show no telomeric signals at chromatid ends, suggesting an aberrant telomere structure. These findings demonstrate that acetylation of TRF2 by p300 plays a crucial role in the maintenance of functional telomeres as well as in the regulation of the telomere-associated DNA-damage response, thus providing a new route for modulating telomere protection function.     

A stable, inducible, dose-responsive ODC overexpression system in human cell lines

ODC is a labile protein subject to rapid turnover, and a conditional expression system providing long-term overexpression may be helpful in further understanding the biochemical properties of this enzyme and elucidating aspects of the polyamine biosynthetic pathway that have otherwise been difficult to study. HEK293 and LNCaP cell lines were engineered to stably and inducibly overexpress ODC using a Tet-on inducible construct. Clones from both cell lines were characterized by evaluating ODC mRNA expression, ODC activity, intracellular and extracellular polyamine levels, SSAT activity and growth kinetics. The ODC-inducible cell lines were time- and dose-responsive providing a mechanism to increase ODC and putrescine accumulation to a desired level in a flexible and controllable manner. The findings demonstrate that LNCaP ODC overexpressing cells maintained over a 100-fold increase in ODC activity and over a 10-fold increase in intracellular putrescine after 6 h. ODC induction at the highest levels was accompanied by a slight decline in intracellular spermidine and spermine levels and this observation was supported by the finding that SSAT activity was induced over 40-fold under these conditions. Growth rate remained unaffected following at least 12 h of ODC overexpression. Similar results were observed in the HEK293 ODC overexpressing cells.     

Involvement of the ubiquitin-proteasome pathway in the degradation of nontyrosine kinase-type cytokine receptors of IL-9, IL-2, and erythropoietin

The ubiquitin-dependent proteasome-mediated (Ub-Pr) degradation pathway has been shown to regulate a large variety of substrates, including nuclear, cytosolic, and membrane proteins. In mammalian systems, polyubiquitin modification has been identified in a number of cell surface receptors for more than a decade; however, its biological significance has remained unclear until recently. For growth factor receptors with intrinsic tyrosine kinase domains, polyubiquitination is believed to trigger the internalization and subsequent degradation via the lysosomal pathway. In this study we provide the first evidence that non-tyrosine kinase-type cytokine surface receptors, IL-9R alpha-chain, IL-2 receptor ss-chain, and erythropoietin receptor, can be polyubiquitinated and degraded by proteasomes. The Ub-Pr pathway regulates both the basal level turnover and the ligand-induced degradation of the receptors. A previously identified putative molecular chaperon, valosin-containing protein, undergoes tyrosine phosphorylation in a cytokine-dependent manner and associates with the receptor complexes following receptor engagement, suggesting that valosin-containing protein may target the ubiquitinated receptors to the proteasome for degradation.     

Investigation of the reciprocal relationship between the expression of two gap junction connexin proteins, connexin46 and connexin43

Connexins are the transmembrane proteins that form gap junctions between adjacent cells. The function of the diverse connexin molecules is related to their tissue-specific expression and highly dynamic turnover. Although multiple connexins have been previously reported to compensate for each other's functions, little is known about how connexins influence their own expression or intracellular regulation. Of the three vertebrate lens connexins, two connexins, connexin43 (Cx43) and connexin46 (Cx46), show reciprocal expression and subsequent function in the lens and in lens cell culture. In this study, we investigate the reciprocal relationship between the expression of Cx43 and Cx46. Forced depletion of Cx43, by tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate, is associated with an up-regulation of Cx46 at both the protein and message level in human lens epithelial cells. An siRNA-mediated down-regulation of Cx43 results in an increase in the level of Cx46 protein, suggesting endogenous Cx43 is involved in the regulation of endogenous Cx46 turnover. Overexpression of Cx46, in turn, induces the depletion of Cx43 in rabbit lens epithelial cells. Cx46-induced Cx43 degradation is likely mediated by the ubiquitin-proteasome pathway, as (i) treatment with proteasome inhibitors restores the Cx43 protein level and (ii) there is an increase in Cx43 ubiquitin conjugation in Cx46-overexpressing cells. We also present data that shows that the C-terminal intracellular tail domain of Cx46 is essential to induce degradation of Cx43. Therefore, our study shows that Cx43 and Cx46 have novel functions in regulating each other's expression and turnover in a reciprocal manner in addition to their conventional roles as gap junction proteins in lens cells.     

Ubc4 and Not4 Regulate Steady-State Levels of DNA Polymerase-�� to Promote Efficient and Accurate DNA Replication

The accurate duplication of chromosomal DNA is required to maintain genomic integrity. However, from an evolutionary point of view, a low mutation rate during DNA replication is desirable. One way to strike the right balance between accuracy and limited mutagenesis is to use a DNA polymerase that lacks proofreading activity but contributes to DNA replication in a very restricted manner. DNA polymerase-α fits this purpose exactly, but little is known about its regulation at the replication fork. Minichromosome maintenance protein (Mcm) 10 regulates the stability of the catalytic subunit of pol-α in budding yeast and human cells. Cdc17, the catalytic subunit of pol-α in yeast, is rapidly degraded after depletion of Mcm10. Here we show that Ubc4 and Not4 are required for Cdc17 destabilization. Disruption of Cdc17 turnover resulted in sensitivity to hydroxyurea, suggesting that this pathway is important for DNA replication. Furthermore, overexpression of Cdc17 in ubc4 and not4 mutants caused slow growth and synthetic dosage lethality, respectively. Our data suggest that Cdc17 levels are very tightly regulated through the opposing forces of Ubc4 and Not4 (destabilization) and Mcm10 (stabilization). We conclude that regular turnover of Cdc17 via Ubc4 and Not4 is required for proper cell proliferation.     

The Ubiquitin System in Higher and Lower Plants — Pathways in Protein Metabolism

The multiple biological functions of the small polypeptide ubiquitin are mirrored by its unparalleled conservation on the amino acid and gene organization level. During the last years, it has become widely accepted that ubiquitin is an essential component in the ATP-dependent nonlysosomal protein degradation pathway occurring in all eukaryotic organisms. As turnover, consisting of protein synthesis and disassembly, is a central and vital process for each living cell, ubiquitin-mediated proteolysis is of enormous physiological value. The components of the ubiquitin ligation system have been characterized skillfully in plant and animal cells, but at the moment many questions remain as to how the high degree of specificity that is necessary for the regulation of intracellular breakdown is ensured. The recent hypotheses and models proposed for the basic mechanisms of protein recognition, conjugation and degradation will be discussed in detail. The existence of ubiquitin-protein conjugates which are not rapidly degraded clearly suggested that the role of ubiquitin is not restricted in its implication for protein turnover. Alterations of DNA structure, specific cell recognition mechanisms and cytoskeletal variations were observed as further ubiquitin-dependent processes which are not directly coupled to protein degradation.     

Targeting BAX ubiquitin-binding sites reveals that BAX activation is essential for its ubiquitin-dependent degradation

BAX is an important proapoptotic protein of the BCL-2 family, and its stability is essential for the regulation of the mitochondrial apoptotic pathway. A previous study revealed that BAX could undergo degradation through the ubiquitin-proteasome pathway. In this study, we identified two lysine sites, K21 and K123, that were critical ubiquitin-binding sites in BAX. Mutation of these two sites prolonged the half-life of BAX and also affected its proapoptotic ability. Intriguingly, we found that ABT-737, a BCL-2 inhibitor, significantly enhanced TRAIL-induced BAX degradation in HCT116 cells and increased TRAIL-induced apoptosis in the HCT116 only with the BAX K21R/K123R mutant, not other BAX mutants. In addition, overexpression of PARKIN, an E3 ubiquitin ligase targeting BAX, dramatically decreased BAX protein level when only treated with ABT-737 in HCT116 cells. Therefore, we speculated that BAX activation is essential for its ubiquitin-dependent degradation.     

Cell cycle-dependent regulation of N-acetylglucosaminyltransferase-III in a human colon cancer cell line, Colo201

The mechanism for cell-cycle-dependent regulation of N-acetylglucosaminyltransferase III (GnT-III) activity was investigated using synchronized culture of Colo201, a human colon cancer cell line. In the synchronized culture, it was found that GnT-III activity rapidly increased in the M phase and the maximal activity was five times higher than the basal level found in the G1 phase. Northern blot and Western blot analyses revealed that the increase in the activity is due not to an increase in expression level of its mRNA but, rather, to the level of protein. Furthermore, it was shown by a pulse-chase experiment that the increased protein level of GnT-III is the result of its prolonged turnover rate. Lectin blotting with erythroagglutinating phytohemagglutinin showed that the content of bisecting N-acetylglucosamine structure in glycoproteins was transiently increased during the M phase in conjunction with the increased activity of GnT-III. These results suggest that GnT-III activity undergoes a cell-cycle-dependent regulation and thereby oligosaccharide structures of N-glycans vary specifically during the M phase of the cell cycle. Thus, it is possible that the cell-cycle-dependent alteration of N-glycans by GnT-III might play a role in biological events, such as the progression of cell cycle and cell division.