Requirement of phosphatase of regenerating liver-3 for the nucleolar localization of nucleolin during the progression of colorectal carcinoma.

Phosphatase of regenerating liver-3 (PRL-3) is a protein tyrosine phosphatase (PTP) that is frequently overexpressed in liver metastases of colorectal carcinomas (CRCs). The PTP activity of the PRL-3 protein is indispensable for the promotion of distant metastasis of CRC; however, little is known about the effect of PRL-3 on cell growth. In this study, we investigated a novel protein that can connect to PRL-3 to modulate the proliferation of CRC cells. In CRC-derived SW480 cells, transduction of ectopic wild-type PRL-3, but not the C104S catalytic "dead" mutant, up-regulated cell proliferation and increased the population of cells at the S and G(2) /M phases. Also, inhibition of PTP activity of the PRL-3 protein by treatment with the PRL-3 inhibitor suppressed cell proliferation in a dose-dependent manner as well as PRL-3 knockdown by RNA interference. Using a comparative study of monodimensional gel electrophoresis of immunoprecipitates from PRL-3-transfected SW480 cells and subsequent mass spectrometry analysis, nucleolar-specific protein nucleolin (NCL) was identified as a novel PRL-3-binding protein. We confirmed physiological interaction between PRL-3 and NCL, and found that PRL-3 phosphatase activity was associated with the suppression of the phospho-NCL levels and nucleolar assembly of NCL protein. In CRC cases, nucleolar NCL expression was correlated not only with higher levels of PRL-3 expression but also with frequent incidence of lymph node metastasis and a higher clinicopathologic stage. These findings suggest that NCL is involved in PRL-3-mediated cancer progression/metastasis signaling, which plays an important role in the acceleration of CRC growth.

Negative regulation of the tight junction protein tricellulin by snail-induced epithelial-mesenchymal transition in gastric carcinoma cells.

OBJECTIVE: Tricellulin plays a central role in the sealing of epithelia at tricellular contacts. We examined the effects of Snail, an epithelial-mesenchymal transition (EMT)-related transcription factor, on the regulation of tricellulin expression in human gastric carcinoma (GC)-derived cells. METHOD: Six human GC-derived cell lines were used in this study. Expression and localization of tricellulin was analyzed by reverse transcription (RT)-PCR and immunohistochemistry. Also, a Snail expression vector was transfected into HSC-45 cells to examine altered mRNA levels of tricellulin,E-cadherin, vimentin, N-cadherin and several EMT transcription factors by quantitative real-time RT-PCR. RESULTS: Abundant tricellulin expression was detected in all GC-derived cells examined. In HSC-45 cells, transduction of Snail decreased the expression levels of tricellulin and E-cadherin but increased vimentin and N-cadherin, which was accompanied by induction of EMT transcription factors such as Twist1, Twist2 and Slug. In normal gastric mucosa, tricellulin protein was localized at the tricellular tight junction; however, in HSC-45 cells, tricellulin protein was distributed in the cytoplasm. In GC tissues, tricellulin expression at the cellular membrane was retained in a subset of EMT-negative GCs, and it disappeared in EMT-positive GCs. CONCLUSIONS: The findings in the present study suggest that repression of tricellulin expression may be related to Snail-induced EMT in human GCs.

Down-modulation of keratin 8 phosphorylation levels by PRL-3 contributes to colorectal carcinoma progression.

Phosphatase of regenerating liver-3 (PRL-3) is a member of the PRL protein tyrosine phosphatase family and has been proposed to promote the invasiveness and metastastic capability of colorectal cancers (CRCs); however, the underlying mechanisms and target molecules of PRL-3 protein remain unknown. On the basis of the biological significance of PRL-3 phosphatase activity confirmed by the catalytically inactive PRL-3 mutant (C104S) and a PRL-3 inhibitor in CRC-derived SW480 cells, we performed protein expression profiling to search for PRL-3-mediated effector proteins. By a comparative study of phosphorylated proteins that differentially expressed in wild type and C104S mutant PRL-3-transfected SW480 cells; the cytoskeletal intermediate filament keratin 8 (KRT8) was identified as a physiological PRL-3-interacting protein. Indeed, treatment with the PRL-3 inhibitor effectively suppressed the phosphorylation of KRT8 at S73 and S431. Moreover, we detected the physiological interaction between PRL-3 and KRT8 and their colocalization at cellular lamellipodias and ruffles in vivo. In CRC tissue samples, tumor cells with high PRL-3 expression showed reduction or loss of phosphorylated KRT8 expression, particularly at the invasive front and in the liver metastases. In conclusion, our results indicate that PRL-3 may play an important role for the promotion of CRC cell migration and metastatic potential through direct KRT8 dephosphorylation.

Involvement of Rho-type GTPase in control of cell size in Saccharomyces cerevisiae.

Maintaining specific cell size, which is important for many organisms, is achieved by coordinating cell growth and cell division. In the budding yeast Saccharomyces cerevisiae, the existence of two cell-size checkpoints is proposed: at the first checkpoint, cell size is monitored before budding at the G1/S transition, and at the second checkpoint, actin depolymerization occurring in the small bud is monitored before the G2/M transition. Morphological analyses have revealed that the small GTPase Rho1p participates in cell-size control at both the G1/S and the G2/M boundaries. One group of rho1 mutants (rho1A) underwent premature entry into mitosis, leading to the birth of abnormally small cells. In another group of rho1 mutants (rho1B), the mother cells failed to reach an appropriate size before budding, and expression of the G1 cyclin Cln2p began at an earlier phase of the cell cycle. Analyses of mutants defective in Rho1p effector proteins indicate that Skn7p, Fks1p and Mpk1p are involved in cell-size control. Thus, Rho1p and its downstream regulatory pathways are involved in controlling cell size in S. cerevisiae.

Glycosylation of recombinant proforms of major house dust mite allergens Der p 1 and Der f 1 decelerates the speed of maturation.

BACKGROUND: The efficient manufacture of recombinant Der p 1 and Der f 1 has been an important bottleneck in the study of house dust mite allergies and the development of applications for allergen engineering. While Der f 1 has only one N-glycosylation motif in the mature sequence, Der p 1 has two motifs, one in the prosequence and the other in the mature sequence. To test whether inefficient maturation of a recombinant Pro-Der p 1 versus Pro-Der f 1 is due to N-glycosylation, the maturation speed of N-glycosylation motif mutants was compared. METHODS: Expression vectors for the mutants, in which the motif in the Der p 1 prodomain was disrupted or a motif was created within the Der f 1 prodomain, were constructed by site-directed mutagenesis of preproforms with or without the motif within the mature portion. Culture supernatants of yeast Pichia pastoris transfectant cells containing proforms were buffer exchanged by gel filtration and incubated for maturation. Samples from the reactions were collected every 20 min and subjected to electrophoresis. The maturation speed was compared based on the band densities of the pro- and mature forms. RESULTS: Disruption of the motif in the mature portion decreased the productivity and accelerated the maturation. Maturation was also accelerated by disruption of the other motif in the Der p 1 prodomain and slowed down by introduction of the motif into the Der f 1 prodomain. CONCLUSIONS: Maturation systems using Pro-Der p 1 without the prodomain glycosylation are useful for the efficient preparation of a recombinant mature allergen. In addition, these results demonstrated that the maturation of cysteine protease could be controlled through glycosylation of the prodomain.

Multiple-mutation at a potential ligand-binding region decreased allergenicity of a mite allergen Der f 2 without disrupting global structure.

We assessed the effect of multiple-mutations within one IgE-binding area on allergenicity of Der f 2. The triple-mutant of Der f 2, P34/95/99A, exhibited the most significant reduction of allergenicity and circular dichroism analysis showed that the global structure of Der f 2 was maintained in P34/95/99A. These results indicate that such a strategy is effective when designing allergen-vaccines, which achieve less allergenicity for a broad population of patients without disrupting the global structure. Structurally, Der f 2 is a member of the MD-2 related lipid-recognition proteins. The sites for the triple-mutation located on the characteristically charged entrance of a cavity and corresponded to the regions critical to ligand-binding in the Niemann-Pick type 2 disease protein and MD-2.