CKAP2 phosphorylation by CDK1/cyclinB1 is crucial for maintaining centrosome integrity

Previously, we have reported that CKAP2 is involved in the maintenance of centrosome integrity, thus allowing for proper mitosis in primary hepatocytes. To understand this biological process, we identified the mitosis-specific phosphorylation sites in mouse CKAP2 and investigated CKAP’s possible role in cell cycle progression. Because we observed mouse CKAP2 depletion in amplified centrosomes and aberrant chromosomal segregation, which was rescued by ectopic expression of wild-type CKAP2, we focused on the centrosome duplication process among the various aspects of the cell cycle. Among the identified phosphorylation sites, T603 and possibly S608 were phosphorylated by CDK1–cyclin B1 during mitosis, and the ectopic expression of both T603A and S608A mutants was unable to restore the centrosomal abnormalities in CKAP2-depleted cells. These results indicated that the phosphorylation status of CKAP2 during mitosis is critical for controlling both centrosome biogenesis and bipolar spindle formation.

Clinical implications of proliferation activity in T1 or T2 male gastric cancer patients

Proliferation activity has already been established as a prognostic marker or as a marker for anticancer drug sensitivity. In gastric cancer, however, the prognostic significance of proliferation activity is still being debated. Several studies evaluating proliferation activity using Ki-67 have shown controversial results in terms of the relationship between proliferation activity and overall survival (OS) or drug sensitivity in gastric cancer patients. Because cytoskeleton-associated protein 2 (CKAP2) staining has recently been introduced as a marker of proliferation activity, we analyzed 437 gastric cancer tissues through CKAP2 immunohistochemistry, and we evaluated the chromatin CKAP2-positive cell count (CPCC) for proliferation activity. Although the CPCC did not show any significant correlation with OS in the male, female or total number of cases, it did show a significant correlation in the T1 or T2 male patient subgroup, according to log-rank tests (P=0.001) and univariate analysis (P=0.045). Additionally, multivariate analysis with the Cox proportional hazard regression model showed a significant correlation between the CPCC and OS (P=0.039) for the co-variables of age, gender, T stage, N stage, histology, tumor location, tumor size and adjuvant chemotherapy. In male gastric cancer cell lines, faster-growing cancer cells showed higher sensitivity to cisplatin than slow-growing cells. Thus our study indicates that CPCC-measured proliferation activity demonstrates a significantly worse prognosis in T1 or T2 male gastric cancer patients. The CPCC will help to more precisely classify gastric cancer patients and to select excellent candidates for adjuvant chemotherapy, which in turn will facilitate further clinical chemotherapeutic trials.

Characterization of mitosis-specific phosphorylation of tumor-associated microtubule-associated protein

Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton associated protein 2 (CKAP2), has been recently shown to be involved in the assembly and maintenance of mitotic spindle and also plays an essential role in maintaining the fidelity of chromosome segregation during mitosis. We have previously reported that TMAP is phosphorylated at multiple residues specifically during mitosis, and characterized the mechanism and functional importance of phosphorylation at one of the mitosis-specific phosphorylation residues (i.e., Thr-622). However, the phosphorylation events at the remaining mitotic phosphorylation sites of TMAP have not been fully characterized in detail. Here, we report on generation and characterization of phosphorylated Thr-578- and phosphorylated Thr-596-specific antibodies. Using the antibodies, we show that phosphorylation of TMAP at Thr-578 and Thr-596 indeed occurs specifically during mitosis. Immunofluorescent staining using the antibodies shows that these residues become phosphorylated starting at prophase and then become rapidly dephosphorylated soon after initiation of anaphase. Subtle differences in the kinetics of phosphorylation between Thr-578 and Thr-596 imply that they may be under different mechanisms of phosphorylation during mitosis. Unlike the phosphorylation-deficient mutant form for Thr-622, the mutant in which both Thr-578 and Thr-596 had been mutated to alanines did not induce significant delay in progression of mitosis. These results show that the majority of mitosis-specific phosphorylation of TMAP is limited to pre-anaphase stages and suggest that the multiple phosphorylation may not act in concert but serve diverse functions.

Protective Effects of Gabapentin on Allodynia and alpha2delta1-Subunit of Voltage-dependent Calcium Channel in Spinal Nerve-Ligated Rats

This study was designed to determine whether early gabapentin treatment has a protective analgesic effect on neuropathic pain and compared its effect to the late treatment in a rat neuropathic model, and as the potential mechanism of protective action, the alpha2delta1-subunit of the voltage-dependent calcium channel (alpha2delta1-subunit) was evaluated in both sides of the L5 dorsal root ganglia (DRG). Neuropathic pain was induced in male Sprague-Dawley rats by a surgical ligation of left L5 nerve. For the early treatment group, rats were injected with gabapentin (100 mg/kg) intraperitoneally 15 min prior to surgery and then every 24 hr during postoperative day (POD) 1-4. For the late treatment group, the same dose of gabapentin was injected every 24 hr during POD 8-12. For the control group, L5 nerve was ligated but no gabapentin was administered. In the early treatment group, the development of allodynia was delayed up to POD 10, whereas allodynia was developed on POD 2 in the control and the late treatment group (p<0.05). The alpha2delta1-subunit was up-regulated in all groups, however, there was no difference in the level of the alpha2delta1-subunit among the three groups. These results suggest that early treatment with gabapentin offers some protection against neuropathic pain but it is unlikely that this action is mediated through modulation of the alpha2delta1-subunit in DRG.

Transient phosphorylation of tumor associated microtubule associated protein (TMAP)/cytoskeleton associated protein 2 (CKAP2) at Thr-596 during early phases of mitosis

Tumor associated microtubule associated protein (TMAP), also known as cytoskeleton associated protein 2 (CKAP2) is a mitotic spindle-associated protein whose expression is cell cycle-regulated and also frequently deregulated in cancer cells. Two monoclonal antibodies (mAbs) against TMAP/CKAP2 were produced: B-1-13 and D-12-3. Interestingly, the reactivity of mAb D-12-3 to TMAP/CKAP2 was markedly decreased specifically in mitotic cell lysate. The epitope mapping study showed that mAb D-12-3 recognizes the amino acid sequence between 569 and 625 and that phosphorylation at T596 completely abolishes the reactivity of the antibody, suggesting that the differential reactivity originates from the phosphorylation status at T596. Immunofluorescence staining showed that mAb D-12-3 fails to detect TMAP/CKAP2 in mitotic cells between prophase and metaphase, but the staining becomes evident again in anaphase, suggesting that phosphorylation at T596 occurs transiently during early phases of mitosis. These results suggest that the cellular functions of TMAP/CKAP2 might be regulated by timely phosphorylation and dephosphorylation during the course of mitosis.

PIAS1 interacts with the KRAB zinc finger protein, ZNF133, via zinc finger motifs and regulates its transcriptional activity

Zinc finger protein 133 (ZNF133) is composed of a Kruppel-associated box (KRAB) domain and 14 contiguous zinc finger motifs. ZNF133 is regarded as a transcriptional repressor because the KRAB domain has potent repressor activity and the zinc finger motifs usually act in binding to DNA. However, we found that the zinc finger motifs of ZNF133 also possessed transcriptional repressor activity. By two-hybrid screening assay, we found that the zinc finger motifs of ZNF133 interacted with protein inhibitor of activated STAT1 (PIAS1). PIAS1 enhanced the transcriptional repression activity of ZNF133 through the zinc finger motifs. This effect of PIAS1 was relieved by an inhibitor of the histone deacetylases (HDACs). These results demonstrate that the transcriptional repressor activity of ZNF133 is regulated by both the KRAB domain and the zinc finger motifs, and that the repressive effect by zinc finger motifs is mediated by PIAS1.

Downregulation of regenerating islet-derived 3 alpha (REG3A) in primary human gastric adenocarcinomas

Gastric carcinoma is considered to be one of the most prevalent cancers worldwide. We have performed differential-display polymerase chain reaction (DD-PCR) in order to compare the gene expression profile of gastric carcinoma and that of a normal stomach, in an attempt to identifiy differentially expressed genes associated with primary human gastric cancers. One of the down-regulated genes in gastric cancers was identified as regenerating islet-derived 3 alpha (REG3A), also known as hepatocarcinoma-intestine-pancreas/ pancreatitis-associated protein (HIP/PAP). REG3A exhibited relatively high expression levels in normal gastric mucosa. However, REG3A was found to be down-regulated in 67% (20 out of 30 samples) of primary human gastric cancers, as determined by RT-PCR. In addition, REG3A mRNA expression was not detected in stomach cancer cell lines, SNU cells. Immunohistochemical analysis further confirmed the down-regulation of REG3A expression in primary human gastric cancers. Treatment with the demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-dC) resulted in the restoration of REG3A mRNA expression in the gastric cancer cell line, indicating that the transcriptional silencing of REG3A in SNU cell lines was caused by DNA methylation. Taken together, these data indicate that REG3A is down-regulated in most primary human gastric cancer cells, and might be useful in the diagnosis of gastric cancer. Further characterization of the differentially expressed gene, REG3A, should lead to a better understanding of the changes occurring at the molecular level during the development and progression of primary human gastric cancer.

AMPA, not NMDA, activates RhoA GTPases and subsequetly phosphorylates moesin

Glutamate induced rapid phosphorylation of moesin, one of ERM family proteins involved in the ligation of membrane to actin cytoskeleton, in rat hippocampal cells (JBC, 277:16576-16584, 2002). However, the identity of glutamate receptor has not been explored. Here we show that a-amino- 3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor is responsible for glutamate-induced RhoA activation and phosphorylation of moesin. Glutamate induced phosphorylation at Thr-558 of moesin was still detectible upon chelation of Ca(2+), suggesting involvement of AMPA receptor instead of N-methyl D-Aspartate (NMDA) receptor in this phosphorylation of moesin. AMPA but not NMDA- induced moesin phosphorylation was independent of Ca(2+). Both AMPA and NMDA but not Kainate induced moesin phosphorylation at similar levels. However, the kinetics of phosphorylation varied greatly between AMPA and NMDA where AMPA treatment rapidly increased phosphomoesin, which reached a maximum at 10 min after treatment and returned to a basal level at 30 min. In contrast, NMDA-induced phosphorylation of moesin reached a maximum at 30 min after treatment and was remained at higher levels at 60 min. A possible involvement of RhoA and its downstream effector, Rho kinase in the AMPA receptor-triggered phosphorylation of moesin was also explored. The kinetics for the glutamate- induced membrane translocation of RhoA was similar to that of moesin phosphorylation induced by AMPA. Moreover, Y-27632, a specific Rho kinase inhibitor, completely blocked AMPA-induced moesin phosphorylation but had no effect on NMDA-induced moesin phosphorylation. These results suggest that glutamate-induced phosphorylation of moesin may be mediated through the AMPA receptor/RhoA/Rho kinase pathway.

Post-transcriptional control of c-erb B-2 overexpression in stomach cancer cells

The growth factor receptor oncogene, c-erb B-2, is frequently overexpressed in the adenocarcinomas of breast, ovary, lung and stomach. Although the mechanism of erb B-2 overexpression is thought as the result of transcriptional upregulation in many primary human carcinomas, expression rate of c-erb B-2 at mRNA level is usually lower than the level of translated protein. We also found that the expression of erb B-2 in SNU-1 stomach cancer cells was greater at post-transcription level (Bae et al., 1993). To explore the underlying mechanism of erb B-2 protein overexpression, we have chosen two cells lines, SNU-1 and SNU-16 where transcription rate of erb B-2 was closely resemble to each other while expressed protein levels were quite different. The synthesis rate of erb B-2 protein in SNU-1 cells was faster than SNU-16 cells while levels of erb B-2 mRNA were found to be similar in both cell lines. The half-life of the expressed erb B-2 protein was not significantly different in both cell lines. Analysis of the 5' untranslated region (UTR) of erb B-2 mRNA (-1approximately-323) showed no sequence abnormality in both cell lines. However, ribonuclease protection assay using cloned 5 UTR sequence revealed that the size of 5' UTR of erb B-2 mRNA which associate with transcription initiation site(s) in SNU-1 cells was longer than that in SNU-16. These results suggest that the increased erb B-2 protein synthesis rate possibly due to the redundant selection of transcription initiation might be a mechanism of erb B-2 overexpression in SNU-1 cells.

Activation of JNK and p38 in rat hippocampus after kainic acid induced seizure

Kainic acid, an analogue of glutamate, causes limbic seizures and induces cell death in the rat brain. We examined the activation of MAPK family kinases; ERKs, JNKs and p38 kinase in rat hippocampus after KA treatment. Activation of all three kinases were observed at 30 min after the treatment, but, in contrary to ERK phosphorylation, which lasted up to 3 h, the phosphorylation of JNK and p38 returned to the basal level by 2 h. The phosphorylation of' upstream kinases for the MAPK family was distinct. The phosphorylation of MEK1 clearly increased at 30 min but diminished rapidly thereafter. The phosphorylation of MKK6 was also increased but reached peak at 2 h after KA treatment. However, the phosphorylation of other upstream kinases, SEK1 and MKK3, gradually decreased to 3 h after KA treatment. These results indicate that the KA activates all of the three MAPK family kinases with different time patterns and suggest the possibility that MKK3 and MKK6, and SEK1 may not be the upstream kinases for p38 and JNK in rat hippocampus.

Multi-facet expressions of adenylate cyclase isoforms in B16-F10 melanoma cells differentiated by forskolin treatment

The terminal differentiation of malignant melanoma cells is known to be induced by activating cAMP signaling pathway with alpha-MSH or cAMP analogues. However, sustained activation of cAMP signaling system that induces the differentiation of melanoma cells, also induces the desensitization of the pathway at the receptor level. Nevertheless, the adaptation of adenylate cyclase (AC) expression by sustained activation of cAMP signaling system has not been clearly understood. This study was performed to examine whether the sustained activation of cAMP system induce changes in the expression AC isoforms as an adaptation mechanism. Treatment of B16/F10 murine melanoma cells with 100 mM forskolin for 6 days resulted in differentiation, melanin accumulation and increased expression of tyrosine hydroxylase mRNA. In the forskolin-treated melanoma cells, change in expression of various AC isoform at the transcription level was detected by reverse-transcription polymerase chain reaction (RT-PCR). Expression of AC isoform mRNA: ACI, III, VI, VII, and IX increased to the level of 196-392% of the control whereas the level of ACII was decreased by 30%. The cAMP concentration was increased both in basal and alpha-MSH stimulated cells, but the AC activity was decreased in the forskolin treated cells. Thus, these results suggest that sustained activation of cAMP system induces differential expression of AC isoforms, which results in increase of cAMP accumulation.

Purification and cloning of glyoxalase II from rat liver

Glyoxalase (GLO) II, which is a component of GLO system and catalyze the conversion of S-lactoyl-glutathione to D-lactate, was purified 1488 fold from rat liver by two steps of Affigel blue and carbobenzoxyglutathione-Sepharose 4B affinity chromatography. The molecular weight of the enzyme was estimated to be 29 kDa which is similar to those from other species. The sequence of N-terminal 9 amino acid residues was determined to be MGIRLLPAT. This was then used to synthesize degenerative primers. cDNA clone was isolated by first synthesizing cDNA from RNA and then PCR amplification. The sequence of cDNA clone was determined by serial sequencing analysis.

Calpain inhibitors reduce the cornified cell envelope formation by inhibiting proteolytic processing of transglutaminase 1

Calpain I (mu-calpain) and II (m-calpain) are well known calcium-activated neutral cysteine proteases. Many reports have shown that activation of calpain is related to cataract formation, neuronal degeneration, blood clotting, ischemic injuries, muscular dystrophy and cornified cell envelope (CE) formation. Here, we report that insoluble CE formation was reduced after treatment with calpain I inhibitor (N-acetyl-leucyl-leucyl-norleucinal) on normal human epidermal keratinocytes (NHEK), whereas serine and thiol protease inhibitors had no effect on the reduction of CE. When NHEK cells were confluent, keratinocytes were treated with various concentrations (0.5 microM-0.5 mM) of calpain I inhibitor or serine and thiol protease inhibitors under calcium induced differentiation. Insoluble CE formation was reduced about 90% in the 50 microM calpain inhibitor I treated group by day 9 of culture, whereas insoluble CE was reduced only 10% in the same condition. Interestingly TGase activity was blocked by 90% in the 0.5 mM calpain inhibitor treated group within 72 h, whereas TGase activity was retained by 80% in the 0.5 mM serine protease inhibitor treated group at 7 day treatment. Therefore it can be suggested that cysteine protease calpains might be responsible for the activation of the TGase 1 enzyme to complete insoluble CE formation during epidermal differentiation.

Expression of the EGF Receptor Gene Family in Gastric Cancer

Growth factor receptors play critical roles in the regulation of normal growth and developement. Some of these molecules have been implicated in the neoplastic process as well. In this study, We examined the expression of the EGF receptor gene family in gastric carcinoma by Ribonuclease protection assay and investigated the relationship between the expression of using a mRNA and clinicopathologic parameters. Expression of EGFR mRNA was found in 34 of 59 cases (57.6%), expression of c-erbB-2 mRNA was found in 27 of 57 cases (64.9%), expression of c-erbB-3 mRNA was found in 47 of 60 cases (78.3%), and expression of c-erbB-4 mRNA was found in 39 of 59 cases (66.1%). The expression of EGFR was correlated with the size of the tumor. The expression of c-erbB-2 was correlated with the presence of endolymphatic tumor emboli, Ming's classification, and lymph node matastasis. The expression of c-erbB-3 was correlated with the macroscopic type. Coexpression of c-erbB-2 and c-erbB-3 was correlated with age. These results suggest that the production of the EGF receptor gene family by various tumor cells it may play an important role in the cellular function. Therefore, further studies are currently being carried out to clarify the role of these oncogenes in tumor behavior and gastric carcinogenesis.

Expression of EGF Receptor Family Genes and Proteins in Gastric Cacner ( EGFR , c-erbB-2 , c-erbB-3 , c-erbB-4 ) / 대한암학회지

PURPOSE: to evaluate the relationship between the expression of EGF receptor gene family and clinico-pathologic parameters. MATERIALS AND METHODS: We compared adenocarcinoma tissue with normal mucosa obtained from same patients in 60 cases. The amplifications of DNA was examined by slot blot, while the expression of mRNA by ribonuclease protection assay, and that of protein by immunohistochemistry. RESULTS: Expression of EGFR mRNA was observed in 9 of 59, that of c-erbB-2 mRNA in 8 of 57, that of c-erbB-3 mRNA in 4 of 60, and that of c-erbB-4 mRNA in 13 of 59. The expression of EGFR in expanding type showed a higher tendency than that in infiltrative type. The expression of c-erbB-2 in poorly differentiated adenocarcinoma showed a higher tendency than that in well differentiated adenocarcinoma. And expression of c-erbB-2 was correlated with presence of endolymphatic tumor emboli. No significant correlation was observed between expression of EGFR mRNA and that of its protein or amplification of its DNA. Similarly, No clear relationship between c-erbB-2 gene amplification and expression of mRNA or proteins was detected. CONCLUSION: EGFR, c-erbB-2, c-erbB-3, and c-erbB-4 were expressed in gastric adenocarcinoma in Korea. The presence of EGF receptor gene family by various tumor cells suggested that it may play an important role in adenocarcinoma. Therefore further studies are currently being carried out to clarify the role of these oncogenes in tumor behavior and gastric carcinogenesis.

Retainment of membrane binding capacity of non-palmitoylated Gs alpha mutants expressed in COS-1 cells

Heterotrimeric guanine nucleotide binding regulatory proteins (G proteins) transduce extracellular signals into intracellular signals by coupling receptors and effectors. Because most of the G protein-coupled receptors are integral proteins, the G proteins need to have a membrane binding capacity to receive signals from the receptors. The alpha subunit of G protein binds tightly to the cytoplasmic face of the plasma membrane without any membrane spanning domain. Fatty acylation of G alpha with myristic acid or palmitic acid, in addition to the beta gamma subunits, plays an important role in anchoring the G alpha subunit. The reversible and dynamic palmitoylation of the alpha subunit of stimulatory G protein (Gs alpha) has been suggested as essential for its membrane attachment. However, in our previous experiments, Gs alpha deleted in the amino terminus containing palmitoylation site, retained its binding capacity when expressed in COS cells. Thus, to evaluate the role of palmitoylation in Gs alpha membrane binding, we constructed and expressed non-palmitoylated mutants of Gs alpha and analyzed their subcellular distributions in COS-1 cells. We found that non-palmitoylated mutants of Gs alpha, C3S- and G2A/C3S Gs alpha, retained their membrane binding capacities in COS-1 cells, demonstrating that palmitoylation is not essential for membrane binding of Gs alpha in COS-1 cells. We also found that the palmitoylation did not change significantly the distribution of Gs alpha in Triton X-114 partition. These results suggest that the palmitoylation of Gs alpha may produce different effects on membrane binding depending on cell types.

The mechanism of c-erbB-2 gene product increase in stomach cancer cell lines

c-erbB-2 oncogene encodes a growth factor receptor whose amino acid sequence has extensive homology with human epidermal growth factor receptor. It is frequently overexpressed in human breast, ovary, lung, and stomach cancers, where its overexpression is related significantly to the prognosis. Tl investigate the possible role of c-erbB-2 oncogene in the oncogenesis of stomach cancer, we examined the genetic alterations of c-erbB-2 oncogene in 4 stomach cancer cell lines, SNU-1, SNU-5, SNU-16 and KATO III. There were no differences in c-erbB-2 mRNA level as well as c-erbB-2 gene copy number among them. But gp185-erbB-2, c-erbB-2 gene product, was increased from 2- to 4-fold in SNU-1 and SNU-5 cells, compared with that in SNU-16 or KATO III cells. Our results suggest that post-transcriptional regulation of gp185erbB-2 expression may underlie gp185erbB-2 overexpression in cancer cells.