Mortalin controls centrosome duplication via modulating centrosomal localization of p53.

Abnormal amplification of centrosomes, commonly found in human cancer, is the major cause of mitotic defects and chromosome instability in cancer cells. Like DNA, centrosomes duplicate once in each cell cycle, hence the defect in the mechanism that ensures centrosome duplication to occur once and only once in each cell cycle results in abnormal amplification of centrosomes and mitotic defects. Centrosomes are non-membranous organelles, and undergo dynamic changes in its constituents during the centrosome duplication cycle. Through a comparative mass spectrometric analysis of unduplicated and duplicated centrosomes, we identified mortalin, a member of heat shock protein family, as a protein that associates preferentially with duplicated centrosomes. Further analysis revealed that mortalin localized to centrosomes in late G1 before centrosome duplication, remained at centrosomes during S and G2, and dissociated from centrosomes during mitosis. Overexpression of mortalin overrides the p53-dependent suppression of centrosome duplication, and mortalin-driven centrosome duplication requires physical interaction between mortalin and p53. Moreover, mortalin promotes dissociation of p53 from centrosomes through physical interaction. The p53 mutant that lacks the ability to bind to mortalin remains at centrosomes, and suppresses centrosome duplication in a transactivation function-independent manner. Thus, our present findings not only identify mortalin as an upstream molecule of p53 but also provide evidence for the involvement of centrosomally localized p53 in the regulation of centrosome duplication.

Essential role for extracellular Ca(2+) in JNK activation by mechanical stretch in bladder smooth muscle cells.

Mechanical stretch has been implicated in phenotypic changes as an adaptive response to stretch stress physically loaded in bladder smooth muscle cells (BSMCs). To investigate stretch-induced signaling, we examined the mitogen-activated protein kinase (MAPK) family using rat primary BSMCs. When BSMCs were subjected to sustained mechanical stretch using collagen-coated silicon membranes, activation of c-Jun NH(2)-terminal kinase (JNK) was most relevant among three subsets of MAPK family members: the activity was elevated from 5 min after stretch and peaked at 10 min with an 11-fold increase. Activation of p38 was weak compared with that of JNK, and ERK was not activated at all. JNK activation by mechanical stretch was totally dependent on extracellular Ca(2+) and inhibited by Gd(3+), a blocker of stretch-activated (SA) ion channels. Nifedipine and verapamil, inhibitors for voltage-dependent Ca(2+) channels, had no effect on this JNK activation. Moreover, none of the inhibitors pertussis toxin, genistein, wortmannin, or calphostin C affected stretch-induced JNK activation, indicating that G protein-coupled and tyrosine kinase receptors are unlikely to be involved in this JNK activation. On the other hand, W-7, a calmodulin inhibitor, and cyclosporin A, a calcineurin inhibitor, prevented JNK activation by stretch. These results suggest a novel pathway for stretch-induced activation of JNK in BSMCs: mechanical stretch evokes Ca(2+) influx via Gd(3+)-sensitive SA Ca(2+) channels, resulting in JNK activation under regulation in part by calmodulin and calcineurin.

Induction of transcription factor AP-2 by cytokines and prostaglandins in cultured mesangial cells.

BACKGROUND/AIMS: Activator protein-2 (AP-2) is an important transcription factor for activation of growth- and inflammatory-associated genes. To detect AP-2 in the mesangium, the expression level of AP-2 was examined in cultured mesangial cells in response to various cytokines and prostaglandins. The level was also observed in kidney tissue samples obtained from patients with proteinuria and from a rat nephrosis model. METHODS: AP-2 was immunohistochemically detected with a specific antibody. The expression level was analyzed by immunoblotting. Human tissue samples were obtained from patients with proteinuria. Kidney samples were also obtained from rats with puromycin aminonucleoside-induced nephrosis. RESULTS: Pro-inflammatory cytokines, such as IL-6, IL-1 and IL-2, but not TNF-alpha, induced AP-2 expression in a time- and dose-dependent manner in cultured mesangial cells. PGE(2) and PGI(2) also induced AP-2 expression, while PGF(2 alpha) failed to induce this protein. High expression levels of AP-2 were observed in different cell types including mesangial cells of kidney samples from patients with proteinuria. Similar results were obtained from the rat nephrosis model. CONCLUSION: These findings demonstrate that the primary cytokines induce AP-2 protein in mesangial cells. AP-2 may act as a transcription factor to produce additional cytokines and growth-associated gene products, suggesting an important role for AP-2 for the function of mesangial cells in glomerular disorders.

Involvement of ErbB-2 in rheumatoid synovial cell growth.

OBJECTIVE: The synovial tissue affected by rheumatoid arthritis (RA) is characterized by hyperproliferation of synovial cells. High amounts of epidermal growth factor (EGF) in the synovial fluid of RA patients contribute to the growth of rheumatoid synovial cells. To characterize the receptor for EGF in rheumatoid synovial cells, the expression and function of ErbB family members were examined. METHODS: Synovial tissues were obtained from surgical excisions. The expression of ErbB products was examined by immunohistochemistry and immunoblotting by using specific antibodies. Primary cultures were established from the surgical materials. Cell growth was measured using MTT. The levels and phosphorylation state of the ErbB-2 protein were analyzed by immunoprecipitation and immunoblotting. RESULTS: The expression of ErbB-2, but not other ErbB-related products, was detected in synovium with RA as compared with that with osteoarthritis (OA) and ligament injury. Growth of primary synovial cells with RA was inhibited by genistein, a tyrosine kinase inhibitor, and herceptin, a specific monoclonal antibody against ErbB-2. Herceptin showed a small effect on growth of primary synovial cells with OA. EGF stimulated the phosphorylation of ErbB-2 in primary synovial cells with RA. This EGF-stimulated phosphorylation was completely abrogated by genistein and herceptin. CONCLUSION: ErbB-2 is expressed in rheumatoid synovial cells and may function as the receptor for EGF. Our data suggest that mitotic signals from EGF family members are transduced by ErbB-2 in these cells. Inhibition of ErbB-2 may provide a new approach to the effective treatment for RA.

Wavelength-specific activation of MAP kinase family proteins by monochromatic UV irradiation.

The depletion of stratospheric ozone causes related increase in UV light below about 310 nm, which significantly affects biological and ecological systems. To understand the wavelength-specific effects of UV light, Molt4 cells (human T lymphoma cells) were irradiated with a series of monochromatic UV lights and the activities of three members of the mitogen-activated protein (MAP) kinase group were examined. Extracellular signal-regulated kinase was specifically activated within 1 min after UV irradiation in the range 320-360 nm. In contrast, P38 kinase was activated by 270-280 nm light with a peak at 1 min after irradiation. c-Jun N-terminal kinase activation was observed in a narrow range of UV light with a sharp peak at 280 nm occurring in 10 min. JNK translocated from the cytosol to the nucleus upon irradiation, while P38 remained in the cytosol even after UV irradiation. The activation of three MAP kinases was prevented by antioxidant reagents, suggesting that an oxidative signal initiates these responses. These results confirm that UV light affects various cellular functions through the activation of intracellular signaling systems including MAP kinase family proteins. However, the UV-induced activities of the separate MAP kinases show distinctly different dose, time and wavelength dependencies.

Morphological changes and detachment of adherent cells induced by p122, a GTPase-activating protein for Rho.

We recently cloned a novel signaling molecule, p122, that shows a GTPase-activating activity specific for Rho and the ability to enhance the phosphatidylinositol 4,5-bisphosphate-hydrolyzing activity of phospholipase C delta1 in vitro. Here we analyzed the in vivo function of p122. Microinjection of the GTPase-activating domain of p122 suppressed the formation of stress fibers and focal adhesions induced by lysophosphatidic acid, suggesting a GTPase-activating activity for Rho as in in vitro. Transfection of p122 also induced the disassembly of stress fibers and the morphological rounding of various adherent cells. Analyses using deletion and point mutants demonstrated that the GTPase-activating domain of p122 is responsible for the morphological changes and detachment and that arginine residues at positions 668 and 710 and a lysine residue at position 706 in the GTPase-activating domain are essential. Using Fluo-3-based Ca2+ microscopy, we found that p122 evoked a rapid elevation of intracellular Ca2+ levels, suggesting that p122 stimulates the phosphatidylinositol 4, 5-bisphosphate-hydrolyzing activity of phospholipase C delta1. These results demonstrate that p122 synergistically functions as a GTPase-activating protein specific for Rho and an activator of phospholipase C delta1 in vivo and induces morphological changes and detachment through cytoskeletal reorganization.

VEGF-dependent signaling in retinal microvascular endothelial cells.

We examined the effect of vascular endothelial growth factor (VEGF) on intracellular signal transduction pathways using isolated bovine microvascular endothelial cells (BREC). When cell growth was determined by [3H]thymidine incorporation, it was significantly stimulated by VEGF stimulation. In situ hybridization results also demonstrated that c-fos expression was enhanced by the stimulation. Although BREC expressed Flt-1 and Flk-1 as VEGF receptors at similar levels, VEGF stimulation preferentially enhanced the activity of Flt-1 tyrosine kinase. This stimulation initiated an increase in the level of GTP-form Ras and the activation of mitogen activated protein kinase (MAPK). On the other hand, BREC expressed the Janus kinase (Jak) family members Jak1, Jak2, and Tyk2, and the signal transducers and activators of transcription (Stat) family members Stat1, Stat3, and Stat6. These molecules were tyrosine phosphorylated under culture conditions used, and the phosphorylation of Tyk2 and Stat6 was specifically enhanced by VEGF stimulation. These results demonstrate that, in addition to Ras/MAPK pathways, the Flt-1/Tyk2/Stat6 pathway is important in VEGF signaling in BREC. These signal transduction systems may regulate the growth of retinal endothelial cells.

Role of the inositol phosphatase SHIP in B cell receptor-induced Ca2+ oscillatory response.

Src homology-2 domain-containing inositol polyphosphate 5'-phosphatase (SHIP) is a recently identified protein that has been implicated as an important signaling molecule. Although SHIP has been shown to participate in the FcgammaRIIB-mediated inhibitory signal, the functional role of SHIP in activation responses by immunoreceptor tyrosine-based activation motif-bearing receptors such as B cell receptor (BCR) remains unclear. Indeed, it has been proposed that SHIP serves as a linking molecule for the regulation of the extracellular signal-regulated kinase pathway in BCR signaling, because SHIP associates with Shc. We now report that SHIP-deficient DT40 B cells display enhanced Ca2+ mobilization in response to BCR ligation, whereas extracellular signal-regulated kinase activation is unaffected. This Ca2+ enhancement is due to a sustained intracellular Ca2+ increase or to long-lasting Ca2+ oscillations by loss of SHIP, as revealed by single-cell Ca2+ imaging analysis. These results demonstrate the importance of SHIP in B cell activation by the modulation of Ca2+ mobilization.

Unstable retrovirus mutants with acquired transforming activity: rapid changes in the number of repeats of a specific junD polynucleotide segment.

We have previously reported that the non-transforming jun D (wild type) gene can acquire transforming activity through spontaneous mutations when it is replicated through avian replication-competent retrovirus vectors in chicken embryo fibroblasts. In two of these spontaneous mutants, T1 and T2, which were isolated from proviral DNA in the same transformed cell clone, a specific 48 bp polynucleotide segment of the jun D coding sequence was tandemly repeated three and five times, respectively. We report here that the number of direct repeats in these mutants rapidly changes (mostly decreases) in the context of either RSV-based replication-competent or MLV-based replication-defective retroviruses, most likely during the process of reverse transcription, while these mutations are stable in the cellular chromosome. We also show that the growth conditions of the infected culture modulate the proportions of polymorphic proviral populations in the infected culture. We finally discuss the possible molecular mechanisms that generate genetic diversity in these amplification mutants.

Wavelength specific activation of PI 3-kinase by UVB irradiation.

We investigated the effect of UVB irradiation on phospholipid metabolism using normal human diploid skin fibroblasts. When cells were exposed to monochromatic UV light, the intracellular PIP3 level was increased within 1 min in a wavelength-specific manner. The increase was most marked in the UVB range and was inhibited by a PI 3-kinase inhibitor, tyrosine kinase inhibitor, or antioxidant reagents. Furthermore, the decrease of cell viability induced by UVB irradiation was significantly blocked by wortmannin. These results suggest that PI 3-kinase activation is involved in novel cellular responses specific for UVB that lead to cell death.

Phosphorylation and high level expression of Fra-2 in v-src transformed cells: a pathway of activation of endogenous AP-1.

Chicken embryo fibroblasts (CEF) transformed with v-src were previously reported to revert to normal phenotype after the introduction of dominant-negative mutants of Fos or Jun, indicating that endogenous AP-1 activity is essential for the cellular transformation. The major changes in the expression levels of fos and jun family genes induced by v-src were the elevation of fra-2 and c-jun transcripts. We show here that extensive phosphorylation of the AP-1 component Fra-2 is a major qualitative change in v-src transformed CEF and that several Ser and Thr residues in a C-terminal region of Fra-2 (amino acids 266-323) are phosphorylated specifically. The induced kinase activity was detected at the position of 42 kDa by in gel kinase assay using the Fra-2 C-terminal region as a substrate, and it was identified as chicken ERK2. JNK1 and JNK2, other members of the MAP kinase family, were not significantly activated in v-src transformed CEF and Fra-2 was not a good substrate for JNKs. fra-2 promoter analysis indicated that this promoter activity is elevated in v-src transformed CEF via two AP-1 binding sites and CRE-like sequence. We propose that phosphorylation of Fra-2 by ERK2 converts it from an inefficient transcriptional activator to an active one and further that fra-2 expression is autoregulated in response to the phosphorylation status of its gene product.

Purification and characterization of the phosphatidylinositol-3,4,5-trisphosphate phosphatase in bovine thymus.

Using phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] prepared from phosphatidylinositol 4,5-bisphosphate and inositolphospholipid 3-kinase, we identified in bovine thymus extracts the enzyme activity which catalyzed dephosphorylation of PtdIns(3,4,5)P3, to produce phosphatidylinositol biphosphate. Since bovine thymus exhibited the highest level of activity among tissues screened, we tried to purify this enzyme PtdINs(3,4,5)P3 phosphatase from bovine thymus. After sequential chromatographies using S-Sepharose, heparin-Sepharose, blue Sepharose, and Toyopearl HW55, the enzyme was purified 1875-fold with a yield of 10%. SDS/PAGE analysis revealed that a 120-kDA protein band copurified with the enzyme activity. The apparent molecular mass of the active protein was 120 kDa on size-exclusion chromatography, suggesting that the 120-kDa band on SDS/PAGE is the PtdIns(3,4,5)P3 phosphatase. Since PtdIns(3,4,5)P3 phosphatase seemed to be the only activity that metabolized PtdIns(3,4,5)P3, and the enzyme did not hydrolyze phosphatidylinositol 4,5-biphosphate, the enzyme may play a critical role in the inositolphospholipid 3-kinase signalling.

Wortmannin, a specific inhibitor of phosphatidylinositol-3 kinase, blocks osteoclastic bone resorption.

The biological role of phosphatidylinositol (PI)-3 kinase was examined in osteoclast-like multinucleated cells (OCLs) formed in co-cultures of mouse osteoblastic cells and bone marrow cells. The expression of PI-3 kinase in OCLs was confirmed by Western blot analysis. Wortmannin (WT), a specific inhibitor of PI-3 kinase, inhibited PI-3 kinase activity in OCLs both in vitro and in vivo. WT also inhibited pit-forming activity on dentine slices and disrupted a ringed structure of F-actin-containing dots (an actin ring) in OCLs in a dose-dependent manner. The inhibitory profiles of WT for pit and actin ring formation were similar to that for PI-3 kinase activity in OCLs. Electron microscopic analysis revealed that OCLs treated with WT did not form ruffled borders. Instead, numerous electron lucent vacuoles of differing sizes were found throughout the cytoplasm. These results suggest that PI-3 kinase is important in osteoclastic bone resorption.

Neurite outgrowth of PC12 cells is suppressed by wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase.

The effects of wortmannin (WT), an inhibitor of phosphatidylinositol (PI) 3-kinase, on differentiation of PC12 cells were analyzed. WT inhibited PI 3-kinase activity of PC12 cells at a concentration of 10(-7) M in vivo and in vitro. Transient inhibition of PI 3-kinase activity at the time of nerve growth factor stimulation had no effect on activation of the ras protein or neurite formation by the cells. However, continuous inhibition of PI 3-kinase blocked differentiation at the step just before neurite formation. When WT was applied to cells growing neurites, elongation of the neurites was stopped at that step. These results suggest that PI 3-kinase may be involved in neurite elongation.

SecY is an indispensable component of the protein secretory machinery of Escherichia coli.

Using a reconstitution system for protein translocation, the involvement of SecY in the translocation of secretory proteins across the cytoplasmic membrane of Escherichia coli was studied. Anti-SecY antibodies raised against the N- and C-terminal sequences prevented the functional reconstitution of the translocation system. Depletion of SecY from the solubilized membrane preparation was performed by treatment with anti-SecY IgG, followed by removal of IgG with protein A-agarose. The SecY-depleted preparation was inactive as to functional reconstitution. However, reconstitution with it was demonstrated in the presence of a protein fraction, which was released from the anti-SecY immunoprecipitate upon addition of the SecY fragment used to raise the antibody. Reconstitution with the SecY-depleted membrane fraction was also demonstrated in the presence of a purified SecY preparation. OmpT proteinase specifically cleaved SecY in the solubilized membrane preparation. The cleavage was accompanied by a decrease in the reconstituted activity. Based on these findings we conclude that SecY is an indispensable component of the secretory machinery.