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Intracellular pH (pHi) regulation fundamentally participates in maintaining HCO3- release from HCO3--secreting epithelia. We used parotid intralobular ducts loaded with BCECF to investigate the contributions of a carbonic anhydrase (CA), anion channels and a Na+-H+ exchanger (NHE) to pHi regulation for HCO3- secretion by cAMP and Ca2+ signals. Resting pHi was dispersed between 7.4 and 7.9. Forskolin consistently decreased pHi showing the dominance of pHi-lowering activities, but carbachol gathered pHi around 7.6. CA inhibition suppressed the forskolin-induced decrease in pHi, while it allowed carbachol to consistently increase pHi by revealing that carbachol prominently activated NHE via Ca2+-calmodulin. Under NHE inhibition, forskolin and carbachol induced the remarkable decreases in pHi, which were slowed predominantly by CA inhibition and by CA or anion channel inhibition, respectively. Our results suggest that forskolin and carbachol primarily activate the pHi-lowering CA and pHi-raising NHE, respectively, to regulate pHi for HCO3- secretion.

Cholinergic agonists evoke elevations of the cytoplasmic free-calcium concentration ([Ca2+ ]i ) to stimulate fluid secretion in salivary glands. Salivary flow rates are significantly reduced in diabetic patients. However, it remains elusive how salivary secretion is impaired in diabetes. Here, we used an ex vivo submandibular gland perfusion technique to characterize the dependency of salivary flow rates on extracellular glucose concentration and activities of glucose transporters expressed in the glands. The cholinergic agonist carbachol (CCh) induced sustained fluid secretion, the rates of which were modulated by the extracellular glucose concentration in a biphasic manner. Both lowering the extracellular glucose concentration to less than 2.5 mM and elevating it to higher than 5 mM resulted in decreased CCh-induced fluid secretion. The CCh-induced salivary flow was suppressed by phlorizin, an inhibitor of the sodium-glucose cotransporter 1 (SGLT1) located basolaterally in submandibular acinar cells, which is altered at the protein expression level in diabetic animal models. Our data suggest that SGLT1-mediated glucose uptake in acinar cells is required to maintain the fluid secretion by sustaining Cl- secretion in real-time. High extracellular glucose levels may suppress the CCh-induced secretion of salivary fluid by altering the activities of ion channels and transporters downstream of [Ca2+ ]i signals.

The pluripotency of embryonic stem cells has been well demonstrated by a vast variety of studies showing the induction of differentiation into desired cell types that have the potential to be used not only in basic studies but also in medical applications. The induction of mesodermal cells, especially blood cells, from embryonic stem cells is notable from the point of view of transplantation, and the methods for this induction have improved over the last few years, with more defined culture conditions in place. Concurrently, the generation of induced pluripotent stem cells from somatic cells opens the possibility of autologous transplantation. In fact, there are a growing number of reports demonstrating that several mesodermal cells can be differentiated from induced pluripotent stem cells using the same methods used for embryonic stem cells. This review summarizes recent advances in the differentiation of mesodermal cells from embryonic stem cells and induced pluripotent stem cells.

Intracellular pH (pH

NeuroD-related factor (NDRF)/NeuroD2 is a basic helix-loop-helix (bHLH) protein that plays important roles in neuronal development. To elucidate the NDRF transcription network, we used mouse cDNA microarray analysis combined with a tetracycline-regulatable expression system in P19 embryonal carcinoma cells. Five genes were identified to be up-regulated in the presence of NDRF protein. RNA hybridization analysis confirmed that brain-lipid-binding protein (BLBP) and inhibitor of differentiation 1 (Id1) genes were among the five genes that were rapidly and significantly up-regulated after induction of NDRF. When a dominant negative form of NDRF protein was expressed during retinoic acid-induced neuronal differentiation of P19 cells, the BLBP gene, but not the Id1 gene, was potently repressed. Immunohistochemical analysis revealed that both NDRF and Id1 immunoreactivities were observed in some granule cells of the cerebellum in the postnatal period. These results suggest that NDRF or its related bHLH proteins may act upstream of these genes in a subset of developing neurons.

MCF-2 (DBL) proto-oncogene is a prototype guanine nucleotide exchange factor (GEF) that modulates Rho GTPases such as Rho, Rac, and Cdc42. Although the partial sequence of mouse MCF-2 has been determined, its full-length cDNA and biochemical functions had not been elucidated. We isolated the complete mouse MCF-2 cDNA and obtained recombinant functional protein. Homology between the mouse and human MCF-2 (DBL) cDNAs is 75.08% identity and between the mouse and human amino acid sequences 74.52% identity. Analysis of tissue distribution showed that mouse MCF-2 mRNA is expressed in brain, kidney, intestine, and testis. The brain-specific transcript is an alternatively spliced derivative that omits the 48 bp exon 11. A similar alternatively spliced mRNA product is also found in humans (DBL). Guanine nucleotide exchange activities of the testis-expressed mouse Mcf-2 and human Dbl were analyzed using RhoA, Rac1, and Cdc42 as substrates. RhoA and Cdc42 were activated similarly by both gene products, but Rac1 was activated only by the mouse product. The brain-specific Mcf-2 gene product, and its human counterpart, was less active than the respective testis-specific products. This indicates that the element encoded by the 48 bp exon missing in the brain transcripts is necessary for full GEF activity. This report provides fundamental data on the structure of Mcf-2, which regulates a variety of cellular signaling pathways.

The DBL (MCF-2) proto-oncogene is a prototype guanine nucleotide exchange factor (GEF) that modulates the Rho family of GTPases. In this communication we describe the isolation of three novel splicing variants of Dbl. The prototype Dbl gene (designated var.1 here) contains 25 exons, while splicing variant 2 (var.2) lacks exons 23 and 24. Var.3 contains additional 3 exons from 5(')-UTR in place of exon 1, while var.4, var.2, and var.3 contain a 48bp insertion between exons 10 and 11, resulting in the insertion of 16 amino acids. We found that var.1 was expressed only in brain, whereas var.3 was expressed in heart, kidney, spleen, liver, and testis, and var.4 in brain, heart, kidney, testis, placenta, stomach, and peripheral blood. The Dbl protein was detectable in brain, heart, kidney, intestine, muscle, lung, and testis. An assay for GEF activity revealed that the var.2 exhibits decreased GEF activity towards Cdc42, var.3 exhibits a weak but significant activity toward Rac1 and Cdc42, var.4 exhibits significant activity toward RhoA and Cdc42, while var.1 exhibits no activity toward RhoA, Rac1, or Cdc42. In summary, we describe 4 splicing variants of the human DBL proto-oncogene that show different tissue distributions and GEF specificities.

A possible role for a protein kinase, PKN, a fatty acid-activated serine/threonine kinase with a catalytic domain homologous to the protein kinase C family and a direct target for Rho, was investigated in the pathology of Alzheimer’s disease (AD) using a sensitive immunocytochemistry on postmortem human brain tissues and a kinase assay for human tau protein. The present study provides evidences by light, electron, and confocal laser microscopy that in control human brains, PKN is enriched in neurons, where the kinase is concentrated in a subset of endoplasmic reticulum (ER) and ER-derived vesicles localized to the apical compartment of juxtanuclear cytoplasm, as well as late endosomes, multivesicular bodies, Golgi bodies, secretary vesicles, and nuclei. In AD-affected neurons, PKN was redistributed to the cortical cytoplasm and neurites and was closely associated with neurofibrillary tangles (NFTs) and their major constituent, abnormally modified tau. PKN was also found in degenerative neurites within senile plaques. In addition, we report that human tau protein is directly phosphorylated by PKN bothin vitroandin vivo. Thus, our results suggest a specific role for PKN in NFT formation and neurodegeneration in AD damaged neurons.

PKN is a fatty acid- and Rho-activated serine/threonine protein kinase, having a catalytic domain homologous to protein kinase C family. To identify components of the PKN-signaling pathway such as substrates and regulatory proteins of PKN, the yeast two-hybrid strategy was employed. Using the N-terminal region of PKN as a bait, cDNAs encoding actin cross-linking protein α-actinin, which lacked the N-terminal actin-binding domain, were isolated from human brain cDNA library. The responsible region for interaction between PKN and α-actinin was determined by in vitro binding analysis using the various truncated mutants of these proteins. The N-terminal region of PKN outside the RhoA-binding domain was sufficiently shown to associate with α-actinin. PKN bound to the third spectrin-like repeats of both skeletal and non-skeletal muscle type α-actinin. PKN also bound to the region containing EF-hand-like motifs of non-skeletal muscle type α-actinin in a Ca2+-sensitive manner and bound to that of skeletal muscle type α-actinin in a Ca2+-insensitive manner. α-Actinin was co-immunoprecipitated with PKN from the lysate of COS7 cells transfected with both expression constructs for PKN and α-actinin lacking the actin-binding domain. In vitro translated full-length α-actinin containing the actin-binding site hardly bound to PKN, but the addition of phosphatidylinositol 4,5-bisphosphate, which is implicated in actin reorganization, stimulated the binding activity of the full-length α-actinin with PKN. We therefore propose that PKN is linked to the cytoskeletal network via a direct association between PKN and α-actinin.

Effects of environmental stresses on the subcellular localization of PKN were investigated in NIH 3T3, BALB/c 3T3, and Rat-1 cells. The immunofluorescence of PKN resided prominently in the cytoplasmic region in nonstressed cells. When these cells were treated at 42 degrees C, there was a time-dependent decrease of the immunofluorescence of PKN in the cytoplasmic region that correlated with an increase within the nucleus as observed by confocal microscope. After incubation at 37 degrees C following beat shock, the immunofluorescence of PKN returned to the perinuclear and cytoplasmic regions from the nucleus. The nuclear translocation of PKN by heat shock was supported by the biochemical subcellular fractionation and immunoblotting. The nuclear localization of PKN was also observed when the cells were exposed to other stresses such as sodium arsenite and serum starvation. These results raise the possibility that there is a pathway mediating stress signals from the cytosol to the nucleus through PKN.

PKN is a fatty acid-activated serine/threonine kinase that has a catalytic domain highly homologous to that of protein kinase C in the carboxyl terminus and a unique regulatory region in the amino terminus. Recently, we reported that the small GTP-binding protein Rho binds to the amino-terminal region of PKN and activates PKN in a GTP-dependent manner, and we suggested that PKN is located on the downstream of Rho in the signal transduction pathway (Amano, M., Mukai, H., Ono, Y., Chihara, K., Matsui, T., Hamajima, Y., Okawa, K., Iwamatsu, A., and Kaibuchi, K. (1996) Science 271, 648-650; Watanabe, G., Saito, Y., Madaule, P., Ishizaki, T., Fujisawa, K., Morii, N., Mukai, H., Ono, Y. Kakizuka, A., and Narumiya, S. (1996) Science 271, 645-648). To identify other components of the PKN pathway such as substrates and regulatory proteins of PKN, the yeast two-hybrid strategy was employed. By this screening, a clone encoding the neurofilament L protein, a subunit of neuron-specific intermediate filament, was isolated. The amino-terminal regulatory region of PKN was shown to associate with the head-rod domains of other subunits of neurofilament (neurofilament proteins M and H) as well as neurofilament L protein in yeast cells. The direct binding between PKN and each subunit of neurofilament was confirmed by using the in vitro translated amino-terminal region of PKN and glutathione S-transferase fusion protein containing the head-rod domain of each subunit of neurofilament. PKN purified from rat testis phosphorylated each subunit of the native neurofilament purified from bovine spinal cord and the bacterially synthesized head-rod domain of each subunit of neurofilament. Polymerization of neurofilament L protein in vitro was inhibited by phosphorylation of neurofilament L protein by PKN. The identification and characterization of the novel interaction with PKN may contribute toward the elucidation of mechanisms regulating the function of neurofilament.

We have identified a novel putative protein kinase-encoding gene from Schizosaccharomyces pombe (Sp), designated pskl + , by using a highly conserved amino acid (aa) sequence motif to design amplification of DNA fragments using PCR. The putative translation product of pskl + contains 436 aa, with a molecular mass of 49 317 Da. A single pskl + was identified by genomic Southern blot analysis, and the genomic mapping indicated that psk1 + was localized in Sp chromosome III. Growth of wild-type Sp cells was inhibited by 0.5 μM phenylarsine oxide, a protein tyrosine phosphatase inhibitor, but pskl − cells were relatively resistant to this drug.

The regulation of mesenchymal cell growth by signaling molecules plays an important role in maintaining tissue functions. Aberrant mesenchymal cell proliferation caused by disruption of this regulatory process leads to pathogenetic events such as fibrosis. In the current study we have identified a novel nuclear factor, Phf14, which controls the proliferation of mesenchymal cells by regulating PDGFRα expression. Phf14-null mice died just after birth due to respiratory failure. Histological analyses of the lungs of these mice showed interstitial hyperplasia with an increased number of PDGFRα(+) mesenchymal cells. PDGFRα expression was elevated in Phf14-null mesenchymal fibroblasts, resulting in increased proliferation. We demonstrated that Phf14 acts as a transcription factor that directly represses PDGFRα expression. Based on these results, we used an antibody against PDGFRα to successfully treat mouse lung fibrosis. This study shows that Phf14 acts as a negative regulator of PDGFRα expression in mesenchymal cells undergoing normal and abnormal proliferation, and is a potential target for new treatments of lung fibrosis.

PKN, a novel protein kinase with catalytic domain homologous to PKC family and unique amino terminal leucine zipper-like sequences, was purified partially from COS7 cells transfected with the cDNA construct encoding human PKN for enzymatic characterization of the enzyme. Using serine containing synthetic peptides based on PKC pseudosubstrate sites as the phosphate acceptors, kinase activities estimated from partially purified PKN were not stimulated by Ca2+/phosphatidylserine/diolein but were activated several-fold to several tens-fold by 40 μM unsaturated fatty acids, such as arachidonic acid, linoleic acid, and oleic acid. Autophosphorylation of the immunoprecipitates using anti-PKN antiserum was also stimulated by various unsaturated fatty acids. Limited proteolysis of PKN with trypsin induced an enhancement of the peptide kinase activity that was almost independent of arachidonic acid.

A cDNA clone encoding a novel member of the putative taste receptor T1R family, designated T1R3, was isolated from circumvallate papillae of the mouse tongue using degenerate primers. Reverse transcription–polymerase chain reaction analysis showed predominant expression of the receptor in circumvallate papillae. In situ hybridization analysis revealed that T1R3 was expressed in a subset of taste receptor cells in taste buds and that the topographic distribution of T1R3 in various taste papillae was different from those of the other T1R members. Genetic mapping of T1R3 with a mouse/hamster radiation hybrid panel located the gene on the distal end of mouse chromosome 4 correlated with the Sac locus affecting sweet sensitivity of mice. Our results indicate that T1R3 may serve as the receptor for sweet perception in mice.

We previously reported that PKN, a fatty acid-activated serine/threonine protein kinase, translocates from the cytosol to the nucleus by stresses such as heat shock, sodium arsenite, and serum starvation. To clarify the role of PKN under heat stress, we examined whether PKN regulates the expression of heat shock proteins. Co-expression of heat shock transcription factor 1 (HSF1) and the catalytically active fragment of PKN induced the accumulation of alphaB-crystallin but not HSP27 and HSP70 in HeLa S3 cells. The expression of the reporter gene for alphaB-crystallin promoter was activated by co-expression of HSF1 and the catalytically active fragment of PKN, and this activation was dependent on the protein kinase activity of PKN. Deletion analysis of the alphaB-crystallin promoter region revealed that both the proximal and the distal heat shock elements were necessary for the transactivation. These results raise the possibility that there is a signal transduction pathway mediating stress signals for the accumulation of alphaB-crystallin by HSF1 and PKN.

A novel 65-kDa protein (designated MIPP65), which was phosphorylated by PKNin vitroin a manner highly dependent on arachidonic acid, was partially purified from the heat-stable proteins extracted from a 30,000gprecipitate of rat liver. The cDNA clones were obtained by polymerase chain reaction using oligonucleotides based on partial amino acid sequences. The complete amino acid sequence deduced from the cDNAs contained two homologous regions with the mitochondrial NADH-ubiquinone oxidoreductase 9-kDa subunit precursor at the amino- and carboxyl-termini, whereas the central region was not related to any known proteins and contained a serine cluster. Northern blotting and immunoblotting analyses indicated that MIPP65 was expressed ubiquitously in rat tissues. Immunofluorescence analysis of the endogenous MIPP65 using polyclonal antiserum against MIPP65 showed a predominantly mitochondrial localization in C6 glioma cells. The recombinant MIPP65 expressed in COS7 cells showed a similar pattern of localization to that in C6 glioma cells. On the other hand, deletion of the amino-terminal region of MIPP65 abrogated such localization, indicating that the amino-terminal region contained a mitochondrial-targeting signal. From [32P]orthophosphate-labeled C6 glioma cells, the endogenous MIPP65 could be immunoprecipitated as a phosphoprotein with antiserum against MIPP65. These results suggest that MIPP65 is a novel mitochondrial phosphoprotein that is a candidate substrate for PKN.

PKN, a novel protein kinase with a catalytic domain homologous to that of the protein kinase C (PKC) family and unique N-terminal leucine-zipper-like sequences, was identified by molecular cloning from a human hippocampus cDNA library [Mukai and Ono (1994) Biochem. Biophys. Res. Commun. 199, 897-904]. Recently we partially purified recombinant PKN from COS7 cells transfected with the cDNA construct encoding human PKN, and demonstrated that the recombinant PKN was activated by unsaturated fatty acids and limited proteolysis [Mukai, Kitagawa, Shibata et al. (1994) Biochem. Biophys. Res. Commun. 204, 348-356]. The present work has focused on the further purification and characterization of PKN from native rat tissue. Immunochemical measurement revealed that PKN was found in every tissue, and was especially abundant in testis, spleen and brain; subcellular fractionation of rat brain showed that half of the PKN was localized in the soluble cytosolic fraction. PKN was purified approx. 8000-fold to apparent homogeneity from the cytosolic fraction of rat testis by DEAE-cellulose chromatography, ammonium sulphate fractionation and chromatography on butyl-Sepharose, heparin-Sepharose, Mono Q and protamine-CH-Sepharose. The enzyme migrates as a band of apparent molecular mass 120 kDa. Using serine-containing peptides based on the pseudosubstrate sequence of PKC-delta as phosphate acceptors, the kinase activity was stimulated several-fold by 40 microM unsaturated fatty acids or by detergents such as 0.04% sodium deoxycholate and 0.004% SDS. In the absence of modifiers, protamine sulphate, myelin basic protein and synthetic peptides based on the pseudosubstrate site of PKCs or ribosomal S6 protein were good substrates for phosphorylation by the kinase. In the presence of 40 microM arachidonic acid the kinase activity of PKN for these phosphate acceptors was increased 2-18-fold. The autophosphorylation activity of purified PKN was partially inhibited by pretreatment with alkaline phosphatase. These properties appear to distinguish PKN from many protein kinases isolated previously.

cDNA clone encoding Xenopus laevis PKN has been isolated from Xenopus kidney library. Sequencing of this clone has revealed a single open reading frame encoding a protein of 901 amino acids. Immunoprecipitate from cytoplasmic fraction of COS7 cells transfected with this cDNA construct using antiserum against bacterially expressed Xenopus PKN revealed arachidonic acid-dependent autophosphorylation activity. Comparison of the closely related sequences of human and rat PKN with a protein from evolutionarily distant Xenopus, revealed several highly invariant domains in the NH2-terminal regulatory regions, suggesting that they participate in binding interaction with arachidonic acid.