A Novel cdc2-Related Protein Kinase Expressed in the Nervous System

Abstract: We report the cloning and characterization of a cDNA encoding a cdc2-related protein kinase, named PFTAIRE, that is expressed primarily in the postnatal and adult nervous system. We have demonstrated by in situ hybridization and indirect immunofluorescence that several populations of terminally differentiated neurons and some neuroglia expressed PFTAIRE mRNA and protein. In neurons, PFTAIRE protein was localized in the nucleus and cytoplasm of cell bodies. The anatomical, cellular, and ontogenic patterns of PFTAIRE expression in the nervous system differed from those of p34^cdc2 and cdk5, which are expressed in brain and several other mitotic tissues. Proteins of ～58–60 kDa coprecipitated specifically with PFTAIRE from cytosolic protein preparations of adult mouse brain and transfected cells. These proteins appeared to be the major endogenous substrates associated with this kinase activity. The temporal and spatial expression patterns of PFTAIRE in the postnatal and adult nervous system suggest that PFTAIRE kinase activity may be associated with the postmitotic and differentiated state of cells in the nervous system and that its function may be distinct from those of p34^cdc2 and cdk5.     

Transient expression of a p58 protein kinase cDNA enhances mammalian glycosyltransferase activity

The effect of expression of a p58 protein kinase on mammalian beta-1,4 galactosyltransferase enzyme activity was examined in vitro and in vivo. We found that p58 protein kinase expression enhanced galactosyltransferase enzyme activity approximately three-fold in vivo when compared to reporter gene activity. Galactosyltransferase enzyme activity was also substantially reduced in vitro when dephosphorylated, or when p58 specific antibodies were used to inhibit kinase activity. These results suggest that galactosyltransferase activity is influenced by phosphorylation, and that the p58 protein kinase may mediate this effect.     

Elafin expression in human fetal and adult submandibular glands

Elafin, a bifunctional protein, has the NH(2)-terminal domain functions as a transglutaminase substrate for crosslinking to lysine-containing proteins and the COOH-terminal whey acidic protein domain as a potent anti-elastase. Human fetal submandibular glands (n=100) and adult submandibular glands (n=10) were used to elucidate the expression pattern of elafin in the developmental processes of human submandibular gland by immunohistochemistry, in situ hybridization, and western blot analysis. Elafin mRNA was expressed both in the gland epithelium and intralobular mesenchymal tissue of fetal submandibular gland in an early developmental stage (10-18 weeks) and an early intermediate developmental stage (EIDS; 19-24 weeks). The elafin antigen was also found in the intralobular mesenchyme of submandibular gland in the same stages. Thereafter, elafin was transitionally expressed in the ducts and acini of submandibular glands. In the late intermediate developmental stage (LIDS; 25-32 weeks) and the late developmental stage (LDS; 33-40 weeks), elafin became markedly positive in the excretory and striated ducts but weakly positive in the intralobular mesenchymal tissue. The elafin was heavily present in the excretory and striated ducts of adult submandibular gland, while it was sparse in the intralobular mesenchymal tissues. Western blot analysis showed the protein extracts from submandibular glands of EIDS, LIDS, and LDS, adult submandibular gland, fetal tissues (8 weeks), and adult parotid saliva migrated into multiple bands, about 25, 50, 65, and 140 kDa, which were higher than the putative size of elafin protein, 12 kDa. These data suggest that elafin, anti-elastase, is an essential component highly utilized during the morphogenetic processes of fetal salivary gland development and continuously plays a role for the protection of the tubuloalveolar structures of adult salivary gland.     

Developmental expression of survivin during embryonic submandibular salivary gland development

Background  

The regulation of programmed cell death is critical to developmental homeostasis and normal morphogenesis of embryonic tissues. Survivin, a member of the inhibitors of apoptosis protein (IAP) family primarily expressed in embryonic cells, is both an anti-apoptosis and a pro-survival factor. Since our previous studies have demonstrated the importance of apoptosis during embryonic submandibular salivary gland (SMG) development, we postulated that survivin is a likely mediator of SMG epithelial cell survival.     

Cell surface galactosyltransferase acts as a general modulator of rat acinar cell proliferation

Summary Several physiological parameters were examined for inducing acinar cell proliferation and corresponding increased expression of 1–4 galactosyltransferase. In this study, dietary changes causing acinar cell proliferation included the following: the introduction of animals to a liquid diet (causing gland atrophy) followed by reintroduction of solid chow, gustatory stimulation provided by the introduction of 0.5% citric acid to animal drinking water, and removal of the submandibular gland with subsequent reliance on the parotid gland for salivary protein. Alterations in growth factor levels were produced by injecting animals with a chronic (3 day) regimen of either nerve growth factor or epidermal growth factor. Parotid gland proliferation could be blocked in all cases except EGF by the injection of propranolol, a -adrenoceptor antagonist, or the galactosyltransferase specific modifier protein, -lactalbumin. EGF-induced proliferation could, however, be prevented by treating the animals with monoclonal antibody to EGF receptor or galactosyltransferase modifier protein a-lactalbumin. These results for normal acinar cell proliferation suggest a direct role for cell surface 1–4 galactosyltransferase in signalling and maintaining active cell growth.     

Purification and tissue-specific expression of casein kinase from the lactating guinea-pig mammary gland

A serine-specific casein kinase, an integral membrane protein of the lactating guinea-pig mammary gland, has been purified from a Golgi-enriched membrane fraction, using a combination of sucrose gradient centrifugation and chromatography on ATP-agarose. The enzyme comprises a polypeptide of estimated Mr 74 000 as judged by sodium dodecyl sulphate/polyacrylamide gel electrophoresis, compared with a monomer Mr of 50 000 as determined by sucrose gradient centrifugation in the presence of 500 mM NaCl and 0.1% Triton X-100. Kinetic studies show that the purified enzyme exhibits kinetic constants distinctly different from the rabbit reticulocyte casein kinases I and II, whilst polyclonal antisera raised against the mammary gland enzyme did not cross-react with soluble liver or reticulocyte protein kinase activities. Immunoblotting and immunocytochemical analyses demonstrate the mammary gland enzyme's apparently unique location in lactating mammary gland tissue. Comparative studies with polyclonal antisera raised against bovine galactosyltransferase, show that casein kinase and galactosyltransferase have a similar intracellular localisation in the lactating mammary gland as judged by immunocytochemistry at the light level, but that casein kinase was unique to mammary gland whereas galactosyltransferase could be found in other tissues. The results extended our earlier observations which suggest a Golgi location for casein kinase, and demonstrate that future studies using this enzyme may well prove advantageous for the study of intracellular mechanisms involved in the biogenesis of organelles, in this instance the Golgi apparatus.     

HIF-1α involvement in low temperature and anoxia survival by a freeze tolerant insect

Protein kinases are important signalling molecules critical for normal cell growth and development. CDK11^p58 is a p34^cdc2-related protein kinase, and plays an important role in normal cell cycle progression. In this study, we mainly characterized the protein expression of CDK11^p58 during postnatal development in mouse testes and examined the cellular localization of CDK11^p58 and cyclinD3, which was associated with CDK11^p58 in mammalian cells. Western blot analysis revealed that CDK11^p58 was present in the early stages of development. It gradually increased and reached a peak in adult testes. The protein expression of CDK11^p58 was further analysed by immunohistochemistry due to its developmentally regulated expression. The variable immunostaining patterns of CDK11^p58 were visualized during different developmental periods and, in adult mouse, different stages of seminiferous tubules. CDK11^p58 expression was detected in proliferating germ cells in the early stages of developing testes. In adult testes, the protein was expressed in pachytene primary spermatocytes from stage VII to XI of spermatogenesis and in postmeiotic spermatids in all stages at different levels. The colocalization of CDK11^p58 and cyclinD3 in the adult testeis was revealed by immunofluorescence analysis. (Mol Cell Biochem 270: 99–106, 2005)     

Oxidative stress signalling in the apoptosis of Jurkat T-lymphocytes

Within the first 24 h after castration of an adult male rat, the vascular system of the ventral prostate gland undergoes a degenerative process that drastically reduces blood flow to the tissue. Since the vascular degeneration precedes the loss of the prostatic epithelium (by apoptosis), we have proposed that the onset of epithelial cell apoptosis in this tissue is caused by an ischemic/hypoxic environment resulting from the loss of blood flow. In order to further evaluate the extent to which ischemia/hypoxia might be a factor in apoptosis of the prostate epithelium after castration, we analyzed for biomarkers of cellular hypoxia in rat ventral prostates during the first 3 days following castration. Ventral prostate tissues removed from hypoxyprobe-1-treated adult male rats (uncastrated controls; surgically castrated for 24, 48 or 72 h, or sham-castrated for equivalent times) were directly analyzed for evidence of hypoxia by in situ immunohistochemical evaluation of hypoxyprobe-1 adduct formation in the prostate cells. Protein extracts from these tissues were also tested for expression of the 120 kDa hypoxia-inducible factor-1-alpha (HIF-1-alpha) protein as well as for expression of mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) proteins using a Western blot assay. The tyrosine phosphorylation status of the latter signaling molecules was also evaluated by Western blotting using anti-tyrosine phosphate antibodies. Our results showed that epithelial cells of the rat ventral prostate stained positively for hypoxyprobe-1 adducts at all times after castration, whereas cells in control tissues were unstained by this procedure. In addition, the prostatic expression of HIF-1-alpha protein was increased approximately 20-fold at 48 h after castration compared to control tissues. Finally, although prostatic MAPK and JNK protein expression was unaltered during the early period after castration, phosphorylation of the JUN kinase protein was significantly elevated, indicating that this stress-activated cellular signaling pathway becomes more active subsequent to castration. These results support our proposal that early castration-induced degeneration and constriction of the vascular system of the rat ventral prostate gland leads to reduced oxygenation of prostatic epithelial cells and the activation of hypoxic cellular signaling in these cells through upregulation of HIF-1-alpha expression and stimulation of the JUN kinase signaling pathway.     

Localization of the expressed human p58 protein kinase chromosomal gene to chromosome 1p36 and a highly related sequence to chromosome 15.

The gene for the human p58 protein kinase, a cell division control-related gene, has been mapped by somatic cell hybrid analyses, in situ localization with the chromosomal gene, and nested polymerase chain reaction amplification of microdissected chromosomes. These studies indicate that the expressed p58 chromosomal gene maps to 1p36, while a highly related p58 sequence of unknown nature maps to chromosome 15. Assignment of a p34cdc2-related gene to 1p36 may have implications for numerous tumors that involve deletion of this region, including neuroblastoma, ductal carcinoma of the breast, malignant melanoma, Merkel cell carcinoma, and endocrine neoplasia.     

The timing of drosophila salivary gland apoptosis displays an l(2)gl-dose response

During Drosophila metamorphosis, larval tissues, such as the salivary glands, are histolysed whereas imaginal tissues differentiate into adult structures forming at eclosion a fly-shaped adult. Inactivation of the lethal(2)giant larvae (l(2)gl) gene encoding the cytoskeletal associated p127 protein, causes malignant transformation of brain neuroblasts and imaginal disc cells with developmental arrest at the larval-pupal transition phase. At this stage, p127 is expressed in wild-type salivary glands which become fully histolysed 12 - 13 h after pupariation. By contrast to wild-type, administration of 20-hydroxyecdsone to l(2)gl-deficient salivary glands is unable to induce histolysis, although it releases stored glue granules and gives rise to a nearly normal pupariation chromosome puffing, indicating that p127 is required for salivary gland apoptosis. To unravel the l(2)gl function in this tissue we used transgenic lines expressing reduced ( approximately 0.1) or increased levels of p127 (3.0). Here we show that the timing of salivary gland histolysis displays an l(2)gl-dose response. Reduced p127 expression delays histolysis whereas overexpression accelerates this process without affecting the duration of third larval instar, prepupal and pupal development. Similar l(2)gl-dependence is noticed in the timing of expression of the cell death genes reaper, head involution defective and grim, supporting the idea that p127 plays a critical role in the implementation of ecdysone-triggered apoptosis. These experiments show also that the timing of salivary gland apoptosis can be manipulated without affecting normal development and provide ways to investigate the nature of the components specifically involved in the apoptotic pathway of the salivary glands.     

Hyperglycemic factor in submandibular glands and its etiological relations to diabetes mellitus in mice.

Bilateral ligation of both the submandibular and parotid ducts of adult normal and mutant hyperinsulinemic diabetic mice resulted in a significant hypoglycemic effect. Therefore, we postulated that duct ligation may result in the removal of hyperglycemic factor (Hoshino et al., 1976) rather than a change in insulin sensitivity. Indeed, no change in specific binding of 125I-insulin was observed in membrane fractions from several tissues obtained from mice of either sex or strains before and after duct ligation. After slices of the submandibular gland were incubated for 4 hr in Eagle's medium, an aliquot of the culture medium was injected i.p. into normal adult mice. A signigicant hyperglycemic effect was observed in 30 min in the injected animals. Eluates obtained by gel filtration of the crude extract of the submandibular gland were injected into normal adult mice, and hyperglycemia ensued. Thus, it is postulated that ligation of salivary ducts results in glandular atrophy and disappearance of the hyperglycemic factor which in turn leads to hypoglycemia and amelioration of diabetes mellitus, particularly of hyperinsulinemic type.     

Embigin,a developmentally expressed member of the immunoglobulin super family,is also expressed during regression of prostate and mammary gland

Cell adhesion molecules (CAMs) are intimately involved in a variety of cellular processes, including development, cell growth, apoptosis, and differentiation. Interaction of CAMs with components of the extracellular matrix (ECM), growth factors, and other CAMs provides an intricate regulatory mechanism for a diverse range of cellular responses. Embigin is a developmentally expressed protein that is a member of the immunoglobulin superfamily (IgSF) class of CAMs. We have identified embigin as a gene expressed during tissue regression in rat prostate and lactating mammary gland following hormonal ablation. In the absence of the appropriate hormone, the secretory epithelial cells of these two tissues undergo successive waves of apoptotic cell death co-incident with extensive reorganization of the surviving tissue. Using Northern analysis, in situ hybridization analysis, RT-PCR, and Western analysis, we have characterized the expression of embigin mRNA and protein in both regressing prostate and mammary gland. During development of the prostate gland, increased expression of embigin is correlated with the appearance of highly organized lumenal and ductal structures. Embigin is also expressed in a variety of adult tissues including heart, liver, lung, and brain. Zoo-blot analysis with the rat embigin cDNA indicates that embigin homologs exist in species as diverse as Homo sapiens and Drosophila melanogaster, suggesting that it has been highly conserved during evolution. Embigin protein is expressed at readily detectable levels in a variety of prostate and mammary cancer cell lines, and in some cell lines the expression of embigin appears to be down-regulated in the presence of ECM. Our data have led us to propose a model in which embigin functions as a regulator of cell/ECM interactions during development and in the homeostasis of normal adult tissues. Dev. Genet. 21:268–278, 1997. © 1997 Wiley-Liss, Inc.     

Protein-tyrosine kinase and protein-serine/threonine kinase expression in human gastric cancer cell lines

Protein kinases play key roles in cellular functions. They are involved in many cellular functions including; signal transduction, cell cycle regulation, cell division, and cell differentiation. Alterations of protein kinase by gene amplification, mutation or viral factors often induce tumor formation and tumor progression toward malignancy. The identification and cloning of kinase genes can provide a better understanding of the mechanisms of tumorigenesis as well as diagnostic tools for tumor staging. In this study, we have used degenerated polymerase-chain-reaction primers according to the consensus catalytic domain motifs to amplify protein kinase genes (protein-tyrosine kinase, PTK, and protein-serine/threonine kinase, PSK) from human stomach cancer cells. Following amplification, the protein kinase molecules expressed in the gastric cancer cells were cloned into plasmid vectors for cloning and sequencing. Sequence analysis of polymerase-chain-reaction products resulted in the identification of 25 protein kinases, including two novel ones. Expression of several relevant PTK/PSK genes in gastric cancer cells and tissues was further substantiated by RT-PCR using gene-specific primers. The identification of protein kinases expressed or activated in the gastric cancer cells provide the framework to understand the oncogenic process of stomach cancer.     

Correlation of the expression of double-stranded RNA-dependent protein kinase (p68) with differentiation in head and neck squamous cell carcinoma

p68 is an inducible protein kinase which is believed to be an important factor in the regulation of both viral and cellular protein synthesis. We have produced a monoclonal antibody (TJ4C4) which specifically detects p68, and which can be used to detect this antigen in formalin-fixed, paraffin-embedded tissues. Because p68 plays an important role in cellular protein synthesis, we hypothesized that it may correlate with normal and neoplastic cellular differentiation. One hundred and seventy-seven head and neck squamous cell carcinoma specimens, representing 82 patients, were studied. The relative amount, frequency, and distribution of p68 expression were determined by microscopic evaluation of ABC immunoperoxidase-stained specimens. A spectrum of immunoreactivity was detected in 156 of 177 tumors, as well as within the normal squamous epithelium. Normal, actively proliferating cells, such as the basal layer of squamous epithelium, expressed comparatively little p68. Increased p68 expression was noted to parallel the morphologic features of cellular differentiation. In neoplastic tissue, p68 expression also increased with the degree of cellular differentiation. These data demonstrate that the expression of p68 parallels the degree of cellular differentiation in squamous cell carcinoma of the head and neck region, as well as within normal squamous mucosa. Therefore, p68 may provide an objective biologic measure of cellular differentiation which does not depend on morphologic features.     

Interaction of p58(PITSLRE), a G2/M-specific protein kinase,with cyclin D3

The p58(PITSLRE) is a p34(cdc2)-related protein kinase that plays an important role in normal cell cycle progression. Elevated expression of p58(PITSLRE) in eukaryotic cells prevents them from undergoing normal cytokinesis and appears to delay them in late telophase. To investigate the molecular mechanism of p58(PITSLRE) action, we used the yeast two-hybrid system, screened a human fetal liver cDNA library, and identified cyclin D3 as an interacting partner of p58(PITSLRE). In vitro binding assay, in vivo coimmunoprecipitation, and immunofluorescence cell staining further confirmed the association of p58(PITSLRE) with cyclin D3. This binding was observed only in the G(2)/M phase but not in the G(1)/S phase of the cell cycle; meanwhile, no interaction between p110(PITSLRE) and cyclin D3 was observed in all the cell cycle. The overexpression of cyclin D3 in 7721 cells leads to an exclusively accumulation of p58(PITSLRE) in the nuclear region, affecting its cellular distribution. Histone H1 kinase activity of p58(PITSLRE) was greatly enhanced upon interaction with cyclin D3. Furthermore, kinase activity of p58(PITSLRE) was found to increase greatly in the presence of cyclin D3 using a specific substrate, beta-1,4-galactosyltransferase 1. These data provide a new clue to our understanding of the cellular function of p58(PITSLRE) and cyclin D3.     

Regeneration of acinar cells following ligation of rat submandibular gland retraces the embryonic-perinatal pathway of cytodifferentiation

Rat submandibular gland can regenerate following ligation-induced atrophy, eventually recovering its normal morphology and function. Previous studies have suggested that the regeneration process implies both self-proliferation of existing acini and formation of new acinar cells. One hypothesis is that new acinar cells may differentiate from the ductal cells in a similar fashion to the process of cytodifferentiation occurring during submandibular glandular development. In this study atrophy was induced, under recovery anaesthesia, by applying a metal clip on the main duct of the submandibular gland without including the chorda lingual nerve. After 2 weeks the duct was deligated for 3, 5 or 7 days or 8 weeks and the glands collected. Tissue was prepared for immunohistochemstry, biochemical analysis and RNA extraction. The histology of the regenerated glands shows several normal-looking acini, which have regained their glycoprotein content (AB/PAS positive), data also confirmed by biochemical analysis (SDS-PAGE/PAS). Regenerating tissue was characterized by the presence of embryonic-like branched structures ending with AB/PAS positive acinar cells. The proteins SMG-B and PSP are normally expressed in acinar cell precursors during development but only by intercalated ductal cells in the adult stage. In the adult regenerating gland mRNA levels of both SMG-B and PSP were found to be up-regulated compared to ligated glands and SMG-B expression localized to acinar cells whilst the ductal cells were negative. This study of rat submandibular gland regeneration suggests new acinar cells have differentiated from ducts and express markers of acinar cell precursors in a similar manner to the cytodifferentiation process occurring during glandular development.     

58-kDa microspherule protein (MSP58) is novel Brahma-related gene 1 (BRG1)-associated protein that modulates p53/p21 senescence pathway

The nucleolar 58-kDa microspherule protein (MSP58) protein is a candidate oncogene implicated in modulating cellular proliferation and malignant transformation. In this study, we show that knocking down MSP58 expression caused aneuploidy and led to apoptosis, whereas ectopic expression of MSP58 regulated cell proliferation in a context-dependent manner. Specifically, ectopic expression of MSP58 in normal human IMR90 and Hs68 diploid fibroblasts, the H184B5F5/M10 mammary epithelial cell line, HT1080 fibrosarcoma cells, primary mouse embryonic fibroblasts, and immortalized NIH3T3 fibroblasts resulted in induction of premature senescence, an enlarged and flattened cellular morphology, and increased senescence-associated β-galactosidase activity. MSP58-driven senescence was strictly dependent on the presence of functional p53 as revealed by the fact that normal cells with p53 knockdown by specific shRNA or cells with a mutated or functionally impaired p53 pathway were effective in bypassing MSP58-induced senescence. At least two senescence mechanisms are induced by MSP58. First, MSP58 activates the DNA damage response and p53/p21 signaling pathways. Second, MSP58, p53, and the SWI/SNF chromatin-remodeling subunit Brahma-related gene 1 (BRG1) form a ternary complex on the p21 promoter and collaborate to activate p21. Additionally, MSP58 protein levels increased in cells undergoing replicative senescence and stress-induced senescence. Notably, the results of analyzing expression levels of MSP58 between tumors and matched normal tissues showed significant changes (both up- and down-regulation) in its expression in various types of tumors. Our findings highlight new aspects of MSP58 in modulating cellular senescence and suggest that MSP58 has both oncogenic and tumor-suppressive properties.