Determination of c-Abl tyrosine kinase and mTERT catalytic subunit of telomerase expression level during prenatal-postnatal mouse ovary-testis development

Expression levels of genes involved in the development of germ cells vary throughout the process from bipotential gonadal period to adult gonadal formation. In mice, developments of female and male reproductive system are regulated by germ cell-specific factors and hormones, and determinative days in this regulation are very important. c-Abl is a non-receptor tyrosine kinase with cellular functions including cell proliferation, growth and development. mTERT is involved in maintaining telomerase activity and proliferation of surviving cells. We suggested that c-Abl and mTERT might be important for the healthy development of prenatal and postnatal mouse ovary and testis. We aim to demonstrate localization and expressions of c-Abl and mTERT in crucial days of ovary and testis development in prenatal and postnatal period in mouse by immunofluorescence staining and qRT-PCR, respectively. The importance of c-Abl and mTERT expressions during the healthy gonadal development is indicated in the prenatal and postnatal gonadal development. Also, protein expression levels were detected by Western Blot in only postnatal ovary and testis. Determining the functions of the c-Abl and mTERT throughout the process will be important in terms of understanding the infertility cases in the female and male with future studies.     

Basigin expression and hormonal regulation in mouse uterus during the peri-implantation period

Basigin, a transmembrane glycoprotein belonging to the immunoglobulin superfamily, has been shown to be essential for fertilization and implantation. The aim of this study was to determine the expression and hormonal regulation of basigin gene in mouse uterus during the peri-implantation period. Basigin immunostaining and mRNA were strongly localized in luminal and glandular epithelium on day 1 of pregnancy and gradually decreased to a basal level from day 2-4 of pregnancy. Basigin mRNA expression in the sub-luminal stroma was first detected on day 3 of pregnancy and increased on day 4 of pregnancy. On day 5 of pregnancy, the expression of basigin protein and mRNA was only detected in the implanting embryos, and the luminal epithelium and sub-luminal stroma surrounding the embryos. A similar expression pattern of basigin was also induced in the delayed-implantation uterus which was activated by estrogen injection. On day 6-8 of pregnancy, although a basal level of basigin protein was detected in the secondary decidual zone, basigin mRNA expression was strongly seen in this location. Basigin mRNA was also highly expressed in the decidualized cells under artificial decidualization. Estrogen significantly stimulated basigin expression in the ovariectomized mouse uterus. A high level of basigin immunostaining and mRNA was also seen in proestrus and estrus uteri. These results suggest that basigin expression is closely related to mouse implantation and up-regulated by estrogen.     

Phosphorylation and consequent stimulation of the tyrosine kinase c-Abl by PKA in mouse spermatozoa; its implications during capacitation

Upon ejaculation, spermatozoa undergo a series of post-translational modifications in a process known as capacitation in order to prepare for fertilization. In the absence of capacitation, fertilization cannot occur. Spermatozoa are unusual in that one of the hallmarks of capacitation is a global up-regulation in phosphotyrosine expression, which is known to be mediated upstream by PKA. Little is known about the signaling events downstream of PKA apart from the involvement of SRC, as a key mediator of PKA-induced tyrosine phosphorylation in the sperm tail. Here we describe the presence of c-Abl in mouse spermatozoa. In vitro analysis confirmed that PKA can up-regulate c-Abl kinase activity. In vivo, this tyrosine kinase was found to associate, and become threonine phosphorylated by PKA in the sperm flagellum. By treating spermatozoa with hemolysin we could demonstrate that a significant proportion of the tyrosine phosphorylation associated with capacitation could be suppressed by the c-Abl inhibitor, Gleevac. This is the first report of c-Abl being up-regulated by PKA for any cell type. We present a model, whereby these kinases may operate together with SRC to ensure optimal levels of tyrosine phosphorylation in the sperm flagellum during the attainment of a capacitated state.     

T cell survival and function requires the c-Abl tyrosine kinase

C-Abl (Abl) regulates multiple cellular processes, including proliferation, survival, shape determination and motility, and participates in cellular responses to genotoxic and oxidative stress stimuli. Mice lacking Abl exhibit retarded growth, osteoporosis and defects in the immune system resulting in lymphopoenia and susceptibility to infections, leading to early death. To define the role of Abl in the regulation of adult T cells we ablated Abl exclusively in T cells by generating mice with floxed abl alleles and expressing an Lck-Cre transgene (Abl-T-/-). These mice exhibited thymic atrophy and abnormally reduced T cell numbers in the periphery. The thymic atrophy was caused by increased susceptibility of thymocytes to cell death. Importantly, Abl deficient T cells displayed abnormally reduced response to mitogenic stimulation in vitro. Consequently, Abl-T-/- mice exhibited impaired ability to reject syngeneic tumor, to induce T-mediated tumor cell killing, and to generate anti-tumor antibodies. These results demonstrate a cell-autonomous role for Abl in T cell function and survival.     

Prolin-rich tyrosine kinase 2 (PYK2) expression and localization in mouse testis

Prolin-rich kinase 2 (PYK2) is a nonreceptor tyrosine kinase related to the focal adhesion kinase (FAK) p125(FAK). PYK2 is rapidly phosphorylated on tyrosine residues in response to various stimuli, such as tumor necrosis factor-alpha (TNF-alpha), changes in osmolarity, elevation in intracellular calcium concentration, angiotensin, and UV irradiation. PYK2 has ligand sequences for Src homology 2 and 3 (SH-2 and SH-3), and has binding sites for paxillin and p130(cas). Activation of PYK2 leads to modulation of ion channel function, phosphorylation of tyrosine residues, and activation of the MAP kinase signaling pathways. Immunocytochemistry shows that PYK2 is present in mouse germinal and Sertoli cells (ser). Northern blot and immunoprecipitation analysis demonstrate that, among germinal cells, PYK2 is more abundant in spermatocytes (spc) and spermatids (spt); in addition, immunofluorescence analysis clearly shows that the diffuse cytoplasmic localization of PYK2 changes in a specific cellular compartment in spt and spermatozoa.     

Peroxidase activity in the mouse uterus, placenta and fetus during pregnancy

Changes in peroxidase activity during pregnancy were examined in CD-1 mice. Peroxidase activity was measured with guaiacol as the substrate in uterine extracts of nonpregnant mice and in uterine, placental, and fetal extracts of pregnant mice on days 9, 12, 14, 16, and 18 of gestation. Uterine peroxidase activity in nonpregnant mice was high, but declined logarithmically to only 0.2% by day 18 of pregnancy. In contrast to this decline, a concomitant 50-fold logarithmic increase in fetal peroxidase activity was observed between day 12 and 18. Activity in placental extracts did not change significantly throughout the gestational period examined. These results suggest that membrane bound peroxidase in mouse uterus and fetus undergoes major shifts during pregnancy.     

Cell-type-specific expression of transforming growth factor-alpha in the mouse uterus during the peri-implantation period.

Immunohistochemistry as well as in situ and Northern blot hybridization were employed to determine temporal and cell-type-specific expression of transforming growth factor-alpha (TGF-alpha) in the mouse uterus during the peri-implantation period. The co-localization of TGF-alpha (by immunohistochemistry) with its mRNA (by in situ hybridization) in the luminal and glandular epithelia on Days 1-4 of pregnancy (Day 1 = vaginal plug) and also in many stromal cells on Days 3 and 4 indicates that these cells are the primary sites of TGF-alpha synthesis during the preimplantation period. The higher levels of TGF-alpha mRNA in total uterine RNA on Day 4, as shown by Northern blotting, is consistent with the recruitment of stromal cells expressing this gene. During the post-implantation period (Days 5-8), the co-localization of the mRNA and protein in the decidua at the implantation sites suggests that the decidualizing stromal cells synthesize TGF-alpha. Although in situ hybridization showed the presence of mRNA in embryos on Days 5-8, immunostaining was noted in the embryo only on Days 5 and 6. These results suggest that uterine and embryonic expression of TGF-alpha during the peri-implantation period could be involved in embryonic development, preparation of the uterus for implantation, and decidualization.     

Cell type-specific expression of transforming growth factor-beta 1 in the mouse uterus during the periimplantation period

Immunohistochemistry and in situ and Northern blot hybridization were employed to determine temporal and spatial expression of transforming growth factor-beta 1 (TGF beta 1) in the mouse uterus during the periimplantation period. The polyclonal antisera anti-LC-(1-30) and anti-CC-(1-30), raised against two different preparations of a peptide corresponding to the amino-terminal 30 amino acids of TGF beta 1, were used for histochemical analyses because of their distinct staining patterns. Anti-LC shows intracellular staining, while staining by anti-CC is primarily extracellular. The colocalization of intracellular staining by anti-LC with in situ hybridization of TGF beta 1 mRNA in the luminal and glandular epithelia on days 1-4 of pregnancy (day 1 = vaginal plug) indicates that the epithelial cells are the primary sites of TGF beta 1 synthesis during the preimplantation period. On the other hand, staining of the extracellular matrix of the stroma by anti-CC during this period suggests an active accumulation of TGF-beta 1 that is synthesized in and secreted from the epithelia. While intracellular staining and accumulation of TGF-beta 1 mRNA in the epithelia were clearly evident on days 1-4, the extracellular staining showed temporal fluctuations. The clear extracellular staining of the stroma that was observed on day 1 was absent on day 2; moderate staining was again visualized in the stroma on day 3 and was markedly increased on day 4.(ABSTRACT TRUNCATED AT 250 WORDS)     

ONIOM DFT/PM3 calculation on the interaction between STI-571 and abelson tyrosine kinase

The ONIOM2 (B3LYP/6–31G (d, p): PM3) and B3LYP/6–31G (d, p) methods were applied to investigate the interaction between STI-571 and abelson tyrosine kinase binding site. The complex of N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)- phenyl]-benzamide (part of STI-571) and related 16 amino acid residues were found at B3LYP/6–31G (d, p) level to have hydrogen bonds and π....π stacking interaction, their binding energy via HAF optimization was −20.4 kcal mol⁻¹. The results derived from this study agreed well with the reported observation. Figure Optimized structure of STI-571 and Thr315 in abelson tyrosine kinase based on ONIOM2 method     

Interaction between UV-damaged DNA binding activity proteins and the c-Abl tyrosine kinase

The c-Abl tyrosine kinase is activated by some forms of DNA damage, including ionizing radiation, but not UV radiation. The functions of this activation in the damage response pathways remain obscure. To identify potential targets of c-Abl kinase, we utilized the yeast two-hybrid system to screen a murine cDNA library. One c-Abl binding protein of particular interest was the large subunit (DDB1) of the heterodimeric complex with UV-damaged DNA binding activity (UV-DDB). This complex binds with high specificity to DNA damaged by UV, is absent in a subset of xeroderma pigmentosum group E cells, and is required for global genomic repair of UV-induced damage. The association of c-Abl with DDB1 required the kinase domain of c-Abl and preserved the interaction between DDB1 and the small subunit (DDB2) of the UV-DDB complex. Significantly, overexpression of c-Abl increased tyrosine phosphorylation of DDB2 and suppressed UV-DDB activity. Conversely, a dominant negative, kinase-deficient allele of c-Abl decreased tyrosine phosphorylation of DDB2 and dramatically stimulated UV-DDB activity. These results suggest that one role of c-Abl may be to negatively regulate UV-DDB activity by phosphorylation of DDB2.     

Abl interactor 1 promotes tyrosine 296 phosphorylation of mammalian enabled (Mena) by c-Abl kinase

Mammalian Enabled (Mena) is a mammalian homologue of Drosophila Enabled (Ena), which genetically interacts with Drosophila Abl tyrosine kinase. The signaling pathway involving c-Abl and Mena (Ena) is not fully understood. To find molecules that participate in the c-Abl/Mena pathway, we searched for Mena-binding proteins using a yeast two-hybrid system. We identified Abl interactor 1 (Abi-1), which is known to interact with c-Abl, as a binding protein for Mena. Binding analysis revealed that the Ena/Vasp homology 1 domain of Mena and the polyproline structure of Abi-1 are necessary for the interaction. The interaction between Mena and Abi-1 was also observed in a mammalian expression system. Importantly, Abi-1 dramatically promoted c-Abl-mediated tyrosine phosphorylation of Mena but not other substrates such as c-Cbl. Mutational analysis demonstrated that the phosphorylation site of Mena is Tyr-296. Our results suggest that Abi-1 regulates c-Abl-mediated phosphorylation of Mena by interacting with both proteins.     

Cytoplasmic signalling by the c-Abl tyrosine kinase in normal and cancer cells

c-Abl is a non-receptor tyrosine kinase which is localized both in the nucleus and cytoplasm, and is involved in the regulation of cell growth, survival and morphogenesis. Although c-Abl nuclear function has been extensively studied, recent data also indicate an important role in cytoplasmic signalling through mitogenic and adhesive receptors. Here, we review the mechanisms by which growth factors promote cytoplasmic c-Abl activation and signalling and its function in the induction of DNA synthesis, changes in cell morphology and receptor endocytosis. The importance of de-regulated c-Abl cytoplasmic signalling in solid tumours is also discussed.     

A new link between the c-Abl tyrosine kinase and phosphoinositide signalling through PLC-gamma1

The c-Abl tyrosine (Tyr) kinase is activated after platelet-derived-growth factor receptor (PDGFR) stimulation in a manner that is partially dependent on Src kinase activity. However, the activity of Src kinases alone is not sufficient for activation of c-Abl by PDGFR. Here we show that functional phospholipase C-gamma1 (PLC-gamma1) is required for c-Abl activation by PDGFR. Decreasing cellular levels of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) by PLC-gamma1-mediated hydrolysis or dephosphorylation by an inositol polyphosphate 5-phosphatase (Inp54) results in increased Abl kinase activity. c-Abl functions downstream of PLC-gamma1, as expression of kinase-inactive c-Abl blocks PLC-gamma1-induced chemotaxis towards PDGF-BB. PLC-gamma1 and c-Abl form a complex in cells that is enhanced by PDGF stimulation. After activation, c-Abl phosphorylates PLC-gamma1 and negatively modulates its function in vivo. These findings uncover a newly discovered functional interdependence between non-receptor Tyr kinase and lipid signalling pathways.