ABSTRACT
Xenopus egg extracts provide a powerful tool for studying formation and function of chromosomes. Two alternative protocols are generally used to obtain mitotic chromosomes. The first one employs direct assembly of chromatin from sperm nuclei in CSF-arrested meiotic extracts, while the second is based on transition of sperm DNA through a replication step, followed by re-establishing of CSF arrest. In this study we show that general kinetochore structure is disrupted in chromosomes assembled directly in CSF egg extracts: the amounts of outer kinetochore proteins such as Bub1, BubR1 and Dynactin subunit p150glued are reduced and the components of the inner centromeric region (Aurora B kinase and Survivin) show compromised recruitment to centromeres. In contrast, kinetochores on chromosomes assembled according to the second protocol closely resemble those in somatic cells. Our results argue that transition of sperm nuclei through interphase is an essential step for proper kinetochore assembly.
Subject(s)
Cell Extracts , Kinetochores/metabolism , Oocytes , Spermatozoa/physiology , Animals , Chromosomes/metabolism , Female , Interphase , Male , Xenopus laevisABSTRACT
The present paper deals with two aspects of EGF-induced signal transduction via transcriptional factor STAT1. Utilizing vesicle fractionation in Percoll density gradient followed by co-immunoprecipitation, we observed the association of STAT1 with EGF receptor internalized in early endosomes. Co-immunoprecipitation studies with antikaryopherin alpha antibody showed the binding of activated STAT1 to nuclear import factors--karyopherins alpha. Both complexes demonstrate similar dynamics upon EGF treatment: they are formed at the early times, cannot be detected within 15 min, and reappear in 20 min or later. The complex of STAT1 and karyopherin alpha is localized in the cytoskeletal fraction.
Subject(s)
DNA-Binding Proteins/metabolism , Epidermal Growth Factor/pharmacology , ErbB Receptors/metabolism , Nuclear Proteins/metabolism , Trans-Activators/metabolism , Animals , Cell Line , Endocytosis , Humans , Phosphorylation , Precipitin Tests , Protein Binding , Rabbits , STAT1 Transcription Factor , alpha KaryopherinsABSTRACT
Transcription factor STAT1 (Signal Transducers and Activators of Transcription) takes part in signal transduction from receptors of growth factors and many cytokines, including interferons. In this paper, the role of tyrosinkinases Src and JAK2 was estimated in activation of STAT1 by epidermal growth factor (EGF) and hyperosmotic shock. Using a pharmacological inhibitor of Src kinases CGP77675 and cells with knockout c-src, it was shown that Src activated STAT1 upon stimulation by both epidermal growth factor and hypersomatic shock. In contrast, JAK2 activity exerted no influence on these processes.
Subject(s)
DNA-Binding Proteins/physiology , Epidermal Growth Factor/pharmacology , ErbB Receptors/physiology , Protein-Tyrosine Kinases/physiology , Proto-Oncogene Proteins , Signal Transduction/physiology , Trans-Activators/physiology , src-Family Kinases/physiology , Animals , Cell Line , Humans , Janus Kinase 2 , Mice , Mice, Knockout , Osmotic Pressure , STAT1 Transcription Factor , src-Family Kinases/geneticsABSTRACT
Intracellular distribution of p42/p44 MAP-kinases in HER14 and A431 cell lines was investigated. Using subcellular fractionation and immunofluorescence approaches we have shown that in quiescent cells of both types MAP-kinases are associated with endoplasmic reticulum. Moreover, ER-localized MAP-kinases were shown to exist only in a nonphosphorylated form. In HER14 cells the epidermal growth factor (EGF) elevates the level of the ER-associated MAP-kinases. In contrast, exposure of A431 cells to EGF leads to a significant decrease in the observed association. The physiological role of this association is discussed.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Endoplasmic Reticulum/enzymology , 3T3 Cells , Animals , Blotting, Western , Epidermal Growth Factor/metabolism , Fluorescent Antibody Technique, Indirect , Humans , Mice , Subcellular Fractions/enzymology , Tumor Cells, CulturedABSTRACT
During endocytosis EGF-receptor complexes are transported from early peripheral endosomes to late juxtranuclear-located endosomes to be then degraded in lysosomes. It is suggested that such a spatial organization of endosomal compartments is maintained by microtubule system and is necessary for lysosomal degradation of endocytosed cargo. In the present work, a study was made of the influence of Nocodazole, a microtubule depolymerizing agent, on endocytosis of fluid phase marker HRP and EGF entering the cell via receptor-mediated endocytosis. By subcellular fractionation in Percoll gradient it was shown that Nocodazole did not affect HRP internalization but stimulated its accumulation in a fraction sedimented together with late endosomes, thus preventing HRP delivery to lysosomes. On the contrary, Nocodazole exerted no influence on dynamics of compartmentalization and lysosomal degradation of EGF-receptor complexes. Moreover, no alterations were found in the functioning of a so well-known EGF-stimulated signal transduction pathway as MAP-kinase cascade. At the same time microtubule depolymerization dramatically changed the morphology of endosomal compartments abolishing juxtranuclear localization of late endosomes. Our data suggest that translocation of EGF-receptor complexes is not necessary for their normal lysosomal processing. Rab7, traditionally considered as a marker of late endosomes, has been found to demonstrate in Nocodazole-treated cells, in contrast to the control, a low extent of co-localization with endosomal structures. It could be supposed that the role of Rab7 is not so much to mediate early-to-late endosome transition as to maintain spatial organization of endosomal apparatus by mediating endosome-cytoskeleton interactions.
Subject(s)
Endocytosis/physiology , ErbB Receptors/drug effects , Nocodazole/therapeutic use , 3T3 Cells , Animals , Colloids , Endosomes/drug effects , ErbB Receptors/metabolism , Lysosomes/drug effects , Mice , Povidone , Silicon Dioxide , Subcellular Fractions/metabolismABSTRACT
A study was made of an association of small GTPase Rab7, commonly considered as a marker of late endosomes, with endosomal compartments of cells expressing EGF receptor with different ability to be sorted for degradative pathway. It was found that in cells HER14, expressing normal EGF receptor, Rab7 was associated with both early and late endosomes and the extent of association correlated with the number of EGF-receptor complexes in the specific endosomal fraction. Cels with a receptor, lacking major sites of autophosphorylation by deletion of 126 C-terminal residues (CD126), demonstrated a low efficiency of EGF-receptor sorting to late endosomes and decreased association dynamics with Rab7. Interaction of Rab7 with endosomes of cells expressing kinase negative receptors (K721) was found to be minimal. At the same time, in cells Cd126 and K721 with a low sorting efficiency Rab7 was mainly associated with early endosomes. These data favor Rab7 involvement in mediating early-to-late endosomal transition.
Subject(s)
ErbB Receptors/chemistry , GTP Phosphohydrolases/chemistry , GTP-Binding Proteins/chemistry , rab GTP-Binding Proteins , 3T3 Cells , Animals , Centrifugation, Density Gradient , Colloids , Endosomes/chemistry , ErbB Receptors/genetics , Mice , Mutation , Phosphorylation , Povidone , Silicon Dioxide , rab7 GTP-Binding ProteinsABSTRACT
Nuclear translocation of MAPK in cell lines, expressing normal and mutant forms of EGF-receptors (EGFR), was investigated. Using immunoblotting and immunofluorescence, EGF-induced MAPK transport was discovered in cell lines, expressing both normal receptor and one with deletion of major autophosphorylation sites. The dynamics of MAPK nuclear translocation in these cell lines was alike. Cells bearing EGFR with inactive tyrosine kinase showed no ability to EGF-dependent activation and nuclear translocation of MAPK. A suggestion is made that tyrosine kinase is needed for EGF-induced activation and nuclear import of MAPK.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Epidermal Growth Factor/physiology , ErbB Receptors/physiology , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinases , 3T3 Cells , Animals , Biological Transport , Cell Nucleus/enzymology , Cytoplasm/enzymology , Humans , Mice , Mitogen-Activated Protein Kinase 3ABSTRACT
The influence of cytoskeleton-depolymerising agents (nocodazole and cytochalasine D) on EGF-induced transport of p42/p44MAPK into the cell nucleus was investigated. Using immunoblotting and immunofluorescence methods we have shown that depolymerization of microtubules does not affect EGF-induced nuclear import of MAPK, while actin depolymerization significantly inhibited this process. At the same time, cytochalasine D did not affect the EGF-induced MAPK activation in the cytoplasm. These data indicate that the EGF-induced nuclear import of MAPK depends on the microfilament system.