In many eukaryotes, a significant part of the plasma membrane is closely associated with the dynamic meshwork of cortical endoplasmic reticulum (cortical ER). We mapped temporal variations in the local coverage of the yeast plasma membrane with cortical ER pattern and identified micron-sized plasma membrane domains clearly different in cortical ER persistence. We show that clathrin-mediated endocytosis is initiated outside the cortical ER-covered plasma membrane zones. These cortical ER-covered zones are highly dynamic but do not overlap with the immobile and also endocytosis-inactive membrane compartment of Can1 (MCC) and the subjacent eisosomes. The eisosomal component Pil1 is shown to regulate the distribution of cortical ER and thus the accessibility of the plasma membrane for endocytosis.
- MeSH
- buněčná membrána fyziologie MeSH
- endocytóza * MeSH
- endoplazmatické retikulum fyziologie MeSH
- fosfoproteiny fyziologie MeSH
- klathrin fyziologie MeSH
- Saccharomyces cerevisiae - proteiny fyziologie MeSH
- Saccharomyces cerevisiae fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- fosfoproteiny MeSH
- klathrin MeSH
- PIL1 protein, S cerevisiae MeSH Prohlížeč
- Saccharomyces cerevisiae - proteiny MeSH
Beside of the protein crystallography or NMR, another attractive option in protein structure analysis has recently appeared: computer modeling of the protein structure based on homology and similarity with proteins of already known structures. We have used the combination of computer modeling with spectroscopic techniques, such as steady-state or time-resolved fluorescence spectroscopy, and with molecular biology techniques. This method could provide useful structural information in the cases where crystal or NMR structure is not available. Molecular modeling of the ATP site within the H4-H5-loop revealed eight amino acids residues, namely besides the previously reported amino acids Asp443, Lys480, Lys501, Gly502 and Arg544, also Glu446, Phe475 and Gln482, which form the complete ATP recognition site. Moreover, we have proved that a hydrogen bond between Arg423 and Glu472 supports the connection of two opposite halves of the ATP-binding pocket. Similarly, the conserved residue Pro489 is important for the proper interaction of the third and fourth beta-strands, which both contain residues that take part in the ATP-binding. Alternatively, molecular dynamics simulation combined with dynamic fluorescence spectroscopy revealed that 14-3-3 zeta C-terminal stretch is directly involved in the interaction of 14-3-3 protein with the ligand. Phosphorylation at Thr232 induces a conformational change of the C-terminus, which is presumably responsible for observed inhibition of binding abilities. Phosphorylation at Thr232 induces more extended conformation of 14-3-3zeta C-terminal stretch and changes its interaction with the rest of the 14-3-3 molecule. This could explain negative regulatory effect of phosphorylation at Thr232 on 14-3-3 binding properties.
- MeSH
- adenosintrifosfát chemie metabolismus MeSH
- alkoholoxidoreduktasy MeSH
- DNA vazebné proteiny fyziologie MeSH
- fluorescenční spektrometrie metody MeSH
- fosfoproteiny fyziologie MeSH
- fosforylace MeSH
- konformace proteinů * MeSH
- konzervovaná sekvence MeSH
- molekulární modely * MeSH
- počítačová simulace * MeSH
- proteiny 14-3-3 chemie metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- sodíko-draslíková ATPasa chemie fyziologie MeSH
- threonin fyziologie MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- adenosintrifosfát MeSH
- alkoholoxidoreduktasy MeSH
- C-terminal binding protein MeSH Prohlížeč
- DNA vazebné proteiny MeSH
- fosfoproteiny MeSH
- proteiny 14-3-3 MeSH
- sodíko-draslíková ATPasa MeSH
- threonin MeSH
Engagement of the Fcepsilon receptor I (FcepsilonRI) on mast cells and basophils initiates signaling pathways leading to degranulation. Early activation events include tyrosine phosphorylation of two transmembrane adaptor proteins, linker for activation of T cells (LAT) and non-T cell activation linker (NTAL; also called LAB; a product of Wbscr5 gene). Previous studies showed that the secretory response was partially inhibited in bone marrow-derived mast cells (BMMCs) from LAT-deficient mice. To clarify the role of NTAL in mast cell degranulation, we compared FcepsilonRI-mediated signaling events in BMMCs from NTAL-deficient and wild-type mice. Although NTAL is structurally similar to LAT, antigen-mediated degranulation responses were unexpectedly increased in NTAL-deficient mast cells. The earliest event affected was enhanced tyrosine phosphorylation of LAT in antigen-activated cells. This was accompanied by enhanced tyrosine phosphorylation and enzymatic activity of phospholipase C gamma1 and phospholipase C gamma2, resulting in elevated levels of inositol 1,4,5-trisphosphate and free intracellular Ca2+. NTAL-deficient BMMCs also exhibited an enhanced activity of phosphatidylinositol 3-OH kinase and Src homology 2 domain-containing protein tyrosine phosphatase-2. Although both LAT and NTAL are considered to be localized in membrane rafts, immunogold electron microscopy on isolated membrane sheets demonstrated their independent clustering. The combined data show that NTAL is functionally and topographically different from LAT.
- MeSH
- adaptorové proteiny signální transdukční fyziologie MeSH
- adaptorové proteiny vezikulární transportní fyziologie MeSH
- degranulace buněk MeSH
- fosfatidylinositol-3-kinasy fyziologie MeSH
- fosfolipasa C gama MeSH
- fosfolipasy typu C metabolismus MeSH
- fosfoproteiny fyziologie MeSH
- fosforylace MeSH
- mastocyty fyziologie MeSH
- membránové proteiny fyziologie MeSH
- myši MeSH
- proteiny fyziologie MeSH
- receptory IgE fyziologie MeSH
- signální transdukce * MeSH
- tyrosin metabolismus MeSH
- vápník metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- adaptorové proteiny signální transdukční MeSH
- adaptorové proteiny vezikulární transportní MeSH
- fosfolipasa C gama MeSH
- fosfolipasy typu C MeSH
- fosfoproteiny MeSH
- LAB protein, mouse MeSH Prohlížeč
- Lat protein, mouse MeSH Prohlížeč
- LAT2 protein, mouse MeSH Prohlížeč
- membránové proteiny MeSH
- proteiny MeSH
- receptory IgE MeSH
- tyrosin MeSH
- vápník MeSH
Patients with secondary myelodysplasias and acute myeloid leukemias (MDS/AML) frequently exhibit interstitial deletions of the chromosome-5q resulting in hemizygous loss of the transcription transactivator Smad5. Smad5 is a member of the signal transducer family conveying the pleiotropic TGF-gb/BMP cytokine signals with roles in development, cell growth control, and tumor progression. Here we present a study of the Smad5 expression and its functional role in leukemia cell lines as well as in primary CD34+ progenitors of MDS/AML patients and healthy individuals. Consistent Smad5 gene expression in these cell types and the gradual increase in its mRNA and protein levels in a model of induced erythroid differentiation of murine erythroleukemia (MEL) cells suggest a role of the gene in hematopoiesis. We show that bone morphogenetic protein 4 (BMP4) directs Smad5 activation in human hematopoietic cells, as monitored at the levels of protein phosphorylation, nuclear translocation, and specific transcription response. In vitro induction of normal human CD34+ cells by BMP4 results in significantly increased proliferation of erythroid progenitors (BFU-E) and formation of glycophorin-A+ cells, whereas perturbation of Smad5 expression by antisense oligonucleotides causes significantly decreased rates of BMP4-induced erythroid differentiation. We have not detected any effects of Smad5 inhibition on BMP4-stimulated progenitors of the granulocyteNmacrophage lineage. We propose that the BMP4/Smad5 signal transduction pathway activates hematopoietic differentiation programs that may be impaired in anemia manifestations in MDS and AML patients with Smad5 haploinsufficiency.
- MeSH
- aktivní transport - buněčné jádro MeSH
- antigeny CD34 MeSH
- antisense oligonukleotidy farmakologie MeSH
- buněčná diferenciace MeSH
- DNA vazebné proteiny antagonisté a inhibitory genetika metabolismus fyziologie MeSH
- erytroidní prekurzorové buňky cytologie MeSH
- fosfoproteiny antagonisté a inhibitory genetika metabolismus fyziologie MeSH
- glykoforin biosyntéza MeSH
- hematopoetické kmenové buňky cytologie MeSH
- kostní morfogenetické proteiny fyziologie MeSH
- kostní morfogenetický protein 4 MeSH
- lidé MeSH
- myelodysplastické syndromy etiologie metabolismus patologie MeSH
- myeloidní leukemie etiologie metabolismus patologie MeSH
- myši MeSH
- nádorové buňky kultivované MeSH
- protein Smad5 MeSH
- regulace genové exprese MeSH
- sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
- signální transdukce MeSH
- studie případů a kontrol MeSH
- trans-aktivátory antagonisté a inhibitory genetika metabolismus fyziologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- antigeny CD34 MeSH
- antisense oligonukleotidy MeSH
- BMP4 protein, human MeSH Prohlížeč
- Bmp4 protein, mouse MeSH Prohlížeč
- DNA vazebné proteiny MeSH
- fosfoproteiny MeSH
- glykoforin MeSH
- kostní morfogenetické proteiny MeSH
- kostní morfogenetický protein 4 MeSH
- protein Smad5 MeSH
- SMAD5 protein, human MeSH Prohlížeč
- Smad5 protein, mouse MeSH Prohlížeč
- trans-aktivátory MeSH
Stimulation of human T cell line Jurkat results in rapid tyrosine phosphorylation of a 35-38 kDa protein that is found in large and buoyant detergent-resistant membrane microdomains containing also glycosylphosphatidylinositol (GPI)-anchored proteins, glycolipids and Src-family protein tyrosine kinases ("GPI-microdomains"). The pp35-38 was found to be identical to LAT, a recently cloned key component of the T-cell receptor signalling pathway. Moreover, a modified form of protein tyrosine kinase Lck (pp60) was newly detected in the GPI-microdomains of the anti-CD3-stimulated Jurkat cells. These data support the idea that GPI-microdomains play important roles in immunoreceptor signalling.
- MeSH
- adaptorové proteiny signální transdukční * MeSH
- detergenty farmakologie MeSH
- fosfoproteiny fyziologie MeSH
- fosforylace MeSH
- glykosylfosfatidylinositoly metabolismus MeSH
- imunoblotting MeSH
- intracelulární signální peptidy a proteiny MeSH
- Jurkat buňky MeSH
- kinetika MeSH
- lidé MeSH
- membránové proteiny * MeSH
- proteiny analýza MeSH
- receptory antigenů T-buněk fyziologie MeSH
- signální transdukce fyziologie MeSH
- skupina kinas odvozených od src-genu fyziologie MeSH
- transportní proteiny fyziologie MeSH
- tyrosin metabolismus MeSH
- tyrosinkinasa p56(lck), specifická pro lymfocyty fyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
- Názvy látek
- adaptorové proteiny signální transdukční * MeSH
- detergenty MeSH
- fosfoproteiny MeSH
- glykosylfosfatidylinositoly MeSH
- intracelulární signální peptidy a proteiny MeSH
- LAT protein, human MeSH Prohlížeč
- membránové proteiny * MeSH
- proteiny MeSH
- receptory antigenů T-buněk MeSH
- SH2B3 protein, human MeSH Prohlížeč
- skupina kinas odvozených od src-genu MeSH
- transportní proteiny MeSH
- tyrosin MeSH
- tyrosinkinasa p56(lck), specifická pro lymfocyty MeSH
