Transient activation of Ras-dependent signalling at the early stages of Herbimycin A induced erythroid differentiation of human K562 cells.
OBJECTIVE
To study the dynamics of Ras-dependent signalling in the course of Herbimycin A induced erythroid differentiation of human erythroleukemia K562 cells.


METHODS
p21Ras functional activity was analyzed by direct measurement of GTP/GDP ratio in anti-p21Ras immunoprecipitates of K562 cells previously incubated with H3(32)PO4. Dynamics of protein tyrosine phosphorylation was studied using Western blotting. Electrophoretic mobility shift assay was used to monitor Erk2 activation. Phosphotyrosine (pY)-containing proteins bound to recombinant glutathione-S-tranferase (GST)-fused form of adaptor protein Grb2 were identified using GST in vitro binding assay.


RESULTS
It was shown that the relative quantity of GTP associated with Ras protein in non-induced cells varied from 27% to 37% upon 72 h of cell culturing. Herbimycin A caused 15% increase of GTP/GDP ratio at 3rd h. This index decreased during further investigated periods, although it did not reach control values even at 72nd h. Transient rise of Ras-GTP level at 3rd h of incubation in the presence of Herbimycin A correlated with the increase in tyrosine phosphorylation of proteins with apparent molecular weight of 210, 160, 140, 116 and 42 kDa, as well as with the activation of Erk2 and increase of binding of a set of pY-containing proteins with recombinant GST-fusion form of Ras activator, adaptor protein Grb2. Dramatic inhibition of interaction between docking protein Shc and GST-Grb2 was observed at late stages of cell induction (48-72 h) while binding of pY-containing proteins during this period did not differ significantly in control and differentiated cells.


CONCLUSIONS
The obtained results suggest that time-dependent changes in Grb2-mediated network of protein-protein interaction events might define implication of Ras-dependent signalling in Herbimycin A-induced erythroid differentiation of K562 cells.
Drobot
Lyudmyla
L
Institute of Cell Biology, NAS of Ukraine, Lviv 79005, Ukraine. drobot@biochem.lviv.ua
Husak
Zvenyslava
Z
Ilnytska
Olga
O
Igumentseva
Natalia
N
Oleksyn
Halyna
H
Kusen'
Stepan
S
D048868:Adaptor Proteins, Signal Transducing / Q000187:drug effects / Q000378:metabolism; D016227:Benzoquinones; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects; D045744:Cell Line, Tumor; D024202:Electrophoretic Mobility Shift Assay; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D041905:Erythroid Cells / Q000166:cytology* / Q000187:drug effects; D051380:GRB2 Adaptor Protein; D005982:Glutathione Transferase / Q000187:drug effects / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D047029:Lactams, Macrocyclic; D010766:Phosphorylation / Q000187:drug effects; D016283:Proto-Oncogene Proteins p21(ras) / Q000187:drug effects / Q000378:metabolism*; D011809:Quinones / Q000494:pharmacology*; D017828:Rifabutin / Q000031:analogs & derivatives; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D014443:Tyrosine / Q000187:drug effects / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D016227:Benzoquinones; D004791:Enzyme Inhibitors; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D047029:Lactams, Macrocyclic; D011809:Quinones; D006153:Guanosine Diphosphate; D017828:Rifabutin; D014443:Tyrosine; C020754:herbimycin; D006160:Guanosine Triphosphate; D005982:Glutathione Transferase; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)