Institute of Cell Biology, NAS of Ukraine, Lviv 79005, Ukraine
Abstract. Aim: 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 H332PO4.
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. Conclusion: 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.
Key Words:Ê562, differentiation, Herbimycin A, ð210Bcr-Abl, ð21Ras, Grb2, Shc.
