INHIBITORS OF MAP-KINASE PATHWAY U0126 AND PD98059
DIFFERENTLY AFFECT ORGANIZATION OF TUBULIN CYTOSKELETON
AFTER STIMULATION OF EGF RECEPTOR ENDOCYTOSIS
M.V. Zlobina,1 Yu.Yu. Steblyanko,1,2 M.A. Shklyaeva,1,2 V.V. Kharchenko,1
A.V. Salova,1 E.S. Kornilova 1,2,*
1 Institute of Cytology RAS, St. Petersburg,
and 2 Cytology and Histology Department of St. Petersburg State University;
* e-mail: elena.kornilova@gmail.com
To confirm the hypothesis about the involvement of EGF-stimulated MAP-kinase ERK1/2 in the regulation
of microtubule (MT) system, the influence of two widely used ERK1/2 inhibitors, U0126 and PD98059, on
the organization of tubulin cytoskeleton in interphase HeLa cells during EGF receptor endocytosis has been investigated.
We have found that addition of U0126 or PD98059 to not-stimulated with EGF ells for 30 min has
no effect on radially organized MT system. However, in the case of U0126 addition before EGF endocytosis
stimulation, the number of MT per cell decreased within 15 min after such stimulation and was followed by
complete MT depolymerization by 60—90 min. Stimulation of EGF endocytosis in the presence of PD98059
resulted only in insignificant depolymerization of MT and it could be detected mainly from their minus-ends.
At the same time, MT regions close to plasma membrane became stabilized, which was proved by increase in
tubulin acetylation level. This situation was characteristic for all period of the experiment. It has been also found
that the inhibitors affect endocytosis dynamics of EGF-receptor complexes. Quantitative analysis demonstrated
that the stimulation of endocytosis in the presence of U0126 generated a greater number of endosomes
compared to control cells, and their number did not change significantly during the experiment. All these endosomes
were localized peripherally. Effect of PD98059 resulted in the formation of lower number of endosomes
that in control, but they demonstrated very slow clusterization despite the presence of some intact MT. Both inhibitors
decreased EGFR colocolization with early endosomal marker EEA1, which indicated a delay in endosome
fusions and maturation. The inhibitors were also shown to affect differently phospho-ERK 1 and 2 forms:
U0126 completely inhibited phospho-ERK1 and 2, white, in the presence of PD98059, the two ERK forms demonstrated
sharp transient activation in 15 min after stimulation, but only phospho-ERK2 could be detected after
60 min of endocytosis. In both cases, MAP-kinase activation dynamics was significantly different from the
control. Our results suggest involvement of EGF-stimulated MAP-kinase pathway in cytoskeleton regulation.
At the same time, they demonstrate that the two studied and widely used inhibitors are not equivalent with respect
to not only the effect on MAP-kinase activity but also to such interdependent processes such as changes in
cytoskeleton organization and signaling receptor’ endocytosis.
Key words:
microtubules, endocytosis, endosomes, EGF receptor, ERK1/2, U0126, PD98059, HeLa cell line
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