Glycobiology, 2001, Vol. 11, No. 4 335-343
© 2001 Oxford University Press
Engagement of endogenous ganglioside GM1a induces tyrosine phosphorylation involved in neuron-like differentiation of PC12 cells
Department of Biochemistry, University of Shizuoka Pharmaceutical Sciences, 52-1 Yada, Shizuoka-shi, Shizuoka 422-8526, Japan
Using the cholera toxin B subunit (CTB) that specifically binds to ganglioside GM1a on the plasma membrane, we investigated intracellular signaling mediated by endogenous GM1a involved in neuronal differentiation of PC12 cells. The treatment with CTB induced morphological alternations of PC12 cells, such as augmentation of the cell body, neurite extension, and branched spikes of tips of neurites. The neurite extension induced with CTB was strongly suppressed by the pretreatment of tyrosine kinase inhibitors in a dose-dependent manner. Western blotting analysis showed that CTB induced tyrosine phosphorylation of several cellular proteins with molecular masses around 120, 70, and 45–40 kDa in PC12 cells. Some of the proteins identified were extracellular-signal regulated kinase (ERKs) (ERK1 and ERK2). The peak activation of ERKs lasted for 60–90 min and gradually decreased thereafter.
Immunoprecipitation analysis demonstrated that the intracellular events induced with CTB are not related with the activation of Trk proteins, suggesting that signals evoked by ligation of endogenous GM1a are unique and distinct from those induced with exogenous GM1a.
Although the presence of a tyrosine kinase inhibitor, genistein, at a concentration of 10 µM diminished the neurite extension of PC12 cells induced with CTB, ERK activation was still observed. However, pretreatment with a MEK inhibitor, PD98059, abolished the activation of ERKs induced with CTB in a dose-dependent manner and only attenuated the morphological alternations of PC12 cells.
Considered together, we concluded that tyrosine phosphorylation induced with CTB was responsible for neuron-like differentiation of PC12 cells and that the MEK–ERK cascade is part of the biological signals mediated by endogenous ganglioside GM1a on PC12 cells.
1 These authors equally contributed to this work.
2 To whom correspondence should be addressed
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