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Cancer Cell death Cell cycle Cytoskeleton Exo/endocytosis Differentiation Division Organelles Signalling Stem cells Trafficking
Cell Biology International (2007) 31, 1–10 (Printed in Great Britain)
Proteomic approach to substrates related to MAPK pathway in 293T cells
Guolin Zhang and Sanying Wang*
Center for Proteomics and Department of Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, PR China


Abstract

Abundant evidence indicates that potential scaffold proteins and adaptor or linker molecules organize and specify various MAP kinase cascades. In the present study, proteomic methodologies were applied to screen these potential molecules in combination with cell morphology and cell cycle analysis. MEK1E, MKK3b, MKK5D and MKK7D were selected as representative MKKs of four main MAPK pathways. Our results showed that similar morphological transformation and G2/M cell cycle arrest were promoted by the over-expressed four kinases. Furthermore, global change in response to the over-expressed four kinases was characterized by differential proteomics. Eleven distinctly changed proteins were detected, in which four proteins (serine/threonine kinase 4, glutathione S-transferase p1-1, glycoprotein IX and soluble inorganic pyrophosphatase) were reported to be relative to MAPK pathways, while the other seven proteins may be new elements of substrates of the kinases. In our experiment, the expression of platelet glycoprotein IX precursor, glutathione S-transferase p1-1, peroxiredoxin 6, Ras-related protein Rab-34 and arginase II, mitochondrial precursor was up-regulated, while the expression of serine/threonine kinase 4 (MST1) was down-regulated by the four kinases. These results suggest that these six proteins may be common targets of all the MAPK pathways in 293T cell line. Interestingly, the expression of splicing factor 3B subunit 4 and soluble inorganic pyrophosphatase (Ppase) was specifically up-regulated by MEK1E and MKK5D, and by MEK1E, MKK3b and MKK5D, respectively. The expression of methylglyoxalase was down-regulated by MEK1E and MKK7D. Furthermore, the expression of ADP-ribosylation factor-like protein 1 was up-regulated by MKK5D but down-regulated by MKK3b and MKK7D. These findings revealed the characteristic molecular responses to four MKKs. In conclusion, our study not only confirms that MST1, glutathione S-transferase p1-1, glycoprotein IX and soluble PPase belong to MAPK pathways, but also provides seven novel molecules for the further study of the pathways.


Key words: MAPK kinase, Differential proteomics, Morphological transformation, G2/M-phase arrest.

*Corresponding author. Tel.: +86 592 218 1140; fax: +86 592 218 1015.


Received 12 June 2006/8 July 2006; accepted 7 August 2006

doi:10.1016/j.cellbi.2006.08.004


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)