Abstract
BackgroundPrevious studies by our group and others have shown that C3G interacts with Bcr-Abl through its SH3-b domain.ResultsIn this work we show that C3G and Bcr-Abl form complexes with the focal adhesion (FA) proteins CrkL, p130Cas, Cbl and Abi1 through SH3/SH3-b interactions. The association between C3G and Bcr-Abl decreased upon Abi1 or p130Cas knock-down in K562 cells, which suggests that Abi1 and p130Cas are essential partners in this interaction. On the other hand, C3G, Abi1 or Cbl knock-down impaired adhesion to fibronectin, while p130Cas silencing enhanced it. C3G, Cbl and p130Cas-SH3-b domains interact directly with common proteins involved in the regulation of cell adhesion and migration. Immunoprecipitation and immunofluorescence studies revealed that C3G form complexes with the FA proteins paxillin and FAK and their phosphorylated forms. Additionally, C3G, Abi1, Cbl and p130Cas regulate the expression and phosphorylation of paxillin and FAK. p38α MAPK also participates in the regulation of adhesion in chronic myeloid leukemia cells. It interacts with C3G, CrkL, FAK and paxillin and regulates the expression of paxillin, CrkL and α5 integrin, as well as paxillin phosphorylation. Moreover, double knock-down of C3G/p38α decreased adhesion to fibronectin, similarly to the single silencing of one of these genes, either C3G or p38α. These suggest that C3G and p38α MAPK are acting through a common pathway to regulate cell adhesion in K562 cells, as previously described for the regulation of apoptosis.ConclusionsOur results indicate that C3G-p38αMAPK pathway regulates K562 cell adhesion through the interaction with FA proteins and Bcr-Abl, modulating the formation of different protein complexes at FA.
Highlights
Previous studies by our group and others have shown that C3G interacts with Bcr-Abl through its SH3-b domain
The Bcr-Abl SH3-domain interacts with C3G, Abi1, Cbl and p130Cas Previous studies by our group and others have demonstrated the existence of functional interactions between, either C3G and/or its isoform p87C3G, and Bcr-Abl in K562 chronic myeloid leukemia (CML) cells [8,10]
C3G, Cbl, Abi1 and p38α MAPK knock-down expression inhibits K562 adhesion to fibronectin, while p130Cas silencing increases it We studied the ability of K562 clones with silenced expression of C3G, p38α MAPK, Cbl, Abi1 or p130Cas to adhere to fibronectin, which is the optimal adhesion substrate for K562 cells (Additional file 10), as compared to laminin or collagen IV
Summary
Previous studies by our group and others have shown that C3G interacts with Bcr-Abl through its SH3-b domain. C3G is a 140 kDa protein, build up with several modular domains clearly differentiated, both structurally and functionally. These comprise a REM-CDC25-H domain, which contains the catalytic or “GEF” domain, and a large proline-rich domain or SH3-binding (SH3-b) domain that interacts directly with Crk isoforms and other SH3-containing proteins such as p130Cas, Hck and c-Abl [1,2,3]. C3G-mediated Rap activation plays critical roles in adhesion. C3G-Rap pathway is essential during early mouse embryogenesis, due to its role in integrin- and paxillinmediated cellular adhesion and spreading [4]. C3G has been implicated in Rap1-dependent adhesion in many hematopoietic-cell types [6,7]
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