- Paper Report
- Open Access
The effect of BRK on EGF signalling via erbB3 in mammary epithelial cells
- Jenny Gomm1
© Biomed Central Ltd 2001
- Published: 1 December 2001
- epithelial cells
- PI 3-kinase
BRK is an intracellular protein-tyrosine kinase that is expressed in 60% of human breast cancers but is undetectable in normal and benign breast tissues. The introduction of the BRK gene into the normal breast epithelial cell lines HB4a and MCF10-A leads to a potentiation of their growth response to the mitogen EGF and their ability to proliferate in an anchorage-independent manner. BRK and the EGF receptor (EGFR) have also been shown to form a constitutive association in mammary epithelial cells.
To investigate how BRK influences EGF downstream signalling in human mammary epithelial cells.
HB4a cells were transiently transfected with empty or brk expression vectors. Following overnight quiescence in 0.5% FCS, cells were treated with EGF (100 ng/ml) for 5 minutes. BRK or erbB3 was then immunoprecipitated from lysates of stimulated or mock-stimulated cells, resolved by SDS-PAGE, Western blotted and probed with antibodies to BRK, phosphotyrosine, erbB3, the p85 subunit of PI 3-kinase (PI 3-kinase p85) or Akt/protein kinase B (Akt). PI 3-kinase activity was assayed in erbB3 precipitates. BRK and erbB3 were also co-expressed in human embryonic kidney (HEK293) cells.
Mock stimulated BRK transfected cells showed an increase in phosphorylation of erbB3 above control cells together with the recruitment of the PI 3-kinase p85. Treatment of BRK-transfected HB4a cells with EGF led to enhanced tyrosine phosphorylation of both BRK and erbB3 and an increased association of PI 3-kinase p85. In HEK293 kidney cells overexpressing both erbB3 and BRK, immunoprecipitation of erbB3 demonstrated not only enhanced phosphorylation but also the formation of erbB3/BRK complexes. Mutant BRK, lacking kinase activity, failed to induce erbB3 phosphorylation but still co-precipitated with it. PI 3-kinase assays showed that there was an increase in enzyme activity associated with erbB3 in response to EGF in HB4a control cells. This increased twofold in BRK-expressing cells. A potential downstream target of PI 3-kinase, Akt, was also found to exhibit a low level of phosphorylation at serine 473 following EGF treatment of BRK-expressing cells. This is one of two sites whose phosphorylation is required for full activation of Akt.
This study shows that the expression of BRK in human mammary epithelial cells significantly enhanced the degree to which erbB3 became phosphorylated in response to EGF. Results using the expression of a kinase inactive BRK mutant suggests that BRK may directly phosphorylate erbB3, possibly by physical interaction. The recruitment of the p85 subunit of PI 3-kinase, its increased activity and the phosphorylation of Akt are further evidence of a role for BRK in EGF downstream signalling. Thus the expression of BRK in human breast cancer cells could lead to a greater efficiency of mitogens such as EGF.
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