The effect of BRK on EGF signalling via erbB3 in mammary epithelial cells

Introduction

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.

Aims

To investigate how BRK influences EGF downstream signalling in human mammary epithelial cells.

Comments

The ability of breast receptor kinase (BRK) to deregulate cellular proliferation together with its expression in 60% of breast cancers and the documented importance of protein-tyrosine kinases in tumorigenesis makes this an important study. BRK is shown to provide a link between epidermal growth factor (EGF) binding to its receptor and the phosphorylation of erbB3 which leads to an increase in cell number, either by increased proliferation or decreased apoptosis. As erbB2 is thought to mediate in the activation of erbB3 and, as both of these receptors have been shown to be overexpressed in a proportion of breast cancers, further studies will be needed to determine the role of erbB2 in this BRK/erbB3 interaction. Also, does neu differentiation factor/heregulin, which is known to bind directly to erbB3 leading to both the recruitment of phosphoinositide (PI) 3-kinase's p85 subunit and increased mitogenesis, also interact with BRK?

Methods

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.

Results

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.

Discussion

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.