Casitas B-cell lymphoma (Cbl) proteins protect mammary epithelial cells from proteotoxicity of active c-Src accumulation

Significance

Casitas B-cell lymphoma (Cbl) family proteins are RING finger-containing E3 ubiquitin ligases involved in degradation of activated tyrosine kinases. Previous studies in Cbl-deficient models focused primarily on the consequences of persistent tyrosine kinase signaling resulting in uncontrolled cell activation and proliferation. In the present study, we provide evidence that, in the complete absence of Cbl family proteins, failure to turn over active tyrosine kinases induces irreparable breakdown of the homeostasis of the protein milieu in primary mouse mammary epithelial cells and triggers stress-mediated cell death. Thus, our data reveal that well-regulated removal of active tyrosine kinases is essential for cell survival, an aspect of Cbl family protein functions that has not been previously fully appreciated.

Abstract

Casitas B-cell lymphoma (Cbl) family ubiquitin ligases negatively regulate tyrosine kinase-dependent signal transduction by promoting degradation of active kinases. We and others previously reported that loss of Cbl functions caused hyperproliferation in lymphoid and hematopoietic systems. Unexpectedly, Cbl deletion in Cbl-b–null, Cbl-c–null primary mouse mammary epithelial cells (MECs) (Cbl triple-deficiency) induced rapid cell death despite enhanced MAP kinase and AKT activation. Acute Cbl triple-deficiency elicited distinct transcriptional and biochemical responses with partial overlap with previously described cellular reactions to unfolded proteins and oxidative stress. Although the levels of reactive oxygen species were comparable, detergent-insoluble protein aggregates containing phosphorylated c-Src accumulated in Cbl triple-deficient MECs. Treatment with a broad-spectrum kinase inhibitor dasatinib blocked protein aggregate accumulation and restored in vitro organoid formation. This effect is most likely mediated through c-Src because Cbl triple-deficient MECs were able to form organoids upon shRNA-mediated c-Src knockdown. Taking these data together, the present study demonstrates that Cbl family proteins are required to protect MECs from proteotoxic stress-induced cell death by promoting turnover of active c-Src.