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Expression of the Notch ligand JAG1 and Notch pathway activation promote poor prognosis, basal-like breast cancer. We have recently shown that the pseudokinase Tribbles homolog 3 (TRB3) regulates JAG1 expression in this malignancy. TRB3 is a stress and metabolic sensor, and here we show that nutrient deprivation or endoplasmic reticulum stress markedly upregulate TRB3, which serves as a scaffold for the deubiquitinase USP9x. USP9x in turn stimulates JAG1 activity through two mechanisms: (1) through TRB3 deubiquitination and stabilization, and (2) through deubiquitination and activation of Mind Bomb 1, an E3 ligase required for JAG1 ubiquitination-mediated endocytosis and Notch activation. These USP9x activities are confined to the signal-sending cell of a cell pair undergoing Notch signaling. We demonstrate that USP9x is required for TRB3 upregulation and Notch activation in response to cellular stress in basal-like breast cancer cells. These data suggest that TRB3 functions as a sensor of tumor microenvironmental stress and together with USP9x induces the cell survival and tumor-promoting activities of Notch. These findings identify a novel mechanism by which cancer cells survive in their hostile environment and provide potential therapeutic targets in breast cancer.

CaLB was originally observed as a conserved sequence motif in various calcium-responsive signalling proteins and also in p120 Ras GTPase activating protein (p120GAP) (Clark et al. Cell 65: 1043-1051, 1991). Here we show the 43 residue CaLB motif in p120GAP is a functional protein domain that when expressed as a fusion protein in vitro confers Ca(2+)-dependent interactions with cellular membranes and phosphatidylserine and phosphatidylinositol vesicles. p120GAP, but not a mutant lacking the CaLB domain, associates with the particulate fraction of cells in response to elevated intracellular Ca2+ suggesting that p120GAP may be regulated in part by calcium signals. Addition of the p120GAP CaLB domain was able to restore transforming activity and particulate localization to an otherwise transformation-defective and cytosolic mutant v-Sre tyrosine kinase. The CaLB domain appears to be a prevalent protein module that may affect the molecular interactions and subcellular localization of signalling proteins.

Efficient binding of active phosphatidylinositol (PI) 3'-kinase to the autophosphorylated macrophage colony stimulating factor receptor (CSF-1R) requires the noncatalytic kinase insert (KI) region of the receptor. To test whether this region could function independently to bind PI 3'-kinase, the isolated CSF-1R KI was expressed in Escherichia coli, and was inducibly phosphorylated on tyrosine. The tyrosine phosphorylated form of the CSF-1R KI bound PI 3'-kinase in vitro, whereas the unphosphorylated form had no binding activity. The p85 alpha subunit of PI 3'-kinase contains two Src homology (SH)2 domains, which are implicated in the interactions of signalling proteins with activated receptors. Bacterially expressed p85 alpha SH2 domains complexed in vitro with the tyrosine phosphorylated CSF-1R KI. Binding of the CSF-1R KI to PI 3'-kinase activity, and to the p85 alpha SH2 domains, required phosphorylation of Tyr721 within the KI domain, but was independent of phosphorylation at Tyr697 and Tyr706. Tyr721 was also critical for the association of activated CSF-1R with PI 3'-kinase in mammalian cells. Complex formation between the CSF-1R and PI 3'-kinase can therefore be reconstructed in vitro in a specific interaction involving the phosphorylated receptor KI and the SH2 domains of p85 alpha.

The receptors for colony stimulating factor-1 (CSF-1), platelet derived growth factor and the c-kit protein tyrosine kinase (PTK) contain within their catalytic domains a stretch of 60-100 residues, largely unrelated in sequence, with no counterpart in other PTKs. Of the 64 amino acids within this kinase insert, 58 were deleted from the mouse CSF-1 receptor by oligonucleotide-directed mutagenesis. The mutant CSF-1 receptor was not markedly affected in its kinase activity, post-translational processing or its ability to induce autocrine transformation of NIH 3T3 mouse fibroblasts. Similarly, retention of kinase and transforming activities were observed following deletion of part or all of the kinase insert from the v-fms oncoprotein. The c- and v-fms kinase inserts were probed using monoclonal and polyclonal antibodies and were found to be highly antigenic. Two monoclonal antibodies raised to the v-fms cytoplasmic domain both recognized epitopes within the insert, and bound enzymatically active v-fms glycoproteins. These results indicate that the fms kinase insert is located on the surface of the protein and folds separately from the rest of the catalytic domain, but is not required for the biological activity of fms PTKs ectopically expressed in mouse fibroblasts. The insert may therefore play a specific function in cells such as monocytes and trophoblasts that normally express the CSF-1 receptor.