SUMMARY The tuberous sclerosis complex (TSC) tumor suppressors form the TSC1-TSC2 complex, which limits cell growth in response to poor growth conditions. Through its GTPase-activating protein (GAP) activity toward Rheb, this complex inhibits the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1), a key promoter of cell growth. Here, we identify and biochemically characterize TBC1D7 as a stably-associated and ubiquitous third core subunit of the TSC1-TSC2 complex. We demonstrate that the TSC1-TSC2-TBC1D7 (TSC-TBC) complex is the functional complex that senses specific cellular growth conditions and possesses Rheb-GAP activity. Sequencing analyses of samples from TSC patients suggest that TBC1D7 is unlikely to represent TSC3. TBC1D7 knockdown decreases the association of TSC1 and TSC2 leading to decreased Rheb-GAP activity, without effects on the localization of TSC2 to the lysosome. Like the other TSC-TBC components, TBC1D7 knockdown results in increased mTORC1 signaling, delayed induction of autophagy, and enhanced cell growth under poor growth conditions.
HER2/HER3 dimerization resulting from overexpression of HER2 or neuregulin (NRG1) in cancer leads to HER3-mediated oncogenic activation of PI3K signaling. Although ligand-blocking HER3 antibodies inhibit NRG1-driven tumor growth, they are ineffective against HER2-drive tumor growth because HER2 activates HER3 in a ligand-independent manner. In this study, we describe a novel HER3 monoclonal antibody (LJM716) that can neutralize multiple modes of HER3 activation, making it a superior candidate for clinical translation as a therapeutic candidate. LJM716 was a potent inhibitor of HER3/AKT phosphorylation and proliferation in HER2-amplified and NRG1-expressing cancer cells and it displayed single agent efficacy in tumor xenograft models. Combining LJM716 with agents that target HER2 or EGFR produced synergistic antitumor activity in vitro and in vivo. In particular, combining LJM716 with trastuzumab produced a more potent inhibition of signaling and cell proliferation than trastuzumab/pertuzumab combinations and was similarly active in vivo. To elucidate its mechanism of action, we solved the structure of LJM716 bound to HER3, finding that LJM716 bound to an epitope within domains 2 and 4 that traps HER3 in an inactive conformation. Taken together, our findings establish that LJM716 possesses a novel mechanism of action that in combination with HER2 or EGFR-targeted agents may leverage their clinical efficacy in ErbB-driven cancers.
Mast cell degranulation is induced by multivalent allergens which cross-link immunoglobulin E (IgE) molecules that are bound to high-affinity IgE receptors (FcεR1) at the cell surface (3). Receptor aggregation leads subsequently to the activation of phosphatidylinositol 3-kinase (PI3K), generating phosphatidylinositol-3,4,5-trisphosphate (PIP3) at the plasma membrane. PIP3 then attracts various intracellular proteins with pleckstrin homology (PH) domains that play critical roles in triggering degranulation. These include phospholipase C-␥ (PLC-␥) (12) and the tyrosine kinase Btk (44). PLC-␥ hydrolyzes PI-4,5-biophosphate, thereby generating diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (the latter triggering the release of intracellular calcium from the endoplasmic reticulum). The tyrosine kinase Btk (44) phosphorylates and activates PLC-␥, thereby sustaining calcium release from the endoplasmic reticulum and promoting the influx of extracellular calcium through I CRAC channels in the plasma membrane (45). The Src homology 2 (SH2) domaincontaining inositol-5Ј-phosphatase (SHIP) acts as a gatekeeper of antigen-induced degranulation by hydrolyzing PIP3 (18), and there is considerable interest in identifying molecules that interact with SHIP in the hope that some of these molecules might modulate its activity and, in turn, regulate mast cell degranulation. , that become phosphorylated and targets of SH2-containing proteins upon cell stimulation; and a C-terminal SH2 domain. SHIP appears to interact with Shc via different intermolecular mechanisms, depending on the cell type involved. In myeloid cells, for example, SHIP appears to interact with Shc in a bidentate manner, with SHIP's SH2 domain binding to one of the three phosphorylated tyrosines within Shc's CH domain while SHIP's two phosphorylated NPXY motifs bind to Shc's PTB domain (34). In B lymphocytes, on the other hand, the adapter protein Grb2 appears to be required for an efficient association between Shc and SHIP, and a ternary complex of SHIP, Shc, and Grb2 is formed (16). Furthermore, studies with T cells suggest that Shc interacts solely via its PTB domain with one of SHIP's two phosphorylated NPXY motifs (29). Common to all three models, however, the SH2 domain of Shc is not involved in the Shc-SHIP interaction and therefore might be available to recruit one or more regulatory proteins into the Shc-SHIP complex.To date, very few proteins have been shown to bind to the SH2 domain of Shc. These include the signal-transducing subunits of the B-cell receptor (2) and the -chain of FcεR1 (23), following receptor activation. Interestingly, unlike the B-cell receptor system, where both signaling components, Ig-␣ and Ig-, interact with Shc, only the -subunit of FcεR1 appears to associate with this adapter protein. In addition, the Shc SH2 domain has been shown to bind to the adapter protein Gab2 (4) and mPAL, a protein whose expression is restricted to tissues containing actively dividing cells (47).
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