We have, for the first time, ordered a pathway from agonist stimulation to integrin activation and established the Rap1-induced formation of an "integrin activation complex," containing RIAM and talin, that binds to and activates the integrin.

In vitro analysis confirms talin binding is sufficient for activation and extension of membrane-embedded integrin.

No abstract

Cell invasion requires cooperation between adhesion receptors and matrix metalloproteinases (MMPs). Remodeling of the extracellular matrix (ECM)1 is critical for cancer cell invasion and tumorigenesis (1-5). Membrane type matrix metalloproteinases (MT-MMPs) localized to the invasive front of highly motile cancer cells (6, 7) were shown to be directly involved in matrix breakdown (8 -13). A cooperation involving MT-MMPs and cell adhesion receptors is likely to be essential to migrating cells (3, 14 -16). So far, six members of the MT-MMP subfamily have been identified and partially characterized (2, 17-22). MT1-, MT2-, and MT3-MMP strongly contribute to tumor cell invasion (12). Recent studies demonstrated a functional significance and a direct role of MT1-, MT2-, and MT3-MMP in cell locomotion on laminin-5 (11) and three-dimensional collagen type I lattice (8, 9, 12). In addition, MT-MMPs contribute indirectly to cell invasion by activating soluble secretory MMP-2 (23) and MMP-13 (24), which further cleave multiple matrix substrates (2, 5, 25-29).Integrin adhesion receptors dynamically regulate cell-matrix interactions by the binding to matrix proteins and inside-out signaling (30,31). This allows cells to discriminate any subtle alteration of the environment and to adjust cell locomotion accordingly. Direct interactions with multiple transmembrane and cell surface proteins (32) including integrin-associated protein-50 (33), TM4SF proteins (tetraspanins) (34) and tTG (35) further attenuate adhesive and signaling efficiency of integrins.Cell surface tTG (protein-glutamine ␥-glutamyltransferase, EC 2.3.2.13) promotes integrin-dependent adhesion and spreading of cells. By both direct associations with multiple ␤ 1 and ␤ 3 integrins and the binding with Fn, tTG independently mediates the interactions of integrins with Fn (35). The high affinity binding of tTG with Fn specifically involves the 42 kDa gelatin-binding domain of the Fn molecule, which consists of modules I 6 II 1,2 I 7-9 (36). The enhancement of integrin-mediated adhesion and spreading of cells on Fn is independent from the enzymatic activity of surface tTG (35). Intriguingly, reduced expression of tTG has been linked to aggressiveness and high metastatic potential of tumors, whereas overexpression of tTG in fibrosarcomas inhibited primary tumor growth (37, 38). Proteolysis of tTG at the normal tissue/tumor boundary was observed in invasive tumors (38).Here, we report that depending on the structure of the ECM, MT-MMPs are capable of both positively and negatively regulating locomotion of cancer cells. Matrix-dependent proteolysis of surface tTG by MT1-MMP occurs on tumor cells of a diverse tissue origin, thereby representing a general phenomenon and a novel MT-MMP function. Our data suggest an existence of an unexpected link between tumor cell locomotion, the ECM and membrane-anchored MMPs. Regulatory proteolysis of cell surface adhesion proteins by the adjacent MT-MMP molecules is likely to play a significant functional role in cancer cell invasion.

Summary Many tumor cells are fueled by altered metabolism and increased glutamine (Gln) dependence. We identify regulation of the L-glutamine carrier proteins SLC1A5 and SLC38A2 (SLC1A5/38A2) by the ubiquitin ligase RNF5. Paclitaxel-induced ER stress to breast cancer (BCa) cells promotes RNF5 association, ubiquitination and degradation of SLC1A5/38A2. This decreases Gln uptake, levels of TCA cycle components, mTOR signaling and proliferation while increasing autophagy and cell death. Rnf5-deficient MMTV-PyMT mammary tumors were less differentiated and showed elevated SLC1A5 expression. Whereas RNF5 depletion in MDA-MB-231 cells promoted tumorigenesis and abolished paclitaxel responsiveness, SLC1A5/38A2 knockdown elicited opposing effects. Inverse RNF5HI/SLC1A5/38A2LO expression was associated with positive prognosis in BCa. Thus, RNF5 control of Gln uptake underlies BCa response to chemotherapies.