No abstract

DNA-dependent protein kinase (DNA-PK), consisting of Ku and DNA-PKcs subunits, is the key component of the non-homologous end-joining (NHEJ) pathway of DNA double strand break (DSB) repair. Although the kinase activity of DNA-PKcs is essential for NHEJ, thus far, no in vivo substrate has been conclusively identified except for an autophosphorylation site on DNA-PKcs itself (threonine 2609). Here we report the ionizing radiation (IR)-induced autophosphorylation of DNA-PKcs at a novel site, serine 2056, the phosphorylation of which is required for the repair of DSBs by NHEJ. Interestingly, IR-induced DNA-PKcs autophosphorylation is regulated in a cell cycle-dependent manner with attenuated phosphorylation in the S phase. In contrast, DNA replication-associated DSBs resulted in DNA-PKcs autophosphorylation and localization to DNA damage sites. These results indicate that although IR-induced DNAPKcs phosphorylation is attenuated in the S phase, DNA-PKcs is preferentially activated by the physiologically relevant DNA replication-associated DSBs at the sites of DNA synthesis.Repair of DNA double strand breaks (DSBs) 1 is critical for the maintenance of genome integrity, cell survival, and prevention of tumorigenesis (1, 2). In higher eukaryotes, non-homologous end joining (NHEJ) and homologous recombination (HR) are the two major pathways for DSB repair (3). HR requires the presence of a sister chromatid and is operational in the late S and G 2 phases of the cell cycle because of the availability of an optimally positioned sister chromatid (4). NHEJ, on other hand, does not depend on the presence of homologous DNA sequences and is the predominant pathway for DSB repair in mammalian cells (5). It was proposed that NHEJ is preferentially used in G 1 and early S phases of the cell cycle (6, 7). However, a recent report indicating that NHEJ-deficient cell lines are radiation-sensitive in all phases of the cell cycle suggests that NHEJ is important throughout the cell cycle (8). Clearly, the exact contribution of NHEJ in different phases of the cell cycle needs to be defined further.The NHEJ pathway of DSB repair requires both the DNAdependent protein kinase (DNA-PK) complex and the XRCC4/ DNA ligase IV complex, as well as possible additional accessory factors (5, 9, 10). DNA-PK, the key component of the NHEJ pathway, is composed of the Ku70/80 heterodimer and the catalytic subunit DNA-PKcs (11). Ku binds to DNA ends with very high affinity and is believed to function as the DNAbinding and regulatory subunit that recruits DNA-PKcs to breaks and stimulates its kinase activity (12, 13). DNA-PKcs is a member of the phosphatidylinositol-3-like kinase family that includes ATM (ataxia-telangiectasia mutated) and 15). Although the biochemical properties of DNA-PK have been extensively studied in vitro, it is still not clear how it functions in vivo in the context of NHEJ. Wild type DNA-PKcs, but not a kinase-dead mutant, is able to rescue the radiation sensitivity and DSB repair defect of DNA-PKcs-defective V3 cells demonstrati...

SUMMARY In vitro, topographical and biophysical cues arising from the extracellular matrix (ECM) direct skeletal stem cell (SSC) commitment and differentiation. However, the mechanisms by which the SSC-ECM interface is regulated and the outcome of such interactions on stem cell fate in vivo remain unknown. Here we demonstrate that conditional deletion of the membrane-anchored metalloproteinase MT1-MMP (Mmp14) in mesenchymal progenitors, but not in committed osteoblasts, redirects SSC fate decisions from osteogenesis to adipo- and chondrogenesis. By effecting ECM remodeling, MT1-MMP regulates stem cell shape, thereby activating a β1-integrin/RhoGTPase signaling cascade and triggering the nuclear localization of the transcriptional coactivators YAP and TAZ, which serve to control SSC lineage commitment. These data identify a critical MT1-MMP/integrin/YAP/TAZ axis operative in the stem cell niche that oversees SSC fate determination.

SUMMARY Notch signaling induced by cell surface ligands is critical to development and maintenance of many eukaryotic organisms. Notch and its ligands are integral membrane proteins that facilitate direct cell-cell interactions to activate Notch proteolysis and release the intracellular domain that directs Notch-specific cellular responses. Genetic studies suggest Notch ligands require endocytosis, ubiquitylation and epsin endocytic adaptors to activate signaling, yet the exact role ligand endocytosis serves remains unresolved. Here we characterize a molecularly distinct mode of clathrin-mediated endocytosis requiring ligand ubiquitylation, epsins and actin for ligand cells to activate signaling in Notch cells. Using a cell-bead optical tweezers system, we obtained evidence for cell-mediated mechanical force dependent on this distinct mode of ligand endocytosis. We propose mechanical pulling force produced by endocytosis of Notch-bound ligand drives conformational changes in Notch that permit activating proteolysis.