Ribosome stalling is a signal for metabolic regulation by the ribotoxic stress response.

Impairment of translation can lead to collisions of ribosomes, which constitute an activation platform for several ribosomal stress-surveillance pathways. Among these is the ribotoxic stress response (RSR), where ribosomal sensing by the MAP3K ZAKα leads to activation of p38 and JNK kinases. Despite these insights, the physiological ramifications of ribosomal impairment and downstream RSR signaling remain elusive. Here, we show that stalling of ribosomes is sufficient to activate ZAKα. In response to amino acid deprivation and full nutrient starvation, RSR impacts on the ensuing metabolic responses in cells, nematodes, and mice. The RSR-regulated responses in these model systems include regulation of AMPK and mTOR signaling, survival under starvation conditions, stress hormone production, and regulation of blood sugar control. In addition, ZAK-/- male mice present a lean phenotype. Our work highlights impaired ribosomes as metabolic signals and demonstrates a role for RSR signaling in metabolic regulation.

ZAKß is activated by cellular compression and mediates contraction-induced MAP kinase signaling in skeletal muscle.

Mechanical inputs give rise to p38 and JNK activation, which mediate adaptive physiological responses in various tissues. In skeletal muscle, contraction-induced p38 and JNK signaling ensure adaptation to exercise, muscle repair, and hypertrophy. However, the mechanisms by which muscle fibers sense mechanical load to activate this signaling have remained elusive. Here, we show that the upstream MAP3K ZAKß is activated by cellular compression induced by osmotic shock and cyclic compression in vitro, and muscle contraction in vivo. This function relies on ZAKß's ability to recognize stress fibers in cells and Z-discs in muscle fibers when mechanically perturbed. Consequently, ZAK-deficient mice present with skeletal muscle defects characterized by fibers with centralized nuclei and progressive adaptation towards a slower myosin profile. Our results highlight how cells in general respond to mechanical compressive load and how mechanical forces generated during muscle contraction are translated into MAP kinase signaling.

Regulation of the Golgi Apparatus by p38 and JNK Kinases during Cellular Stress Responses.

p38 and c-Jun N-terninal kinase (JNK) are activated in response to acute stress and inflammatory signals. Through modification of a plethora of substrates, these kinases profoundly re-shape cellular physiology for the optimal response to a harmful environment and/or an inflammatory state. Here, we utilized phospho-proteomics to identify several hundred substrates for both kinases. Our results indicate that the scale of signaling from p38 and JNK are of a similar magnitude. Among the many new targets, we highlight the regulation of the transcriptional regulators grb10-interacting GYF protein 1 and 2 (GIGYF1/2) by p38-dependent MAP kinase-activated protein kinase 2 (MK2) phosphorylation and 14-3-3 binding. We also show that the Golgi apparatus contains numerous substrates, and is a major target for regulation by p38 and JNK. When activated, these kinases mediate structural rearrangement of the Golgi apparatus, which positively affects protein flux through the secretory system. Our work expands on our knowledge about p38 and JNK signaling with important biological ramifications.

ZAKα Recognizes Stalled Ribosomes through Partially Redundant Sensor Domains.

Impairment of ribosome function activates the MAPKKK ZAK, leading to activation of mitogen-activated protein (MAP) kinases p38 and JNK and inflammatory signaling. The mechanistic basis for activation of this ribotoxic stress response (RSR) remains completely obscure. We show that the long isoform of ZAK (ZAKα) directly associates with ribosomes by inserting its flexible C terminus into the ribosomal intersubunit space. Here, ZAKα binds helix 14 of 18S ribosomal RNA (rRNA). An adjacent domain in ZAKα also probes the ribosome, and together, these sensor domains are critically required for RSR activation after inhibition of both the E-site, the peptidyl transferase center (PTC), and ribotoxin action. Finally, we show that ablation of the RSR response leads to organismal phenotypes and decreased lifespan in the nematode Caenorhabditis elegans (C. elegans). Our findings yield mechanistic insight into how cells detect ribotoxic stress and provide experimental in vivo evidence for its physiological importance.

Metallopeptidase inhibitor 1 (TIMP-1) promotes receptor tyrosine kinase c-Kit signaling in colorectal cancer.

Colorectal cancer (CRC) is the third most prevalent cancer worldwide causing an estimated 700 000 deaths annually. Different types of treatment are available for patients with advanced metastatic colorectal cancer, including targeted biological agents, such as cetuximab, a monoclonal antibody that targets EGFR. We have previously reported a study indicating multiple levels of interaction between metallopeptidase inhibitor 1 (TIMP-1) and the epidermal growth factor (EGF) signaling axis, which could explain how TIMP-1 levels can affect the antitumor effects of EGFR inhibitors. We also reported an association between TIMP-1-mediated cell invasive behavior and KRAS status. To gain insight into the molecular mechanisms underlying the effects of TIMP-1 in CRC, we examined by transcriptomics, proteomics, and kinase activity profiling a matched pair of isogenic human CRC isogenic DLD-1 CRC cell clones, bearing either an hemizygous KRAS wild-type allele or KRAS G13D mutant allele, exposed, or not, to TIMP-1. Omics analysis of the two cell lines identified the receptor tyrosine kinase c-Kit, a proto-oncogene that can modulate cell proliferation and invasion in CRC, as a target for TIMP-1. We found that exposure of DLD-1 CRC cells to exogenously added TIMP-1 promoted phosphorylation of c-Kit, indicative of a stimulatory effect of TIMP-1 on the c-Kit signaling axis. In addition, TIMP-1 inhibited c-Kit shedding in CRC cells grown in the presence of exogenous TIMP-1. Given the regulatory roles that c-Kit plays in cell proliferation and migration, and the realization that c-Kit is an important oncogene in CRC, it is likely that some of the biological effects of TIMP-1 overexpression in CRC may be exerted through its effect on c-Kit signaling.

Osmotic Stress Blocks Mobility and Dynamic Regulation of Centriolar Satellites.

Centriolar satellites (CS) are small proteinaceous granules that cluster around the centrosome and serve as cargo vehicles for centrosomal proteins. It is generally accepted that CS support a number of canonical and specialized centrosome functions. Consequently, these highly dynamic structures are the target of regulation by several cellular signalling pathways. Two decades of research have led to the identification of a large number of molecular components and new biological roles of CS. Here, we summarize the latest advances in the continuous efforts to uncover the compositional, functional, dynamic and regulatory aspects of CS. We also report on our discovery that osmotic stress conditions render CS immobile and insensitive to remodelling. Upon a range of p38-activating stimuli, MK2 phosphorylates the CS component CEP131, resulting in 14-3-3 binding and a block to CS formation. This normally manifests as a rapid cellular depletion of satellites. In the case of osmotic stress, a potent inducer of p38 activity, CS translocation and dissolution is blocked, with the net result that satellites persist in an immobile state directly adjacent to the centrosome. Our results highlight a unique scenario where p38 activation and CS depletion is uncoupled, with potential implications for physiological and pathological osmotic stress responses.

TIMP-1 is under regulation of the EGF signaling axis and promotes an aggressive phenotype in KRAS-mutated colorectal cancer cells: a potential novel approach to the treatment of metastatic colorectal cancer.

It is now widely accepted that therapeutic antibodies targeting epidermal growth factor receptor (EGFR) can have efficacy in KRAS wild-type advanced colorectal cancer (CRC) patients. What remains to be ascertained is whether a subgroup of KRAS-mutated CRC patients might not also derive benefit from EGFR inhibitors. Metalloproteinase inhibitor 1 (TIMP-1) is a pleiotropic factor predictive of survival outcome of CRC patients. Levels of TIMP-1 were measured in pre-treatment plasma samples (n = 426) of metastatic CRC patients randomized to Nordic FLOX (5-fluorouracil and oxaliplatin) +/- cetuximab (NORDIC VII study). Multivariate analysis demonstrated a significant interaction between plasma TIMP-1 protein levels, KRAS status and treatment with patients bearing KRAS mutated tumors and high TIMP-1 plasma level (> 3rd quartile) showing a significantly longer overall survival if treated with cetuximab (HR, 0.48; 95% CI, 0.25 to 0.93). To gain mechanistic insights into this association we analyzed a set of five different CRC cell lines. We show here that EGFR signaling induces TIMP-1 expression in CRC cells, and that TIMP-1 promotes a more aggressive behavior, specifically in KRAS mutated cells. The two sets of data, clinical and in vitro, are complementary and support each other, lending strength to our contention that TIMP- 1 plasma levels can identify a subset of patients with KRAS-mutated metastatic CRC that will have benefit from EGFR-inhibition therapy.