The mechanistic target of rapamycin (mTOR) exists in two complexes that regulate diverse cellular processes. mTOR complex 1 (mTORC1), the canonical target of rapamycin, has been well studied, whereas the physiological role of mTORC2 remains relatively uncharacterized. In mice in which the mTORC2 component Rictor is deleted in liver [ Rictor ‐knockout (RKO) mice], we used genomic and phosphoproteomic analyses to characterize the role of hepatic mTORC2 in vivo . Overnight food withdrawal followed by refeeding was used to activate mTOR signaling. Rapamycin was administered before refeeding to specify mTORC2‐mediated events. Hepatic mTORC2 regulated a complex gene expression and post‐translational network that affects intermediary metabolism, ribosomal biogenesis, and proteasomal biogenesis. Nearly all changes in genes related to intermediary metabolic regulation were replicated in cultured fetal hepatocytes, indicating a cell‐autonomous effect of mTORC2 signaling. Phosphoproteomic profiling identified mTORC2‐related signaling to 144 proteins, among which were metabolic enzymes and regulators. A reduction of p38 MAPK signaling in the RKO mice represents a link between our phosphoproteomic and gene expression results. We conclude that hepatic mTORC2 exerts a broad spectrum of biological effects under physiological conditions. Our findings provide a context for the development of targeted therapies to modulate mTORC2 signaling.—Lamming, D. W., Demirkan, G., Boylan, J. M., Mihaylova, M. M., Peng, T., Ferreira, J., Neretti, N., Salomon, A., Sabatini, D. M., Gruppuso, P. A. Hepatic signaling by the mechanistic target of rapamycin complex 2 (mTORC2). FASEB J . 28, 300–315 (2014). www.fasebj.org

Our understanding of signal transduction networks in the physiological context of an organism remains limited, partly due to the technical challenge of identifying serine/threonine phosphorylated peptides from complex tissue samples. In the present study, we focused on signaling through the mammalian target of rapamycin (mTOR) complex 1 (mTORC1), which is at the center of a nutrient- and growth factor-responsive cell signaling network. Though studied extensively, the mechanisms involved in many mTORC1 biological functions remain poorly understood. We developed a phosphoproteomic strategy to purify, enrich and identify phosphopeptides from rat liver homogenates. Using the anticancer drug rapamycin, the only known target of which is mTORC1, we characterized signaling in liver from rats in which the complex was maximally activated by refeeding following 48 hr of starvation. Using protein and peptide fractionation methods, TiO₂ affinity purification of phosphopeptides and mass spectrometry, we reproducibly identified and quantified over four thousand phosphopeptides. Along with 5 known rapamycin-sensitive phosphorylation events, we identified 62 new rapamycin-responsive candidate phosphorylation sites. Among these were PRAS40, gephyrin, and AMP kinase 2. We observed similar proportions of increased and reduced phosphorylation in response to rapamycin. Gene ontology analysis revealed over-representation of mTOR pathway components among rapamycin-sensitive phosphopeptide candidates. In addition to identifying potential new mTORC1-mediated phosphorylation events, and providing information relevant to the biology of this signaling network, our experimental and analytical approaches indicate the feasibility of large-scale phosphoproteomic profiling of tissue samples to study physiological signaling events in vivo.

Background: Matching into orthopaedic surgery residency in the United States has become an increasingly competitive process because of the large number of well-qualified applicants. Over the past several years, applicants have sought to maximize their chances of matching by submitting an increasing number of applications. The purpose of this study was to assess trends in application numbers, applicant qualifications, and application reviews, with the goal of obtaining data to help inform future improvements in the orthopaedic surgery residency application process. Methods: Applicant data were obtained from the Electronic Residency Application Service (ERAS, www.aamc.org/services/eras/stats) and the National Resident Matching Program (NRMP, www.nrmp.org/report-archives). These included residency application data from 2000 to 2017. In addition, we analyzed available NRMP Applicant Survey Reports between 2008 and 2017, Program Director Survey Reports between 2008 and 2016, and NRMP’s Charting Outcomes in the Match between 2006 and 2016. Results: The number of U.S. senior medical student applicants per orthopaedic surgery residency position was stable from 2000 to 2017 (1.13 vs. 1.16 for 2000 and 2017, respectively). A significant increase in the United States Medical Licensing Examination (USMLE) Step-1 and Step-2 scores and self-reported research activity was present over the same time period. The number of applications submitted per applicant significantly increased, by 71.7%, from 48.4 in 2006 to 83.1 in 2017. Additionally, applications per program increased 46.4% from 457 in 2010 to 669 in 2016. In 2010, programs performed in-depth reviews for 54% of applications; however, in 2016, in-depth reviews had decreased to 45% of applications. Conclusions: Orthopaedic residency applicant USMLE scores and research productivity have increased over time. Concurrently, the average number of applications submitted per applicant has increased, with the average applicant applying to nearly half of all orthopaedic residency programs. Consequently, programs have seen more than double the number of applications over this study period. The accompanying decline in the proportion of applications undergoing in-depth review, along with the applicant and program resources associated with these changes, warrants the development of strategies to enhance the efficiency of the application process for orthopaedic residency.

The liver serves a spectrum of essential metabolic and synthetic functions that are required for the transition from fetal to postnatal life. Processes essential to the attainment of adequate liver mass and function during fetal life include cell lineage specification early in development, enzymic and other functional modes of differentiation throughout gestation, and ongoing cell proliferation to achieve adequate liver mass. Available data in laboratory rodents indicate that the signaling networks governing these processes in the fetus differ from those that can sustain liver function and mass in the adult. More specifically, fetal hepatocytes may develop independent of key mitogenic signaling pathways, including those involving the Erk mitogen-activated protein kinases MAPK1/3 and the mechanistic target of rapamycin (mTOR). In addition, the fetal liver is subject to environmental influences that, through epigenetic mechanisms, can have sustained effects on function and, by extension, contribute to the developmental origin of adult metabolic disease. Finally, the mitogen-independent phenotype of rat fetal hepatocytes in late gestation makes these cells suitable for cell-based therapy of liver injury. In the aggregate, studies on the mechanisms governing fetal liver development have implications not only for the perinatal metabolic transition but also for the prevention and treatment of liver disorders throughout the lifespan.

The introduction of tyrosine kinase inhibitors (TKI) has transformed chronic myeloid leukemia (CML) into a chronic disease with long‐term survival exceeding 85%. However, resistance of CML stem cells to TKI may contribute to the 50% relapse rate observed after TKI discontinuation in molecular remission. We previously described a model of resistance to imatinib mesylate (IM), in which K562 cells cultured in high concentrations of imatinib mesylate showed reduced Bcr‐Abl1 protein and activity levels while maintaining proliferative potential. Using quantitative phosphoproteomic analysis of these IM‐resistant cells, we have now identified significant upregulation of tumor progression locus (Tpl2), also known as cancer Osaka thyroid (COT1) kinase or Map3k8. Overexpression of Tpl2 in IM‐resistant cells was accompanied by elevated activities of Src family kinases (SFKs) and NF‐κB, MEK‐ERK signaling. CD34+ cells isolated from the bone marrow of patients with CML and exposed to IMin vitro showed increased MAP3K8 transcript levels. Dasatinib (SFK inhibitor), U0126 (MEK inhibitor), and PS‐1145 (IκB kinase (IKK) inhibitor) used in combination resulted in elimination of 65% of IM‐resistant cells and reduction in the colony‐forming capacity of CML CD34+ cells in methylcellulose assays by 80%. In addition, CML CD34+ cells cultured with the combination of inhibitors showed reduced MAP3K8 transcript levels. Overall, our data indicate that elevated Tpl2 protein and transcript levels are associated with resistance to IM and that combined inhibition of SFK, MEK, and NF‐κB signaling attenuates the survival of IM‐resistant CML cells and CML CD34+ cells. Therefore, combination of SFK, MEK, and NF‐κB inhibitors may offer a new therapeutic approach to overcome TKI resistance in CML patients.

The transition from undergraduate medical education to graduate medical education (GME) involves a process rooted in the final year of medical school. Students file applications through the Electronic Residency Application Service platform, interview with residency training (i.e., GME) programs from which they have received invitations, and generate a rank-ordered preference list. The National Resident Matching Program reconciles applicant and program rank lists with an eye towards matching students and GME programs. This process has effectively served generations of graduating medical students. However, the past several decades have seen an intensification of the residency placement process that is exemplified by an inexorable increase in the number of applications filed and number of interviews accepted and attended by each student. The authors contend that this trend has untoward effects on both applicants and departments that are home to GME programs. Relevant information in the peer-reviewed literature on the consequences and benefits of the intensification of the residency placement process is scant. The authors address factors that may contribute to the intensity of the residency placement process and the relative paucity of data. They propose approaches to reverse current trends, and conclude that any reevaluation of the process will have to include the generation of outcome data to afford medical educators the opportunity to explore changes in an evidence-based manner.