The expanding landscape of canonical and non-canonical protein phosphorylation.

Protein phosphorylation is a crucial regulatory mechanism in cell signaling, acting as a molecular switch that modulates protein function. Catalyzed by protein kinases and reversed by phosphoprotein phosphatases, it is essential in both normal physiological and pathological states. Recent advances have uncovered a vast and intricate landscape of protein phosphorylation that include histidine phosphorylation and more unconventional events, such as pyrophosphorylation and polyphosphorylation. Many questions remain about the true size of the phosphoproteome and, more importantly, its site-specific functional relevance. The involvement of unconventional actors such as pseudokinases and pseudophosphatases adds further complexity to be resolved. This review explores recent discoveries and ongoing challenges, highlighting the need for continued research to fully elucidate the roles and regulation of protein phosphorylation.

mTORC1 Promotes Metabolic Reprogramming by the Suppression of GSK3-Dependent Foxk1 Phosphorylation.

The mammalian Target of Rapamycin Complex 1 (mTORC1)-signaling system plays a critical role in the maintenance of cellular homeostasis by sensing and integrating multiple extracellular and intracellular cues. Therefore, uncovering the effectors of mTORC1 signaling is pivotal to understanding its pathophysiological effects. Here we report that the transcription factor forkhead/winged helix family k1 (Foxk1) is a mediator of mTORC1-regulated gene expression. Surprisingly, Foxk1 phosphorylation is increased upon mTORC1 suppression, which elicits a 14-3-3 interaction, a reduction of DNA binding, and nuclear exclusion. Mechanistically, this occurs by mTORC1-dependent suppression of nuclear signaling by the Foxk1 kinase, Gsk3. This pathway then regulates the expression of multiple genes associated with glycolysis and downstream anabolic pathways directly modulated by Foxk1 and/or by Foxk1-regulated expression of Hif-1α. Thus, Foxk1 mediates mTORC1-driven metabolic rewiring, and it is likely to be critical for metabolic diseases where improper mTORC1 signaling plays an important role.

Focal Adhesion- and IGF1R-Dependent Survival and Migratory Pathways Mediate Tumor Resistance to mTORC1/2 Inhibition.

Aberrant signaling by the mammalian target of rapamycin (mTOR) contributes to the devastating features of cancer cells. Thus, mTOR is a critical therapeutic target and catalytic inhibitors are being investigated as anti-cancer drugs. Although mTOR inhibitors initially block cell proliferation, cell viability and migration in some cancer cells are quickly restored. Despite sustained inhibition of mTORC1/2 signaling, Akt, a kinase regulating cell survival and migration, regains phosphorylation at its regulatory sites. Mechanistically, mTORC1/2 inhibition promotes reorganization of integrin/focal adhesion kinase-mediated adhesomes, induction of IGFR/IR-dependent PI3K activation, and Akt phosphorylation via an integrin/FAK/IGFR-dependent process. This resistance mechanism contributes to xenograft tumor cell growth, which is prevented with mTOR plus IGFR inhibitors, supporting this combination as a therapeutic approach for cancers.

Improved recurrence-free survival in patients with HCC with post-transplant plasma exchange.

Total plasma exchange (TPE) can play a role in cancer treatment by eliminating immune checkpoint inhibitors. This study investigated whether TPE improved oncological outcomes in patients with HCC who underwent ABO-incompatible living donor liver transplantation (LT). The study included 152 patients who underwent ABO-incompatible living donor LT for HCC between 2010 and 2021 at Samsung Medical Center. Overall survival was analyzed using the Kaplan-Meier curve, whereas HCC-specific recurrence-free survival (RFS) was analyzed using the cumulative incidence curve after propensity score matching. Cox regression and competing risks subdistribution hazard models were used to identify the risk factors associated with overall survival and HCC-specific RFS, respectively. The propensity score matching resulted in 54 matched pairs, grouped according to whether they underwent postoperative TPE [post-transplant TPE(+)] or not [post-transplant TPE(-)]. The 5-year HCC-specific RFS cumulative incidence was superior in the post-transplant TPE (+) group [12.5% (95% CI: 3.1%-21.9%)] compared with the post-transplant TPE(-) group [38.1% (95% CI: 24.4%-51.8%), p = 0.005]. In subgroup analysis for patients with microvascular invasion and those beyond the Milan criteria, the post-transplant TPE(+) group showed significantly superior HCC-specific survival. The multivariable analysis also showed that postoperative TPE had a protective effect on HCC-specific RFS (HR = 0.26, 95% CI: 0.10-0.64, p = 0.004) and that the more post-transplant TPE was performed, the better RFS was observed (HR = 0.71, 95% CI: 0.55-0.93, p = 0.012). Post-transplant TPE was found to improve RFS after ABO-incompatible living donor LT for HCC, particularly in advanced cases with microvascular invasion and beyond Milan criteria. These findings suggest that TPE may have a potential role in improving oncological outcomes in patients with HCC undergoing LT.

SKAR links pre-mRNA splicing to mTOR/S6K1-mediated enhanced translation efficiency of spliced mRNAs.

Different protein complexes form on newly spliced mRNA to ensure the accuracy and efficiency of eukaryotic gene expression. For example, the exon junction complex (EJC) plays an important role in mRNA surveillance. The EJC also influences the first, or pioneer round of protein synthesis through a mechanism that is poorly understood. We show that the nutrient-, stress-, and energy-sensing checkpoint kinase, mTOR, contributes to the observed enhanced translation efficiency of spliced over nonspliced mRNAs. We demonstrate that, when activated, S6K1 is recruited to the newly synthesized mRNA by SKAR, which is deposited at the EJC during splicing, and that SKAR and S6K1 increase the translation efficiency of spliced mRNA. Thus, SKAR-mediated recruitment of activated S6K1 to newly processed mRNPs serves as a conduit between mTOR checkpoint signaling and the pioneer round of translation when cells exist in conditions supportive of protein synthesis.

ERK2 Mediates Metabolic Stress Response to Regulate Cell Fate.

Insufficient nutrients disrupt physiological homeostasis, resulting in diseases and even death. Considering the physiological and pathological consequences of this metabolic stress, the adaptive responses that cells utilize under this condition are of great interest. We show that under low-glucose conditions, cells initiate adaptation followed by apoptosis responses using PERK/Akt and MEK1/ERK2 signaling, respectively. For adaptation, cells engage the ER stress-induced unfolded protein response, which results in PERK/Akt activation and cell survival. Sustained and extreme energetic stress promotes a switch to isoform-specific MEK1/ERK2 signaling, induction of GCN2/eIF2α phosphorylation, and ATF4 expression, which overrides PERK/Akt-mediated adaptation and induces apoptosis through ATF4-dependent expression of pro-apoptotic factors including Bid and Trb3. ERK2 activation during metabolic stress contributes to changes in TCA cycle and amino acid metabolism, and cell death, which is suppressed by glutamate and α-ketoglutarate supplementation. Taken together, our results reveal promising targets to protect cells or tissues from metabolic stress.

ERK2 regulates epithelial-to-mesenchymal plasticity through DOCK10-dependent Rac1/FoxO1 activation.

ERK is a key coordinator of the epithelial-to-mesenchymal transition (EMT) in that a variety of EMT-inducing factors activate signaling pathways that converge on ERK to regulate EMT transcription programs. However, the mechanisms by which ERK controls the EMT program are not well understood. Through an analysis of the global changes of gene expression mediated by ERK2, we identified the transcription factor FoxO1 as a potential mediator of ERK2-induced EMT, and thus we investigated the mechanism by which ERK2 regulates FoxO1. Additionally, our analysis revealed that ERK2 induced the expression of Dock10, a Rac1/Cdc42 GEF, during EMT. We demonstrate that the activation of the Rac1/JNK signaling axis downstream of Dock10 leads to an increase in FoxO1 expression and EMT. Taken together, our study uncovers mechanisms by which epithelial cells acquire less proliferative but more migratory mesenchymal properties and reveals potential therapeutic targets for cancers evolving into a metastatic disease state.

Metabolic switching in pluripotent stem cells reorganizes energy metabolism and subcellular organelles.

Metabolic studies of human pluripotent stem cells (hPSCs) have focused on how the cells produce energy through the catabolic pathway. The less-studied anabolic pathway, by which hPSCs expend energy in the form of adenosine triphosphate (ATP), is not yet fully understood. Compared to fully differentiated somatic cells, hPSCs undergo significant changes not only in their gene expression but also in their production and/or expenditure of ATP. Here, we investigate how hPSCs tightly control their energy homeostasis by studying the main energy-consuming process, mRNA translation. In addition, change of subcellular organelles regarding energy homeostasis has been investigated. Lysosomes are organelles that play an important role in the elimination of unnecessary cellular materials by digestion and in the recycling system of the cell. We have found that hPSCs control their lysosome numbers in part by regulating lysosomal gene/protein expression. Thus, because the levels of mRNA translation rate are lower in hPSCs than in somatic cells, not only the global translational machinery but also the lysosomal recycling machinery is suppressed in hPSCs. Overall, the results of our study suggest that hPSCs reprogram gene expression and signaling to regulate energy-consuming processes and energy-controlling organelles.

ERK2 but not ERK1 induces epithelial-to-mesenchymal transformation via DEF motif-dependent signaling events.

Hyperactivation of Ras-ERK1/2 signaling is critical to the development of many human malignancies, but little is known regarding the specific contribution of ERK1 or ERK2 to oncogenic processes. We demonstrate that ERK2 but not ERK1 signaling is necessary for Ras-induced epithelial-to-mesenchymal transformation (EMT). Further, ERK2 but not ERK1 overexpression is sufficient to induce EMT. Many ERK1/2-interacting proteins contain amino acid motifs, e.g., DEF or D-motifs, which regulate docking with ERK1/2. Remarkably, ERK2 signaling to DEF motif-containing targets is required to induce EMT and correlates with increased migration, invasion, and survival. Importantly, the late-response gene product Fra1 is necessary for Ras- and ERK2-induced EMT through upregulation of ZEB1/2 proteins. Thus, an apparent critical role for ERK2 DEF motif signaling during tumorigenesis is the regulation of Fra1 and the subsequent induction of ZEB1/2, suggesting a potential therapeutic target for Ras-regulated tumorigenesis.

Glucose addiction of TSC null cells is caused by failed mTORC1-dependent balancing of metabolic demand with supply.

The mTORC1-signaling pathway integrates environmental conditions into distinct signals for cell growth by balancing anabolic and catabolic processes. Accordingly, energetic stress inhibits mTORC1 signaling predominantly through AMPK-dependent activation of TSC1/2. Thus, TSC1/2-/- cells are hypersensitive to glucose deprivation, and this has been linked to increased p53 translation and activation of apoptosis. Herein, we show that mTORC1 inhibition during glucose deprivation prevented not only the execution of death, but also induction of energetic stress. mTORC1 inhibition during glucose deprivation decreased AMPK activation and allowed ATP to remain high, which was both necessary and sufficient for protection. This effect was not due to increased catabolic activities such as autophagy, but rather exclusively due to decreased anabolic processes, reducing energy consumption. Specifically, TSC1/2-/- cells become highly dependent on glutamate dehydrogenase-dependent glutamine metabolism via the TCA cycle for survival. Therefore, mTORC1 inhibition during energetic stress is primarily to balance metabolic demand with supply.

Glycogen synthase kinase (GSK)-3 promotes p70 ribosomal protein S6 kinase (p70S6K) activity and cell proliferation.

The p70 ribosomal protein S6 kinase 1 (S6K1) plays a key role in cell growth and proliferation by regulating insulin sensitivity, metabolism, protein synthesis, and cell cycle. Thus, deregulation of S6K contributes to the progression of type 2 diabetes, obesity, aging, and cancer. Considering the biological and clinical importance of S6K1, a complete understanding of its regulation is critical. One of the key motifs in the activation of S6K1 is a turn motif, but its regulation is not well understood. Here we provide evidence for two mechanisms of modulating turn motif phosphorylation and S6K1 activity. First, mammalian target of rapamycin regulates turn motif phosphorylation by inhibiting its dephosphorylation. Second, we unexpectedly found that glycogen synthase kinase (GSK)-3 promotes turn motif phosphorylation. Our studies show that mammalian target of rapamycin and GSK-3 cooperate to control the activity of S6K1, an important regulator of cell proliferation and growth. Our unexpected results provide a clear rationale for the development and use of drugs targeting GSK-3 to treat diseases such as diabetes, cancer, and age-related diseases that are linked to improper regulation of S6K1.

mTOR inhibition reprograms cellular proteostasis by regulating eIF3D-mediated selective mRNA translation and promotes cell phenotype switching.

Cells maintain and dynamically change their proteomes according to the environment and their needs. Mechanistic target of rapamycin (mTOR) is a key regulator of proteostasis, homeostasis of the proteome. Thus, dysregulation of mTOR leads to changes in proteostasis and the consequent progression of diseases, including cancer. Based on the physiological and clinical importance of mTOR signaling, we investigated mTOR feedback signaling, proteostasis, and cell fate. Here, we reveal that mTOR targeting inhibits eIF4E-mediated cap-dependent translation, but feedback signaling activates a translation initiation factor, eukaryotic translation initiation factor 3D (eIF3D), to sustain alternative non-canonical translation mechanisms. Importantly, eIF3D-mediated protein synthesis enables cell phenotype switching from proliferative to more migratory. eIF3D cooperates with mRNA-binding proteins such as heterogeneous nuclear ribonucleoprotein F (hnRNPF), heterogeneous nuclear ribonucleoprotein K (hnRNPK), and Sjogren syndrome antigen B (SSB) to support selective mRNA translation following mTOR inhibition, which upregulates and activates proteins involved in insulin receptor (INSR)/insulin-like growth factor 1 receptor (IGF1R)/insulin receptor substrate (IRS) and interleukin 6 signal transducer (IL-6ST)/Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling. Our study highlights the mechanisms by which cells establish the dynamic change of proteostasis and the resulting phenotype switch.

Regulation of endothelial cell morphogenesis by the protein kinase D (PKD)/glycogen synthase kinase 3 (GSK3)ß pathway.

Vascular morphogenesis is a key process for development, reproduction, and pathogenesis. Thus understanding the mechanisms of this process is of pathophysiological importance. Despite the fact that collagen I is the most abundant and potent promorphogenic molecule known, the molecular mechanisms by which this protein regulates endothelial cell tube morphogenesis are still unclear. Here we provide strong evidence that collagen I induces tube morphogenesis by inhibiting glycogen synthase kinase 3ß (GSK3ß). Further mechanistic studies revealed that GSK3ß activity is regulated by protein kinase D (PKD). PKD inhibited GSK3ß activity, which was required for collagen I-induced endothelial tube morphogenesis. We also found that GSK3ß regulated trafficking of integrin α(2)ß(1) in a Rab11-dependent manner. Taken together, our studies highlight the important role of PKD in the regulation of collagen I-induced vascular morphogenesis and show that it is mediated by the modulation of GSK3ß activity and integrin α(2)ß(1) trafficking.

The PIKK-AKT connection in the DNA damage response.

DNA damage and subsequent cellular response are the basis for many cancer treatments. In this issue of Science Signaling, Liu et al. elucidate a mechanism by which cancer cells survive DNA damage induced by radiation and chemotherapy.

Rapamycin differentially inhibits S6Ks and 4E-BP1 to mediate cell-type-specific repression of mRNA translation.

The mammalian translational initiation machinery is a tightly controlled system that is composed of eukaryotic initiation factors, and which controls the recruitment of ribosomes to mediate cap-dependent translation. Accordingly, the mTORC1 complex functionally controls this cap-dependent translation machinery through the phosphorylation of its downstream substrates 4E-BPs and S6Ks. It is generally accepted that rapamycin, a specific inhibitor of mTORC1, is a potent translational repressor. Here we report the unexpected discovery that rapamycin's ability to regulate cap-dependent translation varies significantly among cell types. We show that this effect is mechanistically caused by rapamycin's differential effect on 4E-BP1 versus S6Ks. While rapamycin potently inhibits S6K activity throughout the duration of treatment, 4E-BP1 recovers in phosphorylation within 6 h despite initial inhibition (1-3 h). This reemerged 4E-BP1 phosphorylation is rapamycin-resistant but still requires mTOR, Raptor, and mTORC1's activity. Therefore, these results explain how cap-dependent translation can be maintained in the presence of rapamycin. In addition, we have also defined the condition by which rapamycin can control cap-dependent translation in various cell types. Finally, we show that mTOR catalytic inhibitors are effective inhibitors of the rapamycin-resistant phenotype.

Glycogen synthase kinase-3 is an endogenous inhibitor of Snail transcription: implications for the epithelial-mesenchymal transition.

We report that the activity of glycogen synthase kinase-3 (GSK-3) is necessary for the maintenance of the epithelial architecture. Pharmacological inhibition of its activity or reducing its expression using small interfering RNAs in normal breast and skin epithelial cells results in a reduction of E-cadherin expression and a more mesenchymal morphology, both of which are features associated with an epithelial-mesenchymal transition (EMT). Importantly, GSK-3 inhibition also stimulates the transcription of Snail, a repressor of E-cadherin and an inducer of the EMT. We identify NFkappaB as a transcription factor inhibited by GSK-3 in epithelial cells that is relevant for Snail expression. These findings indicate that epithelial cells must sustain activation of a specific kinase to impede a mesenchymal transition.