Phosphoproteomic Analysis of FLCN Inactivation Highlights Differential Kinase Pathways and Regulatory TFEB Phosphoserines

In Birt–Hogg–Dubé (BHD) syndrome, germline loss-of-function mutations in the Folliculin (FLCN) gene lead to an increased risk of renal cancer. To address how FLCN inactivation affects cellular kinase signaling pathways, we analyzed comprehensive phosphoproteomic profiles of FLCN^POS and FLCN^NEG human renal tubular epithelial cells (RPTEC/TERT1). In total, 15,744 phosphorylated peptides were identified from 4329 phosphorylated proteins. INKA analysis revealed that FLCN loss alters the activity of numerous kinases, including tyrosine kinases EGFR, MET, and the Ephrin receptor subfamily (EPHA2 and EPHB1), as well their downstream targets MAPK1/3. Validation experiments in the BHD renal tumor cell line UOK257 confirmed that FLCN loss contributes to enhanced MAPK1/3 and downstream RPS6K1/3 signaling. The clinically available MAPK inhibitor Ulixertinib showed enhanced toxicity in FLCN^NEG cells. Interestingly, FLCN inactivation induced the phosphorylation of PIK3CD (Tyr524) without altering the phosphorylation of canonical Akt1/Akt2/mTOR/EIF4EBP1 phosphosites. Also, we identified that FLCN inactivation resulted in dephosphorylation of TFEB Ser109, Ser114, and Ser122, which may be linked to increased oxidative stress levels in FLCN^NEG cells. Together, our study highlights differential phosphorylation of specific kinases and substrates in FLCN^NEG renal cells. This provides insight into BHD-associated renal tumorigenesis and may point to several novel candidates for targeted therapies.     

Cancer/testis antigen LDHC promotes proliferation and metastasis by activating the PI3K/Akt/GSK-3β-signaling pathway and the in lung adenocarcinoma

The cancer/testis antigen lactate dehydrogenase-C4 (LDHC) is a specific isoenzyme of the LDH family that regulates invasion and metastasis in some malignancies; however, little is known regarding its role in progression of lung adenocarcinoma (LUAD). Thus, we investigated LDHC expression by immunohistochemistry, and analyzed its clinical significance in 88 LUAD specimens. The role and molecular mechanisms subserving LDHC in cellular proliferation, migration, and invasion were explored both in vitro and in vivo. As a result, we found that high LDHC expression was significantly correlated with clinicopathological features of aggressive LUAD and a poor prognosis. Overexpression of LDHC induced LUAD cells to produce lactate and ATP, increased their metastatic and invasive potential—, and accelerated xenograft tumor growth. We further demonstrated that overexpression of LDHC affected the expression of cell proliferation-related proteins (cyclin D1 and c-Myc) and epithelial-mesenchymal transition (EMT)-related proteins (MMP-2, MMP-9, E-cadherin, Vimentin, Twist, Slug, and Snail) both in vitro and in vivo. Finally, excessive activation of LDHC enhanced the phosphorylation levels of AKT and GSK-3β, revealing activation of the PI3K/Akt/GSK-3β oncogenic-signaling pathways. Treatment with a PI3K inhibitor reversed the effects of LDHC overexpression by inhibiting cellular proliferation, migration, and invasion, with diminished levels of p-Akt and p-GSK3β. PI3K inhibition also reversed cell proliferation-related and EMT-related proteins in LDHC-overexpressing A549 cells. In conclusion, LDHC promotes proliferation, migration, invasion, and EMT in LUAD cells via activation of the PI3K/Akt/GSK-3β pathway.     

The PI3 Kinase Complex II–PI3P–Vps27 Axis on Vacuolar Membranes is Critical for Microautophagy Induction and Nutrient Stress Adaptation

Phosphatidylinositol 3-phosphate (PI3P), a scaffold of membrane-associated proteins required for diverse cellular events, is produced by Vps34-containing phosphatidylinositol 3-kinase (PI3K). PI3K complex I (PI3KCI)-generated PI3P is required for macroautophagy, whereas PI3K complex II (PI3KCII)-generated PI3P is required for endosomal sorting complex required for transport (ESCRT)-mediated multi-vesicular body (MVB) formation in late endosomes. ESCRT also promotes vacuolar membrane remodeling in microautophagy after nutrient starvation and inactivation of target of rapamycin complex 1 (TORC1) protein kinase in budding yeast. Whereas PI3KCI and macroautophagy are critical for the nutrient starvation response, the physiological roles of PI3KCII and microautophagy during starvation are largely unknown. Here, we showed that PI3KCII-produced PI3P on vacuolar membranes is required for microautophagy induction and survival in nutrient-stressed conditions. PI3KCII is required for Vps27 (an ESCRT-0 component) recruitment and ESCRT-0 complex formation on vacuolar surfaces after TORC1 inactivation. Forced recruitment of Vps27 onto vacuolar membranes rescued the defect in microautophagy induction in PI3KCII-deficient cells, indicating that a critical role of PI3P on microautophagy induction is Vps27 recruitment onto vacuolar surfaces. Finally, vacuolar membrane-associated Vps27 was able to recover survival during nutrient starvation in cells lacking PI3KCII or Vps27. This study revealed that the PI3KCII–PI3P–Vps27 axis on vacuolar membranes is critical for ESCRT-mediated microautophagy induction and nutrient stress adaptation.     

Ensemble structural analyses depict the regulatory mechanism of non-phosphorylated human MAP2K4

Mitogen-activated protein kinase kinase 4 (MAP2K4) plays a critical role in regulating the stress-activated protein kinase signaling cascade. A small angle X-ray scattering experiment, a powerful technique for analyzing a solution structure cleared from the structural artifacts due to crystal packing, provided the ensemble structures of human non-phosphorylated MAP2K4 in three states involving the apo form, the binary complex with an ATP analogue, and the ternary complex with the ATP analogue and substrate peptide. These ensemble structures provided more detailed mechanisms for regulating MAP2K4 in addition to those delineated only by the crystal structures in three states.     

Targeting protein kinases benefits cancer immunotherapy

Small-molecule kinase inhibitors have been well established and successfully developed in the last decades for cancer target therapies. However, intrinsic or acquired drug resistance is becoming the major barrier for their clinical application. With the development of immunotherapies, in particular the discovery of immune checkpoint inhibitors (ICIs), the combination of ICIs with other therapies have recently been extensively explored, among which combination of ICIs with kinase inhibitors achieves promising clinical outcome in a plethora of cancer types. Here we comprehensively summarize the potent roles of protein kinases in modulating immune checkpoints both in tumor and immune cells, and reshaping tumor immune microenvironments by evoking innate immune response and neoantigen generation or presentation. Moreover, the clinical trial and approval of combined administration of kinase inhibitors with ICIs are collected, highlighting the precise strategies to benefit cancer immune therapies.     

DIA-Based Proteomics Identifies IDH2 as a Targetable Regulator of Acquired Drug Resistance in Chronic Myeloid Leukemia

Drug resistance is a critical obstacle to effective treatment in patients with chronic myeloid leukemia. To understand the underlying resistance mechanisms in response to imatinib mesylate (IMA) and adriamycin (ADR), the parental K562 cells were treated with low doses of IMA or ADR for 2 months to generate derivative cells with mild, intermediate, and severe resistance to the drugs as defined by their increasing resistance index. PulseDIA-based (DIA [data-independent acquisition]) quantitative proteomics was then employed to reveal the proteome changes in these resistant cells. In total, 7082 proteins from 98,232 peptides were identified and quantified from the dataset using four DIA software tools including OpenSWATH, Spectronaut, DIA-NN, and EncyclopeDIA. Sirtuin signaling pathway was found to be significantly enriched in both ADR-resistant and IMA-resistant K562 cells. In particular, isocitrate dehydrogenase (NADP(+)) 2 was identified as a potential drug target correlated with the drug resistance phenotype, and its inhibition by the antagonist AGI-6780 reversed the acquired resistance in K562 cells to either ADR or IMA. Together, our study has implicated isocitrate dehydrogenase (NADP(+)) 2 as a potential target that can be therapeutically leveraged to alleviate the drug resistance in K562 cells when treated with IMA and ADR.     

Inhibition of MAP4K4 signaling initiates metabolic reprogramming to protect hepatocytes from lipotoxic damage

The primary hepatic consequence of obesity is non-alcoholic fatty liver disease (NAFLD), affecting about 25% of the global adult population. Non-alcoholic steatohepatitis (NASH) is a severe form of NAFLD characterized by liver lipid accumulation, inflammation, and hepatocyte ballooning, with a different degree of hepatic fibrosis. In the light of rapidly increasing prevalence of NAFLD and NASH, there is an urgent need for improved understanding of the molecular pathogenesis of these diseases. The aim of this study was to decipher the possible role of STE20-type kinase MAP4K4 in the regulation of hepatocellular lipotoxicity and susceptibility to NAFLD. We found that MAP4K4 mRNA expression in human liver biopsies was positively correlated with key hallmarks of NAFLD (i.e., liver steatosis, lobular inflammation, hepatocellular ballooning, and fibrosis). We also found that the silencing of MAP4K4 suppressed lipid deposition in human hepatocytes by stimulating β-oxidation and triacylglycerol secretion, while attenuating fatty acid influx and lipid synthesis. Furthermore, downregulation of MAP4K4 markedly reduced the glycolysis rate and lowered incidences of oxidative/endoplasmic reticulum stress. In parallel, we observed suppressed JNK and ERK and increased AKT phosphorylation in MAP4K4-deficient hepatocytes. Together, these results provide the first experimental evidence supporting the potential involvement of STE20-type kinase MAP4K4 as a component of the hepatocellular lipotoxic milieu promoting NAFLD susceptibility.     

Phosphoproteome Profiling of the Receptor Tyrosine Kinase MuSK Identifies Tyrosine Phosphorylation of Rab GTPases

Muscle-specific receptor tyrosine kinase (MuSK) agonist antibodies were developed 2 decades ago to explore the benefits of receptor activation at the neuromuscular junction. Unlike agrin, the endogenous agonist of MuSK, agonist antibodies function independently of its coreceptor low-density lipoprotein receptor–related protein 4 to delay the onset of muscle denervation in mouse models of ALS. Here, we performed dose–response and time-course experiments on myotubes to systematically compare site-specific phosphorylation downstream of each agonist. Remarkably, both agonists elicited similar intracellular responses at known and newly identified MuSK signaling components. Among these was inducible tyrosine phosphorylation of multiple Rab GTPases that was blocked by MuSK inhibition. Importantly, mutation of this site in Rab10 disrupts association with its effector proteins, molecule interacting with CasL 1/3. Together, these data provide in-depth characterization of MuSK signaling, describe two novel MuSK inhibitors, and expose phosphorylation of Rab GTPases downstream of receptor tyrosine kinase activation in myotubes.     

EGFR pathway targeting drugs in head and neck cancer in the era of immunotherapy

Receptor tyrosine kinases (RTKs) are cell surface receptors that bind growth factor ligands and initiate cellular signaling. Of the 20 classes of RTKs, 7 classes, I-V, VIII, and X, are linked to head and neck cancers (HNCs). We focus on the first class of RTK, epidermal growth factor receptor (EGFR), as it is the most thoroughly studied class. EGFR overexpression is observed in 20% of tumors, and expression of EGFR variant III is seen in 15% of aggressive chemoradiotherapy resistant HNCs. Currently, the EGFR monoclonal antibody (mAb) cetuximab is the only FDA approved RTK-targeting drug for the treatment of HNCs. Clinical trials have also included EGFR mAbs, with tyrosine kinase inhibitors, and small molecule inhibitors targeting the EGFR, MAPK, and mTOR pathways. Additionally, Immunotherapy has been found to be effective in 15 to 20% of patients with recurrent or metastatic HNC as a monotherapy. Thus, attempts are underway for the combinatorial treatment of immunotherapy and EGFR mAbs to determine if the recruitment of immune cells in the tumor microenvironment can overcome EGFR resistance.     

SKAP2 Modular Organization Differently Recognizes SRC Kinases Depending on Their Activation Status and Localization

Dimerization of SRC kinase adaptor phosphoprotein 2 (SKAP2) induces an increase of binding for most SRC kinases suggesting a fine-tuning with transphosphorylation for kinase activation. This work addresses the molecular basis of SKAP2-mediated SRC kinase regulation through the lens of their interaction capacities. By combining a luciferase complementation assay and extensive site-directed mutagenesis, we demonstrated that SKAP2 interacts with SRC kinases through a modular organization depending both on their phosphorylation-dependent activation and subcellular localization. SKAP2 contains three interacting modules consisting in the dimerization domain, the SRC homology 3 (SH3) domain, and the second interdomain located between the Pleckstrin homology and the SH3 domains. Functionally, the dimerization domain is necessary and sufficient to bind to most activated and myristyl SRC kinases. In contrast, the three modules are necessary to bind SRC kinases at their steady state. The Pleckstrin homology and SH3 domains of SKAP2 as well as tyrosines located in the interdomains modulate these interactions. Analysis of mutants of the SRC kinase family member hematopoietic cell kinase supports this model and shows the role of two residues, Y390 and K7, on its degradation following activation. In this article, we show that a modular architecture of SKAP2 drives its interaction with SRC kinases, with the binding capacity of each module depending on both their localization and phosphorylation state activation. This work opens new perspectives on the molecular mechanisms of SRC kinases activation, which could have significant therapeutic impact.     

Tripartite motif 38 alleviates the pathological process of NAFLD–NASH by promoting TAB2 degradation

Nonalcoholic fatty liver disease (NAFLD) has become the most prevalent chronic liver disease worldwide, without any Food and Drug Administration-approved pharmacological intervention in clinic. Trim38, as an important member of the TRIM (tripartite motif-containing) family, was largely reported to be involved in the regulation of innate immune and inflammatory responses. However, the functional roles of TRIM38 in NAFLD remain largely unknown. Here, the expression of TRIM38 was first detected in liver samples of both NAFLD mice model and patients diagnosed with NAFLD. We found that TRIM38 expression was downregulated in NAFLD liver tissues compared with normal liver tissues. Genetic Trim38-KO in vivo showed that TRIM38 depletion deteriorated the high-fat diet and high fat and high cholesterol diet-induced hepatic steatosis and high fat and high cholesterol diet-induced liver inflammation and fibrosis. In particular, we found that the effects of hepatocellular lipid accumulation and inflammation induced by palmitic acid and oleic acid were aggravated by TRIM38 depletion but mitigated by TRIM38 overexpression in vitro. Mechanically, RNA-Seq analysis demonstrated that TRIM38 ameliorated nonalcoholic steatohepatitis progression by attenuating the activation of MAPK signaling pathway. We further found that TRIM38 interacted with transforming growth factor-β-activated kinase 1 binding protein 2 and promoted its protein degradation, thus inhibiting the transforming growth factor-β-activated kinase 1-MAPK signal cascades. In summary, our study revealed that TRIM38 could suppress hepatic steatosis, inflammatory, and fibrosis in NAFLD via promoting transforming growth factor-β-activated kinase 1 binding protein 2 degradation. TRIM38 could be a potential target for NAFLD treatment.     

MBOAT7-driven lysophosphatidylinositol acylation in adipocytes contributes to systemic glucose homeostasis

We previously demonstrated that antisense oligonucleotide-mediated knockdown of Mboat7, the gene encoding membrane bound O-acyltransferase 7, in the liver and adipose tissue of mice promoted high fat diet-induced hepatic steatosis, hyperinsulinemia, and systemic insulin resistance. Thereafter, other groups showed that hepatocyte-specific genetic deletion of Mboat7 promoted striking fatty liver and NAFLD progression in mice but does not alter insulin sensitivity, suggesting the potential for cell autonomous roles. Here, we show that MBOAT7 function in adipocytes contributes to diet-induced metabolic disturbances including hyperinsulinemia and systemic insulin resistance. We generated Mboat7 floxed mice and created hepatocyte- and adipocyte-specific Mboat7 knockout mice using Cre-recombinase mice under the control of the albumin and adiponectin promoter, respectively. Here, we show that MBOAT7 function in adipocytes contributes to diet-induced metabolic disturbances including hyperinsulinemia and systemic insulin resistance. The expression of Mboat7 in white adipose tissue closely correlates with diet-induced obesity across a panel of ∼100 inbred strains of mice fed a high fat/high sucrose diet. Moreover, we found that adipocyte-specific genetic deletion of Mboat7 is sufficient to promote hyperinsulinemia, systemic insulin resistance, and mild fatty liver. Unlike in the liver, where Mboat7 plays a relatively minor role in maintaining arachidonic acid-containing PI pools, Mboat7 is the major source of arachidonic acid-containing PI pools in adipose tissue. Our data demonstrate that MBOAT7 is a critical regulator of adipose tissue PI homeostasis, and adipocyte MBOAT7-driven PI biosynthesis is closely linked to hyperinsulinemia and insulin resistance in mice.     

The Lysine Acetylation Modification in the Porin Aha1 of Aeromonas hydrophila Regulates the Uptake of Multidrug Antibiotics

Protein lysine acetylation (Kac) modification plays important roles in diverse physiological functions. However, there is little evidence on the role of Kac modification in bacterial antibiotic resistance. Here, we compared the differential expressions of whole-cell proteins and Kac peptides in oxytetracycline sensitive and oxytetracycline resistance (OXY^R) strains of Aeromonas hydrophila using quantitative proteomics technologies. We observed a porin family protein Aha1 downregulated in the OXY^R strain, which may have an important role in the OXY resistance. Interestingly, seven of eight Kac peptides of Aha1 decreased abundance in OXY^R as well. Microbiologic assays showed that the K57R, K187R, and K197R Aha1 mutants significantly increased antibiotic resistance to OXY and reduced the intracellular OXY accumulation in OXY stress. Moreover, these Aha1 mutants displayed multidrug resistance features to tetracyclines and β-lactam antibiotics. The 3D model prediction showed that the Kac states of K57, K187, and K197 sites located at the extracellular pore vestibule of Aha1 may be involved in the uptake of specific types of antibiotics. Overall, our results indicate a novel antibiotic resistance mechanism mediated by Kac modification, which may provide a clue for the development of antibiotic therapy strategies.     

A novel PAK4 inhibitor suppresses pancreatic cancer growth and enhances the inhibitory effect of gemcitabine

Over 95% of Pancreatic ductal adenocarcinomas (PDA) carry mutations in the oncogene KRas which has been proven to be a difficult drug target. P21-activated kinase 4 (PAK4), acts downstream of KRas, and is overexpressed in PDA contributing to its growth and chemoresistance, and thus becomes an attractive therapeutic target. We have developed a new PAK4 inhibitor, PAKib and tested its effect on pancreatic cancer (PC) cell growth in vitro and in a syngeneic mouse model of PC. PAKib suppressed PC cell growth by inducing cell death and cycle arrest. PAKib inhibited PC growth and enhanced the inhibition by gemcitabine of PC in cell culture and in PC mouse model. PAKib acted through multiple signaling pathways involved in cell cycle checkpoints, apoptosis, cell junction, and focal adhesion. These proof-of-concept studies demonstrated the anti-cancer effect of PAKib alone and in combination with gemcitabine and warrant a further clinical investigation.     

Depleting deubiquitinating enzymes promotes apoptosis in glioma cell line via RNA binding proteins SF2/ASF1

USP5 and USP8 (Deubiquitinating enzyme) are highly overexpressed and more recognized as poor prognosis marker in various cancers. Depleting USP5 or USP8 to assess the synergism with proteasome inhibitor (Bortezomib) were measured. Furthermore, in present finding USP5 cooperates hnRNPA1 & USP8 cooperate SF2/ASF1, therefore gain in expression of either hnRNPA1 or SF2/ASF1 is sufficient to promote cell survival. On the other side, apoptosis markers were more pronounced in U87 or T98G cells devoid of either USP5 or USP8. However, apparent increase in SF2/ASF1 in absence of USP5, providing resistant factor is new. Antiapoptotic activity due to rise in SF2/ASF1 was validated after co-knock down of SF2/ASF1 in addition to USP5 induces more apoptosis comparing to individual knock down of USP5 or SF2/ASF1. This reveals SF2/ASF1 (RNA binding protein) delayed the apoptotic effect due to loss of USP5, lends ubiquitination of hnRNPA1. In presence of USP5, PI3 kinase inhibition promotes even more interaction between USP5 and hnRNPA1, thereby stabilizes hnRNPA1 in U87MG. In that way hnRNPA1 and SF2/ASF1 impart oncogenic activity. In conclusion, siRNA based strategy against USP5 is not enough to inhibit glioma, moreover targeting additionally SF2/ASF1 by knocking down USP8 is suitably more effective to deal with glioma tumour reoccurrence by indirectly targeting both SF2/ASF1 and hnRNPA1 oncogene.