Macrophage Phosphoproteome Analysis Reveals MINCLE-dependent and -independent Mycobacterial Cord Factor Signaling

Hansen, Madlen; Peltier, Julian; Killy, Barbara; Amin, Bushra; Bodendorfer, Barbara; Härtlová, Anetta; Uebel, Sebastian; Bosmann, Markus; Hofmann, Jörg; Büttner, Christian; Ekici, Arif B.; Kuttke, Mario; Franzyk, Henrik; Foged, Camilla; Beer‐Hammer, Sandra; Schabbauer, Gernot; Trost, Matthias; Lang, Roland

doi:10.1074/mcp.ra118.000929

Cited by 21 publications

(17 citation statements)

References 74 publications

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“…Both the PS1 and PS2 phopshoproteome and total proteome samples are analyzed on an Orbitrap Fusion Tribrid Mass spectrometer (Thermo Fisher Scientific, San Jose, U.S.A.) that was in line with the Dionex 3000 RSLC nano-liquid chromatography system, similar to previously published work [36–38]. The peptide samples were loaded on a nano viper trap column (C18, 5 µm, 100 Å, 100 µm × 2 cm; PN: 164562, Thermo Scientific) and resolved on a 50 cm analytical column (2 µm, 100 Å, 75 µm × 50 cm.…”

Section: Methodsmentioning

confidence: 99%

Phosphoproteomics reveals that the hVPS34 regulated SGK3 kinase specifically phosphorylates endosomal proteins including Syntaxin-7, Syntaxin-12, RFIP4 and WDR44

Malik

Nirujogi

Peltier

et al. 2019

Biochemical Journal

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The serum- and glucocorticoid-regulated kinase (SGK) isoforms contribute resistance to cancer therapies targeting the PI3K pathway. SGKs are homologous to Akt and these kinases display overlapping specificity and phosphorylate several substrates at the same residues, such as TSC2 to promote tumor growth by switching on the mTORC1 pathway. The SGK3 isoform is up-regulated in breast cancer cells treated with PI3K or Akt inhibitors and recruited and activated at endosomes, through its phox homology domain binding to PtdIns(3)P. We undertook genetic and pharmacological phosphoproteomic screens to uncover novel SGK3 substrates. We identified 40 potential novel SGK3 substrates, including four endosomal proteins STX7 (Ser126) and STX12 (Ser139), RFIP4 (Ser527) and WDR44 (Ser346) that were efficiently phosphorylated in vitro by SGK3 at the sites identified in vivo, but poorly by Akt. We demonstrate that these substrates are inefficiently phosphorylated by Akt as they possess an n + 1 residue from the phosphorylation site that is unfavorable for Akt phosphorylation. Phos-tag analysis revealed that stimulation of HEK293 cells with IGF1 to activate SGK3, promoted phosphorylation of a significant fraction of endogenous STX7 and STX12, in a manner that was blocked by knock-out of SGK3 or treatment with a pan SGK inhibitor (14H). SGK3 phosphorylation of STX12 enhanced interaction with the VAMP4/VTI1A/STX6 containing the SNARE complex and promoted plasma membrane localization. Our data reveal novel substrates for SGK3 and suggest a mechanism by which STX7 and STX12 SNARE complexes are regulated by SGK3. They reveal new biomarkers for monitoring SGK3 pathway activity.

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Section: Methodsmentioning

confidence: 99%

Phosphoproteomics reveals that the hVPS34 regulated SGK3 kinase specifically phosphorylates endosomal proteins including Syntaxin-7, Syntaxin-12, RFIP4 and WDR44

Malik

Nirujogi

Peltier

et al. 2019

Biochemical Journal

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“…To date, the ligand- or receptor-specific induction of DUSP family genes and their kinetics in macrophages and dendritic cells has not been investigated in detail. However, while the TLR ligands LPS and CpG induce the strong upregulation of DUSP1, DUSP2 and DUSP16 [11], the mycobacterial cord factor TDM caused a marked induction of DUSP4 and DUSP5 [12]. Such ligand- or receptor-related differential expression of DUSP genes may contribute stimulus-specific transcriptional programs through the selective regulation of individual MAPK substrates and the subcellular localization and activity of the individual DUSP (Figure 2B).…”

Section: Dusp In Immunity and Infectionmentioning

confidence: 99%

Dual-Specificity Phosphatases in Immunity and Infection: An Update

Lang

Raffi

2019

IJMS

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Kinase activation and phosphorylation cascades are key to initiate immune cell activation in response to recognition of antigen and sensing of microbial danger. However, for balanced and controlled immune responses, the intensity and duration of phospho-signaling has to be regulated. The dual-specificity phosphatase (DUSP) gene family has many members that are differentially expressed in resting and activated immune cells. Here, we review the progress made in the field of DUSP gene function in regulation of the immune system during the last decade. Studies in knockout mice have confirmed the essential functions of several DUSP-MAPK phosphatases (DUSP-MKP) in controlling inflammatory and anti-microbial immune responses and support the concept that individual DUSP-MKP shape and determine the outcome of innate immune responses due to context-dependent expression and selective inhibition of different mitogen-activated protein kinases (MAPK). In addition to the canonical DUSP-MKP, several small-size atypical DUSP proteins regulate immune cells and are therefore also reviewed here. Unexpected and complex findings in DUSP knockout mice pose new questions regarding cell type-specific and redundant functions. Another emerging question concerns the interaction of DUSP-MKP with non-MAPK binding partners and substrate proteins. Finally, the pharmacological targeting of DUSPs is desirable to modulate immune and inflammatory responses.

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“…The RNAseq dataset corroborated that stimulation of macrophages with the cord factor TDM induces substantial transcriptome changes, which are both MINCLE-dependent and MINCLE-independent (Hansen et al, 2019). Given the important role of miRNAs in innate immune regulation, the finding that a large fraction of TDM-regulated genes was not affected by DGCR8-deficiency may seem surprising.…”

Section: Discussionmentioning

confidence: 70%

“…Shown are mean + SD of n = 5 mice from five independent experiments. Schoenen et al, 2014;Hansen et al, 2019). Hyperexpression of TDMinduced genes in DGCR8-deficient macrophages may be due to a lack of constitutively expressed miRNAs controlling the initial response or to the absence of inducible miRNAs acting as negative feedback regulators.…”

Section: Kinetics Of Dysregulated Tdm-induced Gene Expression In Dgcrmentioning

confidence: 99%

DGCR8 deficiency impairs macrophage growth and unleashes the interferon response to mycobacteria

Killy

Bodendorfer

Mages³

et al. 2021

Life Sci. Alliance

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The mycobacterial cell wall glycolipid trehalose-6,6-dimycolate (TDM) activates macrophages through the C-type lectin receptor MINCLE. Regulation of innate immune cells relies on miRNAs, which may be exploited by mycobacteria to survive and replicate in macrophages. Here, we have used macrophages deficient in the microprocessor component DGCR8 to investigate the impact of miRNA on the response to TDM. Deletion of DGCR8 in bone marrow progenitors reduced macrophage yield, but did not block macrophage differentiation. DGCR8-deficient macrophages showed reduced constitutive and TDM-inducible miRNA expression. RNAseq analysis revealed that they accumulated primary miRNA transcripts and displayed a modest type I IFN signature at baseline. Stimulation with TDM in the absence of DGCR8 induced overshooting expression of IFNβ and IFN-induced genes, which was blocked by antibodies to type I IFN. In contrast, signaling and transcriptional responses to recombinant IFNβ were unaltered. Infection with live Mycobacterium bovis Bacille Calmette–Guerin replicated the enhanced IFN response. Together, our results reveal an essential role for DGCR8 in curbing IFNβ expression macrophage reprogramming by mycobacteria.

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Macrophage Phosphoproteome Analysis Reveals MINCLE-dependent and -independent Mycobacterial Cord Factor Signaling

Cited by 21 publications

References 74 publications

Phosphoproteomics reveals that the hVPS34 regulated SGK3 kinase specifically phosphorylates endosomal proteins including Syntaxin-7, Syntaxin-12, RFIP4 and WDR44

Phosphoproteomics reveals that the hVPS34 regulated SGK3 kinase specifically phosphorylates endosomal proteins including Syntaxin-7, Syntaxin-12, RFIP4 and WDR44

Dual-Specificity Phosphatases in Immunity and Infection: An Update

DGCR8 deficiency impairs macrophage growth and unleashes the interferon response to mycobacteria

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