The mitochondria-associated membrane (MAM) has emerged as a cellular signaling hub regulating various cellular processes. However, its molecular components remain unclear owing to lack of reliable methods to purify the intact MAM proteome in a physiological context. Here, we introduce Contact-ID, a split-pair system of BioID with strong activity, for identification of the MAM proteome in live cells. Contact-ID specifically labeled proteins proximal to the contact sites of the endoplasmic reticulum (ER) and mitochondria, and thereby identified 115 MAM-specific proteins. The identified MAM proteins were largely annotated with the outer mitochondrial membrane (OMM) and ER membrane proteins with MAM-related functions: e.g., FKBP8, an OMM protein, facilitated MAM formation and local calcium transport at the MAM. Furthermore, the definitive identification of biotinylation sites revealed membrane topologies of 85 integral membrane proteins. Contact-ID revealed regulatory proteins for MAM formation and could be reliably utilized to profile the proteome at any organelle–membrane contact sites in live cells.
The inner mitochondrial membrane (IMM) proteome plays a central role in maintaining mitochondrial physiology and cellular metabolism. Various important biochemical reactions such as oxidative phosphorylation, metabolite production, and mitochondrial biogenesis are conducted by the IMM proteome, and mitochondria-targeted therapeutics have been developed for IMM proteins, which is deeply related for various human metabolic diseases including cancer and neurodegenerative diseases. However, the membrane topology of the IMM proteome remains largely unclear because of the lack of methods to evaluate it in live cells in a high-throughput manner. In this article, we reveal the in vivo topological direction of 135 IMM proteins, using an in situ-generated radical probe with genetically targeted peroxidase (APEX). Owing to the short lifetime of phenoxyl radicals generated in situ by submitochondrial targeted APEX and the impermeability of the IMM to small molecules, the solvent-exposed tyrosine residues of both the matrix and intermembrane space (IMS) sides of IMM proteins were exclusively labeled with the radical probe in live cells by Matrix-APEX and IMS-APEX, respectively and identified by mass spectrometry. From this analysis, we confirmed 58 IMM protein topologies and we could determine the topological direction of 77 IMM proteins whose topology at the IMM has not been fully characterized. We also found several IMM proteins (e.g., LETM1 and OXA1) whose topological information should be revised on the basis of our results. Overall, our identification of structural information on the mitochondrial inner-membrane proteome can provide valuable insights for the architecture and connectome of the IMM proteome in live cells.
3-Phosphoinositide-dependent protein kinase-1 (PDK1) appears to play a central regulatory role in many cell signalings between phosphoinositide-3 kinase and various intracellular serine/threonine kinases. In resting cells, PDK1 is known to be constitutively active and is further activated by tyrosine phosphorylation (Tyr 9 and Tyr 373/376 ) following the treatment of the cell with insulin or pervanadate. However, little is known about the mechanisms for this additional activation of PDK1. Here, we report that the SH2 domain of Src, Crk, and GAP recognized tyrosine-phosphorylated PDK1 in vitro. Destabilization of PDK1 induced by geldanamycin (a Hsp90 inhibitor) was partially blocked in HEK 293 cells expressing PDK1-Y9F. Co-expression of Hsp90 enhanced PDK1-Src complex formation and led to further increased PDK1 activity toward PKB and SGK. Immunohistochemical analysis with anti-phospho-Tyr 9 antibodies showed that the level of Tyr 9 phosphorylation was markedly increased in tumor samples compared with normal. Taken together, these data suggest that phosphorylation of PDK1 on Tyr 9 , distinct from Tyr 373/376 , is important for PDK1/Src complex formation, leading to PDK1 activation. Furthermore, Tyr 9 phosphorylation is critical for the stabilization of both PDK1 and the PDK1/Src complex via Hsp90-mediated protection of PDK1 degradation.One of the key features of multicellular organisms is that all cells are able to adjust to changes in the surrounding environment. A diverse set of environmental cues utilize intracellular protein phosphorylation-dephosphorylation cascades to rapidly and reversibly transduce their signals from their plasma membrane receptors to the cytoplasm and the nucleus. 3-Phosphoinositide-dependent protein kinase-1 (PDK1) 3 was originally identified as an upstream kinase for protein kinase B (PKB/Akt) (1) and is recognized as a master protein kinase for regulating in many cell-signaling pathways (2-5).Targets of PDK1 include many of the AGC family of protein kinases, including protein kinase B (PKB/Akt), p70 ribosomal protein S6 kinase (p70 S6K ), cyclic AMP-dependent protein kinase, protein kinase C, serum and glucocorticoid-inducible kinase (SGK), p90 ribosomal protein S6 kinase (RSK), and p21-activated kinase-1 (PAK1) (4). However, the generation of PDK1-ablated or PDK1-hypomorphic (ϳ10% of PDK1 expression) mice revealed that most of the PDK1 substrates identified in vitro were not physiological targets for PDK1 in vivo, with the exception of PKB, p70 S6K , and RSK (6, 7). PDK1(Ϫ/Ϫ) mice die at embryonic day 9.5 with multiple abnormalities, whereas hypomorphic PDK1 mice are viable (6). Nevertheless, these mice are 40 -50% smaller than control animals due to small cell size, but not cell number, providing genetic evidence that PDK1 is essential for mouse embryonic development and regulates cell size (6).PDK1 possesses an N-terminal kinase domain and a C-terminal pleckstrin homology domain (8, 9). Phosphorylation of PKB by PDK1 is dependent upon prior activation by phosphoinositide 3-kinase a...
Expression of human mitochondrial DNA is indispensable for proper function of the oxidative phosphorylation machinery. The mitochondrial genome encodes 22 tRNAs, 2 rRNAs and 11 mRNAs and their post-transcriptional modification constitutes one of the key regulatory steps during mitochondrial gene expression. Cytosine-5 methylation (m5C) has been detected in mitochondrial transcriptome, however its biogenesis has not been investigated in details. Mammalian NOP2/Sun RNA Methyltransferase Family Member 2 (NSUN2) has been characterized as an RNA methyltransferase introducing m5C in nuclear-encoded tRNAs, mRNAs and microRNAs and associated with cell proliferation and differentiation, with pathogenic variants in NSUN2 being linked to neurodevelopmental disorders. Here we employ spatially restricted proximity labelling and immunodetection to demonstrate that NSUN2 is imported into the matrix of mammalian mitochondria. Using three genetic models for NSUN2 inactivation—knockout mice, patient-derived fibroblasts and CRISPR/Cas9 knockout in human cells—we show that NSUN2 is necessary for the generation of m5C at positions 48, 49 and 50 of several mammalian mitochondrial tRNAs. Finally, we show that inactivation of NSUN2 does not have a profound effect on mitochondrial tRNA stability and oxidative phosphorylation in differentiated cells. We discuss the importance of the newly discovered function of NSUN2 in the context of human disease.
Activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) triggers cellular signals that lead to the activation of the transcription factor NF-kappaB (nuclear factor kappaB) in various cell types. In addition to NF-kappaB activation by short-time PMA treatment, here we report that the prolonged exposure of human colonic cancer epithelial cells treated with PMA can also lead to a persistent inhibition of NF-kappaB activation. PMA selectively causes the degradation of IkappaB kinases (IKKs) including IKK-gamma and IKK-beta, and subsequent inhibition of tumor necrosis factor (TNF) induced IKK and NF-kappaB activation in human colon cancer cell line HCT-116, but not in other gastrointestinal tract cells. The use of Ro-318220 and GO-6983, general PKC inhibitors as well as MG-132, a proteasome-specific inhibitor, abrogated PMA-induced degradation of IKK-gamma and recovered the activation of IKK by TNF, suggesting that IKK complex is predominantly degraded by the proteasome pathway in a PKC-dependent manner. We also found that IKK-gamma strongly associates with heat shock protein 90 (Hsp90) in HCT-116 cells, and that this interaction was dramatically reduced after exposure to PMA. Furthermore, high levels of Hsp90 expression and enhanced association with IKK were observed in human colon cancer tissues. Taken together, these results suggest that long-term activation of PKC by PMA inhibits NF-kappaB system in case of colon cancer cells by disrupting the interaction of IKK-gamma with Hsp90, which may represent a novel regulatory mechanism of PKC-dependent cellular differentiation and limited proliferation of colonic epithelial cells.
Antipsychotic drugs are regularly used for the treatment of many types of psychiatric disorders. The administration of second-generation antipsychotics is often associated with weight gain and the development of diabetes mellitus; however, the molecular mechanisms underlying the effects of these drugs remain poorly understood. Leptin and insulin play key roles in the regulation of energy balance and glucose homeostasis, and resistance to the actions of these hormones can occur with obesity and inflammation, resulting in the pathogenesis of obesity and type 2 diabetes. In this study, the effects of risperidone on the insulin-induced protein kinase B (PKB) phosphorylation and leptin-stimulated signal transducer and activator of transcription 3 (STAT3) phosphorylation were investigated in the human SH-SY5Y neuroblastoma cell line. The treatment of these cells with risperidone induced the activation of extracellular signal-related kinase (ERK) by cellular cyclic adenosine 3-monophosphate (cAMP)-dependent protein kinase (also known as protein kinase A; PKA) and the mechanisms involved include the induction of suppressor of cytokine signaling 3 (SOCS3) and suppressor of cytokine signaling 6 (SOCS6) expression. The risperidone-induced ERK activation induced an upregulation of SOCS3 and SOCS6 mRNA expression levels. Taken together, these results suggest that risperidone modulates SOCS3 and SOCS6 expression through adenylate cyclase-mediated ERK activation, which, in turn, leads to an inhibition of insulin-induced PKB phosphorylation and leptin-stimulated STAT3 phosphorylation. Eventually, these effects result in excessive body weight gain due to the inhibition of both the leptin and insulin signaling pathways.
Protein kinase C (PKC) triggers cellular signals that regulate proliferation or death in a cell-and stimulus-specific manner. Although previous studies have demonstrated that activation of PKC with phorbol 12-myristate 13-acetate (PMA) protects cells from apoptosis induced by a number of mechanisms, including death receptor ligation, little is known about the effect or mechanism of PMA in the necrotic cell death. Here, we demonstrate that PMA-mediated activation of PKC protects against tumor necrosis factor (TNF)-induced necrosis by disrupting formation of the TNF receptor (TNFR)1 signaling complex. Pretreatment with PMA protected L929 cells from TNF-induced necrotic cell death in a PKC-dependent manner, but it did not protect against DNA-damaging agents, including doxorubicin (Adriamycin) and camptothecin. Analysis of the upstream signaling events affected by PMA revealed that it markedly inhibited the TNF-induced recruitment of TNFR1-associated death domain protein (TRADD) and receptor-interacting protein (RIP) to TNFR1, subsequently inhibiting TNF-induced activation of nuclear factor-B and c-Jun NH 2 -terminal kinase (JNK). However, JNK inhibitors do not significantly affect TNF-induced necrosis, suggesting that the inhibition of JNK activation by PMA is not part of the antinecrotic mechanism. In addition, PMA acted as an antagonist of TNF-induced reactive oxygen species (ROS) production, thereby suppressing activation of ROS-mediated poly(ADP-ribose)polymerase (PARP), and thus inhibiting necrotic cell death. Furthermore, during TNF-induced necrosis, PARP was significantly activated in wild-type mouse embryonic fibroblast (MEF) cells but not in RIPϪ/Ϫ or TNFR-associated factor 2Ϫ/Ϫ MEF cells. Taken together, these results suggest that PKC activation ensures effective shutdown of the death receptor-mediated necrotic cell death pathway by modulating formation of the death receptor signaling complex.Members of the tumor necrosis factor (TNF) superfamily are critical regulators of the balance between opposing signals that can trigger either cell survival or death, depending upon the predominating signaling pathway in the particular
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