Project description:Overexpressed either GFP, wild-type (WT) NFIX or a phospho-dead mutant of NFIX in which eight serine residues surrounding S286 were mutated to alanine (S265/267/268/271/272/273/274/275A) was performed C2C12 cells prior to the induction of myogenesis. Cells were harvest 2 days post induction of myogensis with 2% horse serum.
Project description:Pathogenic mutations in alpha kinase 3 (ALPK3) cause cardiomyopathy and a range of musculoskeletal defects. How ALPK3 mutations result in disease remains unclear and little is known about this atypical kinase. Using a suite of engineered human pluripotent stem cells (hPSCs) we show that ALPK3 localizes to the sarcomere, specifically at the M-Band. Both sarcomeric organization and calcium kinetics were disrupted in ALPK3 deficient hPSC derived cardiomyocytes. Further, cardiac organoids derived from ALPK3 knockout hPSCs displayed reduced force generation. Phosphoproteomic profiling of wildtype and ALPK3 null hPSC derived cardiomyocytes revealed ALPK3-dependant phospho-peptides were enriched for proteins involved in sarcomere function and protein quality control. We demonstrate that ALPK3 binds to the selective autophagy receptor SQSTM1 (Sequestome 1) and is required for the sarcomeric localization of SQSTM1. We propose that ALPK3 is a myogenic kinase with an integral role in the intracellular signaling networks underlying sarcomere maintenance required for continued cardiac contractility.
Project description:Myogenesis is governed by signalling networks whose regulations are tightly controlled in a time-dependent manner. While different protein kinases have been identified to regulate various aspects of myogenesis, knowledge on the global signalling networks and their downstream substrates during myogenesis remains incomplete. Here, we map the myogenic differentiation of C2C12 cells using mass spectrometry (MS)-based phosphoproteomics and proteomics. From these data, we infer global kinase activity and predict substrates of key kinases that are involved in myogenesis. We found that multiple mitogen-activated protein kinases (MAPKs) mark the initial wave of signalling cascades. Further phosphoproteomic and proteomic profiling with MAPK1/3 and MAPK8/9 specific inhibitions unveil their shared and distinctive roles on myogenesis.
Project description:Pathogenic mutations in alpha kinase 3 (ALPK3) cause cardiomyopathy and a range of musculoskeletal defects. How ALPK3 mutations result in disease remains unclear and little is known about this atypical kinase. Using a suite of engineered human pluripotent stem cells (hPSCs) we show that ALPK3 localizes to the sarcomere, specifically at the M-Band. Both sarcomeric organization and calcium kinetics were disrupted in ALPK3 deficient hPSC derived cardiomyocytes. Further, cardiac organoids derived from ALPK3 knockout hPSCs displayed reduced force generation. Phosphoproteomic profiling of wildtype and ALPK3 null hPSC derived cardiomyocytes revealed ALPK3-dependant phospho-peptides were enriched for proteins involved in sarcomere function and protein quality control. We demonstrate that ALPK3 binds to the selective autophagy receptor SQSTM1 (Sequestome 1) and is required for the sarcomeric localization of SQSTM1. We propose that ALPK3 is a myogenic kinase with an integral role in the intracellular signaling networks underlying sarcomere maintenance required for continued cardiac contractility.
Project description:To gain insight into possible regulators of liver extracellular vesicle (EV)-mediated glucose effectiveness, we evaluated the phosphoproteomic profile of soleus muscle incubated in low and high glucose, without or with liver EVs
Project description:C18ORF25 is a homolog of Arkadia (RNF111), an E3 ubiquitin ligase with SUMO-interaction motifs (SIMs) (PMID: 31417085). However, C18ORF25 lacks the entire C-terminal RING domain of RNF111 which is required for ubiquitin binding suggesting it lacks ubiquitination activity and may therefore act as an adaptor or signalling scaffold (PMID: 26283374). We have previously shown that mice lacking C18Orf25 throughout the entire body have increased adiposity, decreased lean mass, lower exercise capacity and significantly reduced ex vivo skeletal muscle force production (PMID: 35882232). Skeletal muscle isolated from C18Orf25 knockout (KO) mice have reduced cAMP-dependent protein kinase A (PKA) levels, and reduced phosphorylation of several contractile proteins and proteins involved in calcium handling. Furthermore, analysis of single muscle fibres from C18Orf25 KO mice revealed impaired SR calcium cycling in fast-twitch fibres only (PMID: 35882232). Hence, we investigated these mechanisms by developing an integrated single-fibre physiology and single-fibre proteomic platform. The platform enabled us to identify hundreds of novel phenotype:protein correlations. The analysis also enabled us to identify proteome differences specifically in FT fibres following loss of C18ORF25. Taken together, our data suggest C18ORF25 is likely a multi-functional protein with several underlying mechanisms contributing to skeletal muscle physiology.
Project description:We recently identified C18ORF25 as a new exercise-regulated phophoprotein. To investigate potential in vivo functions of C18ORF25, we used CRISPR/Cas9 to generate a whole-body knock-out (KO) mouse model on a C57BL/6J background. Proteomic analysis was performed to identify potental changes in the proteome. To gain further insights into the possible signalling pathways regulated by C18ORF25, Soleus muscles from WT and KO mice (n=4) were isolated, and the muscle from one leg was maintained at resting tension with no stimulation as a control while the muscle from the contralateral leg was subject to electrical stimulation ex vivo. Muscles were quickly snap frozen and subject to single-shot label-free phosphoproteomic analysis to compare the signalling responses between the genotypes.
Project description:To characterize phosphorylation-based signaling events across different exercise modalities we subjected eight healthy young men (age, 26.3±1.3 years; BMI, 23.5±0.7 kg/m2; maximal oxygen uptake (VO2 max), 42.6±1.5 ml/kg/min) that did not perform regular physical activity apart from local bicycling to an acute bout of endurance (90 min ~60% of VO2 max), sprint (3 x 30-s all-out cycling), or resistance exercise (6 sets of 10 RM knee extensions) in the fasting state. All participants completed the three types of exercise in a randomized crossover design with 14 days washout between each exercise bout.
Project description:MASTL (microtubule-associated serine/threonine kinase-like) has emerged as a critical regulator of mitosis and as a potential oncogene in a variety of cancer types. To date, Arpp-19/ENSA are the only known substrates of MASTL. With the cellular roles of MASTL expanding and increased interest in development of MASTL inhibitors, it has become critical to determine if there are any additional substrates and if so, what the optimal consensus motif for MASTL is. Here we utilised a whole cell lysate (in cellulo) kinase screen approach combined with stable isotope labelling of amino acids in cell culture (SILAC) to identify novel substrates and a consensus motif of MASTL. Using the related AGC kinase family members AKT1/2, the in cellulo assay identified several known and new substrates highly enriched with the validated consensus motif for AKT. Applying this method to MASTL identified 59 phosphosites on 26 novel proteins significantly increased in the presence of active MASTL. Subsequent in vitro kinase assays confirmed that MASTL was capable of phosphorylating YB1, RPS6, TUBA1C, and RPL36A under some conditions, and hnRNPM specifically on a single site (S-633/7). Taken together, these data suggest that MASTL can phosphorylate several additional substrates, which may help provide greater insight into the ever-increasing biological functions and roles MASTL plays in driving cancer progression and therapy resistance.