Analysis of insulin-regulated phosphorylation of AFF4 with Akti or S6ki and S6K invitro kinase
Ontology highlight
ABSTRACT:
INSTRUMENT(S):
ORGANISM(S): Mus Musculus (mouse)
SUBMITTER: 
Benjamin Parker
LAB HEAD: Benjamin Parker
PROVIDER: PXD054247 | Pride | 2025-12-16
REPOSITORIES: Pride
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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.
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Project description:A single bout of exercise followed by intake of carbohydrates leads to glycogen supercompensation in the prior exercised muscle. The molecular mechanisms underlying this well-known phenomenon remain elusive. Here we report that a single bout of exercise induces marked activation of glycogen synthase (GS) and AMP-activated protein kinase (AMPK) for several days beyond normalized muscle glycogen content in man. Acute muscle specific deletion of AMPK activity in mouse muscle abrogated the ability for glycogen supercompensation, providing genetic evidence that AMPK serves as essential driver for glycogen supercompensation. Muscle proteomic analyses revealed elevated glucose uptake capacity in the prior exercised muscle while key proteins in fat oxidation and glycolysis largely remained unchanged. The temporal order of these sustained cellular alterations induced by a single bout of exercise provide a mechanism to offset the otherwise tight feedback inhibition of glycogen synthesis and glucose uptake by glycogen, ultimately leading to muscle glycogen supercompensation.
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Project description:Ubiquitination is a post-translational modification that has been shown to have a range of effects, such as regulating protein function, protein:protein interactions, protein localization, and protein abundance by targeting proteins for degradation by the 26S proteasome. We have previously shown that the muscle-specific ubiquitin E3 ligase, ASB2, is down-regulated in models of muscle growth and that overexpression ASB2 is sufficient to induce muscle atrophy. To gain insight into the effect of increased ASB2 expression on skeletal muscle mass and function, we used 2-dimensional nano-ultra-high-performance mass spectrometry to investigate ASB2-induced changes to the mouse skeletal muscle proteome, and whether these were associated with changes in protein ubiquitination. The results show that in vivo recombinant adeno-associated viral vector-mediated ASB2β overexpression induces impaired intrinsic force production and progressive muscle atrophy over 12 weeks, while ASB2 knockdown induces mild muscle hypertrophy. ASB2-induced muscle atrophy and dysfunction were associated with the early down regulation of mitochondrial and contractile proteins, and the upregulation of proteins involved in proteasome-mediated protein degradation, protein synthesis and the cytoskeleton/sarcomere. The overexpression ASB2β also resulted in marked changes in protein ubiquitination, however, there was no simple relationship between changes in ubiquitination status and protein abundance. To gain insights into proteins that interact with ASB2, and potential ASB2 targets, in muscle cells, flag-tagged wild type ASB2, and a mutant ASB2 with a deleted C-terminal SOCS box domain (dSOCS) were immunoprecipitated from C2C12 myotubes and subjected to label-free proteomic analysis to determine the ASB2 interactome. ASB2β was found to interact with a range of cytoskeletal and nuclear proteins. When combined with the in vivo ubiquitinomic data, we identified several novel potential ASB2β target substrates that await further investigation. Overall, this study reveals for the first time the complexity of changes to the proteome and ubiquitinome during E3 ligase-mediated skeletal muscle atrophy and dysfunction.
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