Knockdown of the C. elegans Kinome identifies Kinases required for normal protein Homeostasis, Mitochondrial network str
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RESEARCH
Open Access
Knockdown of the C. elegans Kinome identifies Kinases required for normal protein Homeostasis, Mitochondrial network structure, and Sarcomere structure in muscle Susann Lehmann, Joseph J Bass and Nathaniel J Szewczyk*
Abstract Background: Kinases are important signalling molecules for modulating cellular processes and major targets of drug discovery programs. However, functional information for roughly half the human kinome is lacking. We conducted three kinome wide, >90%, RNAi screens and epistasis testing of some identified kinases against known intramuscular signalling systems to increase the functional annotation of the C. elegans kinome and expand our understanding of kinome influence upon muscle protein degradation. Results: 96 kinases were identified as required for normal protein homeostasis, 74 for normal mitochondrial networks and 50 for normal sarcomere structure. Knockdown of kinases required only for normal protein homeostasis and/or mitochondrial structure was significantly less likely to produce a developmental or behavioural phenotype than knockdown of kinases required for normal sarcomere structure and/or other sub-cellular processes. Lastly, assessment of kinases for which knockdown produced muscle protein degradation against the known regulatory pathways in C. elegans muscle revealed that close to half of kinase knockdowns activated autophagy in a MAPK dependent fashion. Conclusions: Roughly 40% of kinases studied, 159 of 397, are important in establishing or maintaining muscle cell health, with most required for both. For kinases where decreased expression triggers protein degradation, autophagy is most commonly activated. These results increase the annotation of the C. elegans kinome to roughly 75% and enable future kinome research. As 33% of kinases identified have orthologues expressed in human muscle, our results also enable testing of whether identified kinases function similarly in maintaining human muscle homeostasis. Keywords: Autophagy, C. elegans, Kinome, MAPK, Mitochondria, Muscle, Protein degradation, Proteostasis, Sarcomere
Background Kinases are enzymes that alter proteins and lipids by phosphorylation, the addition of a phosphate group. This modification can influence a protein’s steric structure and cause changes in protein-protein binding affinities and enzyme activities [1]. Kinase encoding genes constitute 2% of the human genome making kinases one of * Correspondence: [email protected] Medical Research Council/Arthritis Research UK Centre for Musculoskeletal Ageing Research, University of Nottingham, Royal Derby Hospital, Derby DE22 3DT, England
the largest protein families, which is termed the kinome. Kinases also appear to play a major role in modulating cellular processes as roughly 30% of intracellular proteins are phosphate bound at any given time [2]. Accordingly, kinase inhibitors account for a large part of drug discovery programs in the pharmaceutical industry. Roughly 150 inhibitors of 42 well validated kinase targets are currently being t
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