Knockdown of the C. elegans Kinome identifies Kinases required for normal protein Homeostasis, Mitochondrial network structure, and Sarcomere structure in muscle

Abstract

BackgroundKinases 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.Results96 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.ConclusionsRoughly 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.

Functional phosphatome requirement for protein homeostasis, networked mitochondria, and sarcomere structure in C. elegans muscle

“…By comparing the developmental phenotypes, such as growth or uncoordinated movement observed in this study to developmental phenotypes observed in RNAi experiments by other investigators using the same RNAi bacteria clone, a potential discrepancy of RNAi results for 17% of total genes screened was identified. This is in concordance with published RNAi screens17, 20 and half of these potential discrepancies are cases in which we identified a developmental phenotype in response to RNAi but for which a wild‐type phenotype was observed in RNAi experiments by others, indicating that either the RNAi was more effective in this study and therefore these results may be new findings, or these results are false positives. This is again consistent with published RNAi screens17, 20 and most likely represents our method producing a first discovery of function rate that is higher than past studies.…”

“…Quality control of our RNAi screens was as previously described and diagrammed for the RNAi screen of the C. elegans kinome requirement for a muscle 20. By comparing the developmental phenotypes, such as growth or uncoordinated movement observed in this study to developmental phenotypes observed in RNAi experiments by other investigators using the same RNAi bacteria clone, a potential discrepancy of RNAi results for 17% of total genes screened was identified.…”

“…Middle (yellow): autophagic degradation is controlled by a balance of signal from insulin/insulin‐like receptor (negative regulator, green lines) and autocrine fibroblast growth factor signal (positive regulator, red lines) 12, 13, 14. Calcium overload, signalling via CaMKII, also promotes autophagic degradation17 as does knockdown of a number of kinases 20. Right (pink): intracellular calcium controlled by a combination of membrane depolarization, and G‐protein signalling events are required to negatively regulate proteasome‐based degradation 15, 17.…”

“…Right (pink): intracellular calcium controlled by a combination of membrane depolarization, and G‐protein signalling events are required to negatively regulate proteasome‐based degradation 15, 17. Displayed model is adapted from models published in Shephard et al 17 and Gaffney et al 19 (B) A schematic of the full RNA interference screen can be found in the kinase screen20 which this phosphatase screen is based upon. Briefly, for identification of phosphatase, genes whose knockdown induced autophagic protein degradation was achieved through four steps: (1) genes for which RNA interference produced decreased amounts of reporter protein in muscle were identified.…”

“…Three recent kinome‐wide RNAi screens performed in C. elegans to identify the kinome requirement for normal muscle development and homeostasis20 identified roughly 40% of the kinome as being important for establishing and/or maintaining proteostasis, mitochondrial structure, or sarcomere structure in muscle. Of these kinases identified in C. elegans , 80 have identified human orthologues and 53 are known to be expressed in skeletal muscle.…”