Kumagai et al. · iScience · 2024
MOTS-c modulates skeletal muscle function by directly binding and activating CK2
Kumagai Hiroshi, Kim Su-Jeong, Miller Brendan, Zempo Hirofumi, Tanisawa Kumpei, Natsume Toshiharu, Lee Shin Hyung, Wan Junxiang, Leelaprachakul Naphada, Kumagai Michi Emma, Ramirez Ricardo, Mehta Hemal H., Cao Kevin, Oh Tae Jung, Wohlschlegel James A., Sha Jihui, Nishida Yuichiro, Fuku Noriyuki, Dobashi Shohei, Miyamoto-Mikami Eri, Takaragawa Mizuki, Fuku Mizuho, Yoshihara Toshinori, Naito Hisashi, Kawakami Ryoko, Torii Suguru, Midorikawa Taishi, Oka Koichiro, Hara Megumi, Iwasaka Chiharu, Yamada Yosuke, Higaki Yasuki, Tanaka Keitaro, Yen Kelvin, Cohen Pinchas
The study
What was asked, and what was found
Kumagai et al., iScience, 2024, set out to find what the mitochondrial microprotein MOTS-c binds. The answer was the kinase CK2. MOTS-c bound CK2 directly and activated it in cell-free systems, and in mice it prevented immobilization-induced muscle wasting and raised muscle glucose uptake, and the rise in glucose uptake was lost when CK2 activity was suppressed. A naturally occurring variant of MOTS-c, K14Q, bound CK2 poorly and did none of this. In Japanese population studies, male carriers of that variant had a higher risk of sarcopenia and type 2 diabetes in an age and activity dependent way.
The AUM material sits at the point where the argument had to be closed. Having shown that a CK2 inhibitor blocked the effect, the group needed to remove the kinase itself. They used AUMsilence sdASOs against CK2 alpha in differentiated human skeletal muscle myotubes, grown from a Lonza primary line taken from a healthy 40 year old male donor. Five sequences were screened by immunoblot at 5 μM over 48 hours against an AUMscramble control, and one of them, ASO number 2, was carried into the functional assay at 5 μM over 24 hours in serum-free medium.
Wild-type MOTS-c raised 2-deoxyglucose uptake in the scramble arm and did nothing in the knockdown arm, and the inactive K14Q variant raised uptake in neither. That is the result that turns CK2 from a binding partner into a required step. The Methods are unusually explicit about why an antisense oligonucleotide was chosen here: the paper writes that these oligos are self-deliverable, need no transfection reagent, and therefore keep toxicity to the cells low.
Key findings
- The paper states plainly in its own Methods that the AUMsilence sdASOs reach the cell on their own, with no transfection reagent, which is why the group chose them for differentiated human myotubes.(Methods, Cell culture)
- Five AUMsilence sdASO sequences against CK2 alpha were screened by immunoblot at 5 μM over 48 hours, and one of them, ASO number 2, was carried into the functional assay at 5 μM over 24 hours.(Supplemental Figure 12A legend, Supplementary Data)
- Knocking down CK2 alpha abolished the rise in glucose uptake that MOTS-c normally produces in human myotubes, while the scramble control left it intact, which is the experiment that made CK2 a required step rather than a bystander.(Results, CK2 activation has a crucial role in MOTS-c-induced improvements in muscle mass and glucose uptake)
- The knockdown result matched the pharmacological one: blocking the kinase and silencing it both lowered glucose uptake in muscle.(Discussion)
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