Casein kinase 2 is a direct binding partner and a functional target of the exercise-mimetic microprotein MOTS-c

• Published in: Physiology (Bethesda, Md.) Md.), 2023-05, Vol.38 (S1)
• Main Authors: Kumagai, Hiroshi, Kim, Su-Jeong, Miller, Brendan, Natsume, Toshiharu, Lee, Shin Hyung, Sato, Ayaka, Ramirez, Ricardo, Wan, Junxiang, Mehta, Hemal H, Yen, Kelvin, Cohen, Pinchas
• Format: Article
• Language: English
• ISSN: 1548-9213; 1548-9221
• DOI: 10.1152/physiol.2023.38.S1.5725846

Abstract only MOTS-c is a novel mitochondrial DNA-encoded microprotein and is expressed in several tissues including skeletal muscle. Since the primary function of MOTS-c is to improve aging- and high-fat diet-induced impaired glucose uptake in the skeletal muscle, MOTS-c has been described as a metabolic regulator and exercise-mimetic microprotein. However, the direct and functional target has not been identified yet. We have previously observed increased casein kinase 2 (CK2) activity in MOTS-c-treated mouse skeletal muscle. Therefore, based on this observation, we hypothesized that CK2 is a direct target of MOTS-c in the skeletal muscle and performed a series of experiments to test our hypothesis. First, dot blot and kinase activity assays showed that MOTS-c directly bound and activated CK2 in vitro. Then, since CK2 is a complex of alpha (CK2α) and beta (CK2β) subunits, we performed a dot blot assay using CK2α and CK2β subunits and observed that MOTS-c bound to the CK2α subunit but not the CK2β subunit. This binding was confidently confirmed using a surface plasmon resonance assay (also called a Biacore assay). On the MOTS-c coding region, there is a naturally occurring variation causing a K14Q amino acid replacement that has been suggested to be a bio-inactive form of MOTS-c and increases type 2 diabetes risk. Interestingly, the Biacore assay demonstrated that the binding affinity of K14Q MOTS-c to CK2α was 16 times less than that of wild type (WT) MOTS-c, and K14Q MOTS-c did not increase CK2 activity in an in vitro kinase assay. Then, we conducted mouse experiments to validate these observations in vivo. A single administration of WT MOTS-c (7.5 mg/kg) into young male C57BL/6J mice (n = 3/group) significantly increased CK2 activity in the skeletal muscle, but not K14Q MOTS-c. Additionally, a western blot following CK2α immunoprecipitation (IP) confirmed the binding between MOTS-c and CK2α in MOTS-c administered mouse skeletal muscle. Importantly, skeletal muscle proteomics analysis following CK2α IP showed that MOTS-c changed the CK2 interactome by recruiting over 30 additional proteins and building a chaperone complex network, which was important for myofibrillogenesis and maintenance of muscle structure and function. Additionally, CK2α no longer binds to PP2A, a protein phosphatase that dephosphorylates AKT, in MOTS-c treated skeletal muscle. Next, we investigated whether CK2 mediates the MOTS-c induced skeletal muscle glucose uptake. A single admin