Casein kinase 2 is a direct binding partner and a functional target of the exercise-mimetic microprotein MOTS-c
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 
Published: May 2023

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 administration of WT MOTS-c (7.5 mg/kg) into young male C57BL/6J mice (n = 10-11/group) significantly increased skeletal muscle 2-Deoxy-D-glucose (2DG) uptake, but not K14Q MOTS-c. As expected, administration of a CK2 inhibitor (CX-4945, 25 mg/kg) with WT MOTS-c eliminates the MOTS-c effect, suggesting that MOTS-c promoted 2DG uptake by activating CK2. Altogether, our study demonstrated that MOTS-c directly binds to and activates CK2, resulting in physiologically crucial effects on skeletal muscle.

NIH grants R01AG061834, R01AG069698, P30AG068345, R01AG068405, and P01AG034906.

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.