Transcriptional, biochemical, and immunohistochemical analyses of CaMKKβ/2 splice variants that co-localize with CaMKIV in spermatids

Satomi Ohtsuka; Yumi Miyai; Hiroyuki Mima; Masaki Magari; Yoichi Chiba; Futoshi Suizu; Hiroyuki Sakagami; Masaki Ueno; Hiroshi Tokumitsu

doi:10.1016/j.ceca.2023.102820

Transcriptional, biochemical, and immunohistochemical analyses of CaMKKβ/2 splice variants that co-localize with CaMKIV in spermatids

Cell Calcium. 2024 Jan:117:102820. doi: 10.1016/j.ceca.2023.102820. Epub 2023 Nov 8.

Authors

Satomi Ohtsuka¹, Yumi Miyai², Hiroyuki Mima¹, Masaki Magari¹, Yoichi Chiba², Futoshi Suizu³, Hiroyuki Sakagami⁴, Masaki Ueno², Hiroshi Tokumitsu⁵

Affiliations

¹ Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University, Okayama, 700-8530, Japan.
² Inflammation Pathology, Department of Pathology and Host Defense, Faculty of Medicine, Kagawa University, Kagawa, 761-0793, Japan.
³ Oncology Pathology, Department of Pathology and Host Defense, Faculty of Medicine, Kagawa University, Kagawa, 761-0793, Japan.
⁴ Department of Anatomy, Kitasato University School of Medicine, Kanagawa, 252-0374, Japan.
⁵ Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University, Okayama, 700-8530, Japan. Electronic address: tokumit@okayama-u.ac.jp.

PMID: 37979343
DOI: 10.1016/j.ceca.2023.102820

Abstract

Ca²⁺/calmodulin-dependent protein kinase kinase (CaMKK) phosphorylates and activates downstream protein kinases, including CaMKI, CaMKIV, PKB/Akt, and AMPK; thus, regulates various Ca²⁺-dependent physiological and pathophysiological pathways. Further, CaMKKβ/2 in mammalian species comprises multiple alternatively spliced variants; however, their functional differences or redundancy remain unclear. In this study, we aimed to characterize mouse CaMKKβ/2 splice variants (CaMKKβ-3 and β-3x). RT-PCR analyses revealed that mouse CaMKKβ-1, consisting of 17 exons, was predominantly expressed in the brain; whereas, mouse CaMKKβ-3 and β-3x, lacking exon 16 and exons 14/16, respectively, were primarily expressed in peripheral tissues. At the protein level, the CaMKKβ-3 or β-3x variants showed high expression levels in mouse cerebrum and testes. This was consistent with the localization of CaMKKβ-3/-3x in spermatids in seminiferous tubules, but not the localization of CaMKKβ-1. We also observed the co-localization of CaMKKβ-3/-3x with a target kinase, CaMKIV, in elongating spermatids. Biochemical characterization further revealed that CaMKKβ-3 exhibited Ca²⁺/CaM-induced kinase activity similar to CaMKKβ-1. Conversely, we noted that CaMKKβ-3x impaired Ca²⁺/CaM-binding ability, but exhibited significantly weak autonomous activity (approximately 500-fold lower than CaMKKβ-1 or β-3) due to the absence of C-terminal of the catalytic domain and a putative residue (Ile478) responsible for the kinase autoinhibition. Nevertheless, CaMKKβ-3x showed the ability to phosphorylate downstream kinases, including CaMKIα, CaMKIV, and AMPKα in transfected cells comparable to CaMKKβ-1 and β-3. Collectively, CaMKKβ-3/-3x were identified as functionally active and could be bona fide CaMKIV-kinases in testes involved in the activation of the CaMKIV cascade in spermatids, resulting in the regulation of spermiogenesis.

Keywords: CaMKIV; CaMKKβ-3/3x; Exons; Phosphorylation; Spermatids.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Calcium-Calmodulin-Dependent Protein Kinase Kinase* / genetics
Calcium-Calmodulin-Dependent Protein Kinase Kinase* / metabolism
Male
Mammals / metabolism
Mice
Phosphorylation
Protein Processing, Post-Translational
Signal Transduction
Spermatids* / metabolism

Substances

Calcium-Calmodulin-Dependent Protein Kinase Kinase