Transcriptional, biochemical, and immunohistochemical analyses of CaMKKβ/2 splice variants that co-localize with CaMKIV in spermatids.
Autor: | Ohtsuka S; Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University, Okayama, 700-8530, Japan., Miyai Y; Inflammation Pathology, Department of Pathology and Host Defense, Faculty of Medicine, Kagawa University, Kagawa, 761-0793, Japan., Mima H; Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University, Okayama, 700-8530, Japan., Magari M; Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University, Okayama, 700-8530, Japan., Chiba Y; Inflammation Pathology, Department of Pathology and Host Defense, Faculty of Medicine, Kagawa University, Kagawa, 761-0793, Japan., Suizu F; Oncology Pathology, Department of Pathology and Host Defense, Faculty of Medicine, Kagawa University, Kagawa, 761-0793, Japan., Sakagami H; Department of Anatomy, Kitasato University School of Medicine, Kanagawa, 252-0374, Japan., Ueno M; Inflammation Pathology, Department of Pathology and Host Defense, Faculty of Medicine, Kagawa University, Kagawa, 761-0793, Japan., Tokumitsu H; 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. |
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Jazyk: | angličtina |
Zdroj: | Cell calcium [Cell Calcium] 2024 Jan; Vol. 117, pp. 102820. Date of Electronic Publication: 2023 Nov 08. |
DOI: | 10.1016/j.ceca.2023.102820 |
Abstrakt: | Ca 2+ /calmodulin-dependent protein kinase kinase (CaMKK) phosphorylates and activates downstream protein kinases, including CaMKI, CaMKIV, PKB/Akt, and AMPK; thus, regulates various Ca 2+ -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 Ca 2+ /CaM-induced kinase activity similar to CaMKKβ-1. Conversely, we noted that CaMKKβ-3x impaired Ca 2+ /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. Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. (Copyright © 2023 Elsevier Ltd. All rights reserved.) |
Databáze: | MEDLINE |
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