精子中CaMKKβ/2剪接变体与CaMKIV共定位的转录、生化和免疫组织化学分析

IF 4.3 2区 生物学 Q2 CELL BIOLOGY Cell calcium Pub Date : 2023-11-08 DOI:10.1016/j.ceca.2023.102820
Satomi Ohtsuka , Yumi Miyai , Hiroyuki Mima , Masaki Magari , Yoichi Chiba , Futoshi Suizu , Hiroyuki Sakagami , Masaki Ueno , Hiroshi Tokumitsu
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引用次数: 0

摘要

Ca2+/钙调素依赖性蛋白激酶(CaMKK)磷酸化并激活下游蛋白激酶,包括CaMKI、CaMKIV、PKB/Akt和AMPK;因此,调节各种Ca2+依赖的生理和病理生理途径。此外,哺乳动物物种中的CaMKKβ/2包括多个可选剪接变体;然而,它们的功能差异或冗余性尚不清楚。在这项研究中,我们旨在表征小鼠CaMKKβ/2剪接变体(CaMKKβ-3和β-3x)。RT-PCR分析显示,小鼠CaMKKβ-1由17个外显子组成,主要在大脑中表达;而小鼠CaMKKβ-3和β-3x分别缺失外显子16和14/16,主要在外周组织中表达。在蛋白水平上,CaMKKβ-3或β-3x变体在小鼠大脑和睾丸中表现出高表达水平。这与CaMKKβ-3/-3x在精细胞精管中的定位一致,而与CaMKKβ-1的定位不一致。我们还观察到CaMKKβ-3/-3x与靶激酶CaMKIV在伸长精子中的共定位。生化表征进一步表明,CaMKKβ-3表现出与CaMKKβ-1相似的Ca2+/ cam诱导的激酶活性。相反,我们注意到CaMKKβ-3x损害了Ca2+/ cam的结合能力,但由于缺乏催化结构域的c末端和负责激酶自抑制的推定残基(Ile478), CaMKKβ-3x表现出明显弱的自主活性(比CaMKKβ-1或β-3低约500倍)。然而,与CaMKKβ-1和β-3相比,CaMKKβ-3x在转染细胞中表现出磷酸化下游激酶的能力,包括camkk α、CaMKIV和AMPKα。总的来说,CaMKKβ-3/-3x被鉴定为具有功能活性,并且可能是睾丸中真正的CaMKIV激酶,参与激活精子中CaMKIV级联反应,从而调节精子发生。
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Transcriptional, biochemical, and immunohistochemical analyses of CaMKKβ/2 splice variants that co-localize with CaMKIV in spermatids

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.

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来源期刊
Cell calcium
Cell calcium 生物-细胞生物学
CiteScore
8.70
自引率
5.00%
发文量
115
审稿时长
35 days
期刊介绍: Cell Calcium covers the field of calcium metabolism and signalling in living systems, from aspects including inorganic chemistry, physiology, molecular biology and pathology. Topic themes include: Roles of calcium in regulating cellular events such as apoptosis, necrosis and organelle remodelling Influence of calcium regulation in affecting health and disease outcomes
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