Transcriptional, biochemical, and immunohistochemical analyses of CaMKKI3/2 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, CaMKKI3/2 in mammalian species comprises multiple alternatively spliced variants; however, their functional differences or redundancy remain unclear. In this study, we aimed to characterize mouse CaMKKI3/2 splice variants (CaMKKI3-3 and I3-3x). RT-PCR analyses revealed that mouse CaMKK beta-1, consisting of 17 exons, was predominantly expressed in the brain; whereas, mouse CaMKK beta-3 and beta-3x, lacking exon 16 and exons 14/16, respectively, were primarily expressed in peripheral tissues. At the protein level, the CaMKKI3-3 or I3-3x variants showed high expression levels in mouse cerebrum and testes. This was consistent with the localization of CaMKKI3-3/-3x in spermatids in seminiferous tubules, but not the localization of CaMKKI3-1. We also observed the co-localization of CaMKKI3-3/-3x with a target kinase, CaMKIV, in elongating spermatids. Biochemical characterization further revealed that CaMKKI3-3 exhibited Ca2+/CaM-induced kinase activity similar to CaMKKI3-1. Conversely, we noted that CaMKKI3-3x impaired Ca2+/CaM-binding ability, but exhibited significantly weak autonomous activity (approximately 500-fold lower than CaMKKI3-1 or I3-3) due to the absence of C-terminal of the catalytic domain and a putative residue (Ile478) responsible for the kinase autoinhibition. Nevertheless, CaMKKI3-3x showed the ability to phosphorylate downstream kinases, including CaMKIa, CaMKIV, and AMPKa in transfected cells comparable to CaMKKI3-1 and I3-3. Collectively, CaMKKI3-3/-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.