Multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) participates in diverse calcium signaling pathways in neurons. The alpha- and beta-CaM kinase isoforms are neuron-specific and highly abundant in rat brain. The variable domain of CaM kinase is a potential site for the generation of isoform diversity by alternative spicing of its N- and/or C-terminal segments. We used specific PCR primers which span the variable domain of either alpha- or beta-CaM kinase and isolated three new isoforms from rat brain, namely alpha B-, beta e- and beta'e-CaM kinase. alpha beta-CaM kinase contains 11 amino acids, likely inserted by alternative splicing, at the C-terminal segment of the variable domain. This insertion introduces a nuclear localization signal (NLS) that targets alpha B-CaM kinase to the nucleus of transfected cells; alpha-CaM kinase is excluded from the nucleus. The mRNA and the protein corresponding to this isoform are detected only in the diencephalon/midbrain regions. We have also identified two alternatively spliced isoforms of beta-CaM kinase that lack the 24 amino acid sequence at the N-terminal segment of the variable domain. Alternative splicing of these two isoforms occurs with a three base pair shift of the 3'-splice site. Our analysis shows that these new beta-CaM kinase isoforms are expressed primarily in early developmental stages, and we therefore term them beta e - (embryonic) and beta' e-CaM kinase. Recombinant alpha B-, beta e and beta' e-CaM kinase expressed in COS-7 cells exhibit characteristic Ca2+/calmodulin-dependent protein kinase activity and autophosphorylation.
The assembly of 6 -12 subunits of Ca 2؉ /calmodulin-dependent kinase II (CaM kinase II) into holoenzymes is an important structural feature of the enzyme and its postulated role as a molecular detector of Ca 2؉ oscillations. Using single cell reverse transcriptase-polymerase chain reaction, we show that ␣-and -CaM kinase II mRNAs are simultaneously present in the majority of hippocampal neurons examined and that co-assembly of their protein products into heteromers is therefore possible. The subunit composition of CaM kinase II holoenzymes was analyzed by immunoprecipitation with subunit-specific monoclonal antibodies. Rat forebrain CaM kinase II consists of heteromers composed of ␣ and  subunits at a ratio of 2:1 and homomers composed of only ␣ subunits. We examined the functional effect of the heteromeric assembly by analyzing the calmodulin dependence of autophosphorylation. Recombinant homomers of ␣-or -CaM kinase II, as well as of alternatively spliced  isoforms, have distinct calmodulin dependences for autophosphorylation based on differences in their calmodulin affinities. Half-maximal autophosphorylation of ␣ is achieved at 130 nM calmodulin, while that for  occurs at 15 nM calmodulin. In CaM kinase II isolated from rat forebrain, however, the calmodulin dependence for autophosphorylation of the  subunits is shifted toward that of ␣ homomers. This suggests that Thr 287 in  subunits is phosphorylated by ␣ subunits present in the same holoenzyme. Once autophosphorylated, -CaM kinase II traps calmodulin by reducing the rate of calmodulin dissociation.Multifunctional Ca 2ϩ /calmodulin-dependent protein kinase II (CaM kinase II) is a major mediator of Ca 2ϩ action whose activation and autophosphorylation appear to regulate numerous neuronal processes, including two forms of synaptic plasticity, long term potentiation and long term depression (1, 2). These two opposing changes in synaptic strength are differentially regulated by the frequency of stimulation of hippocampal neurons. Interestingly, CaM kinase II has been suggested to be a molecular detector of the Ca 2ϩ spike frequency, based on its unique structural and regulatory properties (3) (reviewed in Refs. 4 and 5). The neuronal CaM kinase II consists of two major subunits of 52 and 60 kDa that are encoded by ␣-and
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.