Molecular Identification and Characterization of a Family of Kinases with Homology to Ca2+/Calmodulin-dependent Protein Kinases I/IV

Shogo Ohmae,Sayaka Takemoto-Kimura,Michiko Okamura,Aki Adachi-Morishima,Mio Nonaka,Toshimitsu Fuse,Satoshi Kida,Masahiro Tanji,Tomoyuki Furuyashiki,Yoshiki Arakawa,Shuh Narumiya,Hiroyuki Okuno,Haruhiko Bito

doi:10.1074/jbc.m513212200

Shogo Ohmae, Sayaka Takemoto-Kimura + Show 11 more

Open Access

https://doi.org/10.1074/jbc.m513212200

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Abstract

Despite the critical importance of Ca(2+)/calmodulin (CaM)-dependent protein kinase (CaMK) II signaling in neuroplasticity, only a limited amount of work has so far been available regarding the presence and significance of another predominant CaMK subfamily, the CaMKI/CaMKIV family, in the central nervous system. We here searched for kinases with a core catalytic structure similar to CaMKI and CaMKIV. We isolated full-length cDNAs encoding three mouse CaMKI/CaMKIV-related kinases, CLICK-I (CL1)/doublecortin and CaM kinase-Like (DCAMKL)1, CLICK-II (CL2)/DCAMKL2, and CLICK-I,II-related (CLr)/DCAMKL3, the kinase domains of which had an intermediate homology not only to CaMKI/CaMKIV but also to CaMKII. Furthermore, CL1, CL2, and CLr were highly expressed in the central nervous system, in a neuron-specific fashion. CL1alpha and CL1beta were shorter isoforms of DCAMKL1, which lacked the doublecortin-like domain (Dx). In contrast, CL2alpha and CL2beta contained a full N-terminal Dx, whereas CLr only possessed a partial and dysfunctional Dx. Interestingly, despite a large similarity in the kinase domain, CL1/CL2/CLr had an impact on CRE-dependent gene expression distinct from that of the related CaMKI/CaMKIV and CaMKII. Although these were previously shown to activate Ca(2+)/cAMP-response element-binding protein (CREB)-dependent transcription, we here show that CL1 and CL2 were unable to significantly phosphorylate CREB Ser-133 and rather inhibited CRE-dependent gene expression by a dominant mechanism that bypassed CREB and was mediated by phosphorylated TORC2.

Highlights

As part of the large list of serine/threonine and tyrosine kinase families, the Ca2ϩ/calmodulin (CaM)3-dependent protein kinase (CaMK) group stands out by the large number of its constituent kinases [1,2,3]
Molecular Cloning of CaMK-like Kinases Based on Sequence Similarity around the Conserved Catalytic Domain and Activation Loop of CaMKI/CaMKIV—During a screen for potential cAMP-response elementbinding protein (CREB)-regulatory kinases, we sought for key determinants within the kinase domain of known in vitro CREB kinases such as CaMKI [17, 46, 47] and CaMKIV [11, 33, 34]
The Kinase Activity of CL1/CL2/CLr Targets Components of CREB-dependent Gene Expression in a Manner Distinct from That of CaMKI, CaMKIV, and CaMKII—we addressed whether the kinase activity of CL1/CL2/CLr had an impact to gene transcription that was similar or distinct from that of CaMKI/CaMKIV and CaMKII

Summary

The abbreviations used are

CaM, calmodulin; CaMK, Ca2ϩ/CaM-dependent protein kinase; CLICK, CaMK-like CREB-regulatory kinase candidate; DCAMKL, doublecortin and CaM kinase-Like; CREB, Ca2ϩ/cAMP-response element-binding protein; RACE, rapid amplification of cDNA end; ORF, open reading frame; PBS(Ϫ), Ca2ϩ/Mg2ϩ-free phosphate-buffered saline; CRE, Ca2ϩ/cAMP-response element; PKA, protein kinase A; SRE, serum-response element; IRES, internal ribosome entry sequence; EGFP, enhanced green fluorescent protein; CBP, CREB-binding protein; DA, dominant active; W T, wild-type; EST, expressed sequence tag; indel, insertion and deletion polymorphism; DCK, doublecortin-like kinase; DCLK, doublecortin-like kinase; cpg, candidate plasticity-related gene; Dx, doublecortin-like domain; KID, kinase-inducible domain; TORC, transducer of regulated CREB activity; ANOVA, analysis of variance; aa, amino acid(s). Despite a large structural similarity in the kinase domain, the functional impact of kinase activity of CL1/CL2/CLr kinases appeared distinct from the related CaMKI/CaMKIV and CaMKII. Both CaMKI/CaMKIV and CaMKII branches of the CaMK family are established Ca2ϩ/cAMP-response elementbinding protein (CREB) phosphorylating kinases [11, 31,32,33,34,35,36,37], we show here, using transcriptional readout assays, that CL1/CL2/ CLr kinases were unlikely to target CREB but rather inhibited CREdependent gene expression by a dominant mechanism that bypassed CREB and was mediated by phosphorylated transducer of regulated CREB activity (TORC) 2

EXPERIMENTAL PROCEDURES

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DISCUSSION