Identification of Variants and Dual Promoters of Murine Serine/Threonine Kinase KKIAMRE *²Takayuki Sassa, *Hiroshi Gomi, §William Sun, *Toshio Ikeda, §Richard F. Thompson, and *‡Shigeyoshi Itohara *Laboratory for Behavioral Genetics, Brain Science Institute, RIKEN, Saitama, ² Institute for Virus Research, Kyoto University, Kyoto, and ‡Core Research for Evolutional Science and Technology (CREST), Tokyo, Japan; and §Neuroscience Program, University of Southern California, Los Angeles, California, U.S.A. Abstract: KKIAMRE is a serine/threonine protein kinase whose transcripts increase in the deep cerebellar nuclei of the rabbit after eyeblink conditioning, a model of as- sociative learning and memory. We here characterized the expression, isoforms, and promoters of murine KKI- AMRE gene. The expression of KKIAMRE was detected, by in situ hybridization and immunohistochemistry, in neurons in various brain regions including deep cerebel- lar nuclei. The gene spans 40 kb and consists of 15 exons. Analysis of cDNA clones revealed multiple vari- ants, having diversity in the putative carboxy-terminal regulatory domain, generated by alternative splicing and intraexonal termination. Furthermore, they had alternative 5' noncoding sequences. Primer extension, RNase pro- tection, and transient expression assays revealed that two alternative promoters linked to distinct noncoding exons direct the expression of KKIAMRE. The gene was mapped on chromosomes 5 and 4 in mouse and human, respectively. Key Words: KKIAMRE—Serine/threonine kinase —Promoter—Variants—Eyeblink conditioning. J. Neurochem. 74, 1809 –1819 (2000). Understanding the mechanisms of learning and mem- ory has been a field of intense research in neurobiology. It is now widely accepted that memory storage has short- term and long-term stages (Milner et al., 1998). The latter is dependent on protein and/or RNA synthesis, thus providing the standpoint to analyze these complex mech- anisms in terms of molecular and cellular biology (Davis and Squire, 1984; Montarolo et al., 1986; Tully et al., 1994). Classic conditioning of eyeblink/nictitating has been extensively studied as a useful behavioral model for investigating the neuronal substrates underlying associa- tive learning and memory (Thompson and Krupa, 1994). Extensive studies of lesioning, unit recording, and elec- trical stimulation at defined loci have clearly unraveled the main circuitry for the conditioning. Interestingly, reversible inactivations have demonstrated a very re- stricted locus, the interpositus nucleus and its overlying cortex of the cerebellum, as being essential for the ac- quisition and/or expression of the conditioned responses (Krupa et al., 1993; Thompson and Krupa, 1994). We have recently shown that injection of the RNA synthesis inhibitor actinomycin D into the interpositus nucleus reversibly prevented the conditioning (Gomi et al., 1999), suggesting that this process accompanies changes in gene expression. The differential display method was then applied to compare the difference in the state of gene expression between conditioned and unconditioned rabbits. We found that in the conditioned rabbits, the transcripts of serine/threonine protein kinase KKIAMRE were increased in the deep cerebellar nuclei to which the interpositus nucleus belongs (Gomi et al., 1999). These findings raised the possibility that induced expression of KKIAMRE and subsequent phosphorylation of its sub- strate(s) are important for conditioning. KKIAMRE is one of the most distant members of the cdc2-related serine/threonine protein kinase family (Ta- glienti et al., 1996). Most members of this family were cloned based on the similarity to cdc2 in their kinase (catalytic) domains and named temporarily after the amino acid sequences corresponding to the PSTAIRE motif of cdc2 (Meyerson et al., 1992). Although they have similarity to cdc2, an essential regulator of the cell cycle, some of the members, such as cyclin-dependent kinase 5, PFTAIRE, and KKIALRE, seem to have roles other than regulation of the cell cycle, as they are ex- pressed in terminally differentiated cells in the nervous Received September 1, 1999; revised manuscript received November 30, 1999; accepted December 10, 1999. Address correspondence and reprint requests to Dr. S. Itohara at Laboratory for Behavioral Genetics, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198 Japan. E-mail: sitohara@ brain.riken.go.jp The DDBJ/EMBL/GenBank accession nos. for the sequence re- ported in this article are AB029065–AB029073. Abbreviations used: CRE, cyclic AMP response element; DIG, digoxigenin; LOD, logarithm of the odds; MAP, mitogen-activated protein; NGF, nerve growth factor; PBS, phosphate-buffered saline; PE, primer extension; RH, radiation hybrid; RPA, RNase protection assay; SDS, sodium dodecyl sulfate; SSC, standard saline citrate; UTR, untranslated region. 1809 Journal of Neurochemistry Lippincott Williams & Wilkins, Inc., Philadelphia © 2000 International Society for Neurochemistry