Elsevier

Neuroscience

Volume 148, Issue 2, 24 August 2007, Pages 510-521
Neuroscience

Molecular neuroscience
Structural and functional analysis of the apoptosis-associated tyrosine kinase (AATYK) family

https://doi.org/10.1016/j.neuroscience.2007.05.048Get rights and content

Abstract

Apoptosis-associated tyrosine kinase (AATYK) is a protein kinase that is predominantly expressed in the nervous system and is involved in apoptosis and neurite growth of cerebellar granule cells. In this study, we cloned three new members of the mouse AATYK family, AATYK1B, AATYK2 and AATYK3. AATYK1B is a splicing variant of the previously reported AATYK1 (referred to as AATYK1A hereafter). In comparison with AATYK1A, these three AATYK members were characterized by having an extra N-terminal region that consists of a signal peptide-like sequence and a predicted transmembrane (TM) region, which is followed by a kinase domain and a long C-terminal domain. Both TM-containing AATYK isoforms (AATYK(+)TM: AATYK1B, 2, and 3) and TM-lacking isoform (AATYK(−)TM: AATYK1A) were recovered in membrane fractions, suggesting that AATYK(+)TM and AATYK(−)TM are transmembrane- and peripheral-membrane protein kinases, respectively. AATYK1A was recovered in the soluble fraction when the cells were treated with 2-bromo palmitate, suggesting that AATYK1A associates with membrane via palmitoylation. The kinase domain was highly conserved among all AATYK members and was shown to be catalytically active. Three AATYK family members were predominantly expressed in adult mouse brains with almost similar expression profiles: widespread distribution over the various brain regions, especially in the cerebellum and hippocampus, and up-regulated expression during development of the cerebellum. In cultured cerebellar granule cells, AATYK1 was abundantly localized in both soma and axons, AATYK2 distribution was restricted to soma, and AATYK3 was punctately present over the cells. AATYK1 was concentrated in the central domain of growth cones of dorsal root ganglion neurons. Our results indicate that AATYK family members are brain-dominant and membrane-associated kinases with slightly different distribution patterns in the developing and adult mouse brain, which may be involved in fine regulation of neuronal functions including neurite extension and apoptosis.

Section snippets

Experiments on animals

All animal experiments were carried out in accordance with the RIKEN guidelines for animal use and the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize the number of animals used and their suffering.

Identification and cloning of the AATYK family genes

For the cloning of the 5′ region of AATYK1 mRNA, we screened the mouse adult brain library, 5′ rich-pME18S library, which was constructed by Oligo-Capping method (Suzuki et al., 1997), based on the polymerase chain reaction (PCR)

Structure of the AATYK family members

We previously isolated the AATYK1 gene from cultured Purkinje cells and showed the existence of sequence diversity in the 5′ region among several cloned cDNAs (Tomomura et al., 2001). The major form of AATYK1 clones was AATYK1A, which encodes the same open reading frame (ORF) as that cloned in myeloid cells (Gaozza et al 1997, Baker et al 2001). An alternative-splicing variant form of the AATYK1 (B-AATYK) clone containing an additional in-frame 5′-end sequence was also isolated from mouse

Discussion

In this study, we characterized the mouse AATYK family proteins, AATYK1A, AATYK1B, AATYK2, and AATYK3. AATYK1A and AATYK1B are produced from the same AATYK1 gene by alternative splicing. We showed that AATYK1B, 2, and 3 have the SP and TM domains at the N-terminal region, whereas AATYK1A lacks the SP and TM region. TM(+)AATYKs (1B, 2, and 3) are integral membrane proteins, whereas TM(−)AATYK1A is a peripheral membrane protein anchoring via the N-terminal palmitoylation and is the predominant

Conclusion

In conclusion, our study indicates that the AATYK family consists of three distinct types, AATYK1, 2, and 3, and two alternative splicing types in AATYK1, TM(+) and TM(−) types, with slightly different patterns in their regional and subcellular localization in the brain as well as in their developmental and tissue-specific expression. These subtle differences in the expression patterns and functional properties within the AATYK family may be involved in the fine regulation of neuronal functions

Acknowledgments

We thank Professor Shinichi Hisanaga (Tokyo Metropolitan University), Dr. Akira Sato (RIKEN Genome Center) and Dr. Liria M. Masuda-Nakagawa (University of Tokyo) for their critical comments and reading. We also thank Dr. Masaki Kumai (RIKEN BSI Research resource center) for his excellent service in antibody production and Ms. Emiko Suga for her technical assistance. This research was supported by a grant-in-aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports,

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