Molecular Characterization And Function Analysis Of AMP-activated Protein Kinase In Artemia Franciscana During Development And In Response To Stress

AMP-activated protein kinase (AMPK), as an important energy sensor, is crucial for organism survival under adverse conditions. AMPK homologous exist as heterotrimeric complexes consisting of a catalytic a-subunit and regulatoryβ- andγ-subunits. AMPK families are found in many eukaryotes, including plants, yeast, nematode, insects, and mammals. So far, members of AMPK families have not been reported in crustaceans yet. Artemia is a species of primitive crustacean capable of producing diapause encysted embryos to survive adverse conditions. Encysted embryos of Artemia can remain in a dormant state for long periods with a very low metabolic rate, only resuming their development with the approach of favorable conditions. However, the intrinsic mechanisms of Artemia diapause and development are still unclear. As an important stress sensor and energy regulator, AMPK would probably involve in Artemia surviving from environmental stresses and choosing reproduction mode.To investigate AMPK functions in Artemia life cycle, we isolated two cDNAs of AMPKa encoding genes from the brine shrimp, Artemia franciscana. We have also investigated the gene expression patterns in different stages of Artemia life cycles and also under stressed conditions in adults and cysts. Furthermore, using an antibody that specific binds to phospho-AMPKa, we have also investigated pssible functions of AMPK in Artemia post-diapause developmental process. The main results include the following two parts:1. By using degenerated primer PCR strategy, AMPKa genes were isolated from the Artemia franciscana, named Afr-AMPKalpha1 and Afr-AMPKalpha2 respectively. The Afr-AMPKalpha1 clone was 1858 bp long with a 1548 bp open reading frame (ORF) which was conceptually translated into a 515-amino acid protein (termed Afr-AMPKALPHA1). The basic serine/threonine protein kinase domain was identified. The nucleotide sequence of Afr-AMPKalpha2 was found to be identical to that of Afr-AMPKalpha1 except a 244-bp deletion in the ORF region. Compared with Afr-AMPKalpha1, this 244-bp deletion caused a frameshift, introduced a new translation stop codon and shortened the C terminal of the Afr-AMPKALPHA2 protein. It is possible that the two cDNAs are originated by alternative splicing of a single gene. The basic serine/threonine protein kinase domain of both proteins showed > 64% identity among species. In addition, the genes expressions were characterized by semiquantitive RT-PCR. Afr-AMPKalpha1 was differentially expressed during Artemia developmental stages as well as in response to stresses, such as heat shock, starvation and hyperosmotic stress. Afr-AMPKalpha1 mRNA level in adult Artemia decreased under heat shock, but not in a time-and temperature-dependent manner. By contrast, the transcript sharply decreased in heat shocked cysts with time dependent manner. Under hyperosmotic stress, however, the mRNA level in adults first declined and then increased with prolonged exposure. And in the case of starvation, the gene expression in adults decreased and was undetectable after day 9. Furthermore, our results demonstrate that the mRNA level of Afr-AMPKalpha1 sharply decreases in head region of Artemia carrying diapause-destined embryos, suggesting a role of Artemia AMPK in reproductive mode transition. Additionally, mRNA level of Afr-AMPKalpha2 was too low to be detected unless using nested PCR. Southern blot analysis indicates the possibility that the Artemia AMPKa gene is present in multiple copies.2. Our results demonstrated the specific activation of AMPK in post-diapause developmental process of Artemia using a phospho-AMPKa antibody. By whole-mount immunohistochemistry, phosphorylated AMPK is shown to be predominantly located in ectoderm of the early developed embryos in a ring shape, and the location and shape of the activation region changes as the development proceeds. Additionally, Western blotting analysis on different portions of cyst extracts shows that phosphorylated AMPKa binds to nuclei and this binding is not affected by intracellular pH. This result was further confirmed by immunofluorescent staining of the cyst nuclei, suggesting possible roles of Artemia AMPK in regulating gene expression during resume of the development. Moreover, cellular AMP, ADP and ATP levels in developing cysts were determined by HPLC, and the results suggest that the activation of Artemia AMPK is not associated with cellular AMP: ATP ratios.In summary, we isolated and characterized AMPKa gene in Artemia and thus demonstrated the existence of AMPK in crustacean for the first time. Moreover, our results suggest a significant role of AMPK in Artemia in response to stress and during development, especially in post-diapause developmental process. Our research thus provides valuable information for further investigation of AMPK functions and Artemia developmental biology.