Involvement of p90 Ribosomal S6 Kinase in Termination of Cell Cycle Arrest during Development of Artemia-encysted Embryos

Abstract

Artemia has evolved a unique developmental pattern of encysted embryos to cope with various environmental threats. Cell divisions totally cease during the preemergence developmental stage from gastrula to prenauplius. The molecular mechanism of this, however, remains unknown. Our study focuses on the involvement of p90 ribosomal S6 kinase (RSK), a family of serine/threonine kinase-mediating signal transduction downstream of mitogen-activated protein kinase cascades, in the termination of cell cycle arrest during the post-embryonic development of Artemia-encysted gastrula. With immunochemistry, morphology, and cell cycle analysis, the identified Artemia RSK was established to be specifically activated during the post-embryonic and early larval developmental stages when arrested cells of encysted embryos resumed mitoses. In vivo knockdown of RSK activity by RNA interference, kinase inhibition, and antibody neutralization consistently induced defective larvae with distinct gaps between the exoskeleton and internal tissues. In these abnormal individuals, mitoses were detected to be largely inhibited in the affected regions. These results display the requirement of RSK activity during Artemia development and suggest its role in termination of cell cycle (G 2 /M phase) arrest and promotion of mitogenesis. Our findings may, thus, provide insights into the regulation of cell division during Artemia post-embryonic development and reveal further aspects of RSK functions. p90 ribosomal S6 kinase (p90 RSK , also known as RSK) is a family of serine/threonine kinase that mediates signal transduction downstream of mitogen-activated protein kinase cascades (1). RSKs 2 consist of two distinct and functional kinase domains, the N-terminal kinase domain and the C-terminal kinase domain connected by a regulatory linker (2). The typical RSK possesses six phosphorylation sites, and its in vivo activation is achieved upon their consecutive phosphorylations. Mutation analysis reveals four of the six sites are essential for the kinase activation (Ser-221 in N-terminal kinase domain, Ser-363 and Ser-380 in the linker, and Thr-573 in C-terminal kinase domain, according to the human RSK1 sequence) (3).RSKs are present in most vertebrate species (1, 4), and RSKrelated molecules have also been identified in several invertebrates (5, 6). RSK family members have been reported to be multifunctional in the regulation of diverse cellular processes including transcriptional regulation, cell cycle control, cell survival, and many others (4). Within mammalian cells, RSKs are thought to promote G 1 progression by stimulating growth-related protein synthesis and inhibiting negative regulation (7). It is, thus, shown that the RSK inhibitor SL0101 can effectively inhibit proliferations of the human breast cancer cell and the hamster ovary cell (8, 9).Artemia possesses powerful adaptations to extreme environments that often include aspects of hypersalinity, anoxia, and large changes in ionic composition and temperature. As a strategy to cope with such en...

Involvement of Polo-like Kinase 1 (Plk1) in Mitotic Arrest by Inhibition of Mitogen-activated Protein Kinase-Extracellular Signal-regulated Kinase-Ribosomal S6 Kinase 1 (MEK-ERK-RSK1) Cascade

“…We previously showed that RSK plays an important role in the termination of cell cycle arrest by its activation in nauplius larvae when previously arrested cells resumed mitosis (30). By contrast, RSK is not active in diapause cysts, suggesting a possible role for RSK in the initiation and maintenance of cell cycle arrest.…”

“…dsRNA Synthesis and Microinjection-Reconstructed plasmids that contained two inverted T7 polymerase sites flanking the cloning region were obtained (named pET-T7) as described previously (30). Next, a 442-bp fragment including the 5Ј-UTR and parts of the 5Ј coding region of the Plk1 gene and a fragment of the Aurora A gene were both amplified by PCR with primers iF and iR and primers AiF and AiR, respectively (Table 1).…”

CHH binding protein (CHHBP): a newly identified receptor of crustacean hyperglycemic hormone (CHH)

“…Reconstructed plasmids that contained two inverted T7 promoter sites flanking the cloning region were obtained (named as pET-T7) as described previously (Dai et al, 2008). Next, a 667 bp fragment containing part of the coding region of the CHHBP gene was amplified by PCR with primers iF and iR (Table 1).…”

Two p90 Ribosomal S6 Kinase Isoforms Are Involved in the Regulation of Mitotic and Meiotic Arrest in Artemia

“…We previously reported that an Ar-RSK isoform, termed Ar-Rsk1, plays an important role in diapause termination in Artemia (29). This isoform lacks a typical ERK-docking motif.…”

“…In these embryos, metabolic activity is greatly reduced and RNA/protein synthesis does not occur (25)(26)(27)(28). Post-diapause embryos, in which the cell cycle is arrested at the G 2 /M phase (29), are obtained by storing diapause embryos at Ϫ20°C for at least 3 months (30). In favorable conditions, post-diapause embryos hatch, the diapause is terminated, and cell division and development resume, resulting in the generation of nauplii (30 -32).…”