Functional Characterization of a Maize Ribosomal S6 Protein Kinase (ZmS6K), a Plant Ortholog of Metazoan p70^S6K

Abstract

Ribosomal protein S6 (S6rp) is phosphorylated by the p70S6K enzyme in mammals, under mitogen/IGF regulation. This event has been correlated with an increase in 5'TOP mRNA translation. In this research, a maize S6 kinase (ZmS6K) was isolated from maize (Zea mays L.) embryonic axes by human p70S6K antibody immunoprecipitation. This enzyme, a 62 kDa peptide, proved to be specific for S6rp phosphorylation, as revealed by in vivo and in vitro kinase activity using either the 40S ribosomal subunit or the RSK synthetic peptide as the substrates. ZmS6K activation was achieved by phosphorylation on serine/threonine residues. Specific phospho-Threo recognition by the p70S6K antibody directed to target phospho-Threo residue 389 correlated with ZmS6K activation. The ZmS6K protein content remained almost steady during maize seed germination, whereas the ZmS6K activity increased during this process, consistent with Zm6SK phosphorylation. Addition of insulin to germinating maize axes proved to increase ZmS6K activity and the extent of S6rp phosphorylation. These events were blocked by rapamycin, an inhibitor of the insulin signal transduction pathway in mammals, at the TOR (target of rapamycin) enzyme level. We conclude that ZmS6K is a kinase, structurally and functionally ortholog of the mammalian p70S6K, responsible for in vivo S6rp phosphorylation in maize. Its activation is induced by insulin in a TOR-dependent manner by phosphorylation on conserved serine/threonine residues.

Phospholipase Dα3 Is Involved in the Hyperosmotic Response in Arabidopsis

“…To detect phosphorylated S6K, proteins were transferred to nitrocellulose membranes and blotted with an anti-phospho-p70 S6K (Thr-389) antibody (Cell Signaling Technology), followed by a secondary antibody conjugated with horseradish peroxidase. The rabbit polyclonal antibodies were raised against human p70 S6K and have been shown to react with plant S6K proteins (Reyes de la Cruz et al, 2004). The membranes were preblotted with TBS/T containing 5% BSA and then were incubated with the first antibody (1:1000) in TBS/T buffer.…”

Possible Dual Regulatory Circuits Involving AtS6K1 in the Regulation of Plant Cell Cycle and Growth

“…We think that one possible explanation for this apparent discrepancy is the presence of high concentration of auxin, which was included as a standard component of the culture media for maintenance of the suspension cells' proliferative potency. Auxin has been shown to stimulate AtS6K activity as well as the maize RPS6 phosphorylation (Reyes de la Cruz et al, 2004;Turck et al, 2004), thus it is conceivable that the activities phosphorylating AtS6K1 and AtRPS6 in the suspension cells remain high even under stress conditions. What is still intriguing, however, is that either osmotic stress or starvation alone did not have any effect on these phosphorylation activities, but when these stresses were combined together, they were able to override the stimulatory signal for the phosphorylation of AtS6K1 and AtRPS6, which suggests the two stress signals operate independently but converge on to inhibit this stimulatory signal.…”

Oligogalacturonides inhibit growth and induce changes in S6K phosphorylation in maize (Zea mays L. var. Chalqueño)

“…Rapamycin 100 nM was used because this concentration was reported to inhibit maize S6K (Reyes de la Cruz et al 2004). Five millilitres of medium containing different substances were placed into 20 9 150 mm tubes in sterile conditions.…”

“…The growth hormones, auxins and cytokinins enhance S6rp phosphorylation in Arabidopsis cell cultures (Turck et al 2004), whereas stress factors, such as heat and oxidative stress rapidly block it (Williams et al 2003). Additionally, there are several reports about the conserved signaling pathway in maize, including the existence of an insulin-like growth factor (IGF; García Flores et al 2001;Rodriguez-Lopez et al 2011); and the maize S6K (ZmS6K; Reyes de la Cruz et al 2004). This last protein is responsible for in vivo S6rp phosphorylation in an insulin dependent manner through TOR signaling pathway and this process was rapamycin sensitive (Reyes de la Cruz et al 2004).…”