Abstract
14-3-3 proteins regulate diverse cellular processes in eukaryotes by binding to phospho-serine or threonine of target proteins. One of the physiological functions of 14-3-3 is to bind and protect phosphate groups of the target proteins against phosphatases. REPRESSION OF SHOOT GROWTH (RSG) is a tobacco (Nicotiana tabacum) transcription factor that is involved in the feedback regulation of biosynthetic genes of plant hormone gibberellin. 14-3-3 binds to phospho-Ser-114 in RSG. Ca2+-dependent protein kinase NtCDPK1 was identified as a kinase that phosphorylates Ser-114 of RSG. Our recent study revealed that NtCDPK1 forms a heterotrimer with RSG and 14-3-3 and that 14-3-3 was transferred from NtCDPK1 to phosphorylated RSG (Ito et al., 2014). In the course of the study, we found that 14-3-3 protects the phosphate group of RSG from λ protein phosphatase in vitro. Here, we describe a protocol for in vitro phosphatase protection assay. To detect the phosphorylation state of proteins, we used Phos-tag SDS-PAGE and autoradiography. This protocol can be adapted for the examinations whether the phosphoprotein-binding proteins protect the phosphate group of target proteins from phosphatases although protein kinases may be required for the phosphorylation of target proteins.
Keywords: Phosphatase, 14-3-3, Phosphorylation, Protection, Phosphate
Materials and Reagents
Equipment
Procedure
Representative data
Figure 1. The phosphorylation state of Ser-114 in RSG is protected from λ protein phosphatase by 14-3-3. MBP-RSG proteins phosphorylated with cold ATP were resolved on Phos-tag SDS-PAGE and visualized by coomassie brilliant blue (CBB) staining. MBP-RSG proteins phosphorylated with [γ-32P] ATP were resolved on SDS-PAGE and detected by using imaging system. Several amino acids of RSG are phosphorylated by NtCDPK1. When His-14-3-3 does not bind to MBP-RSG, phosphate groups of RSG are completely removed by λ protein phosphatase. On the other hand, when MBP-RSG that bound to His-14-3-3 is treated with λ protein phosphatase, the phosphorylation state of Ser-114 in RSG is protected from λ protein phosphatase. Top, middle and bottom arrowheads represent multiply phosphorylated, Ser-114 phosphorylated and dephosphorylated MBP-RSG, respectively. For more information, please see Ito et al. (2014).
Notes
Recipes
Acknowledgments
This work was supported by the Japan Society of the Promotion of Science (grant no. 23657038 to Y. T.) and the Ministry of Education, Culture, Sports, Science, and Technology of Japan (grant no. 24118004 to Y. T.).
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.