A novel C-terminal proteolytic processing of cytosolic pyruvate kinase, its phosphorylation and degradation by the proteasome in developing soybean seeds
Article first published online: 28 MAR 2003
The Plant Journal
Volume 34, Issue 1, pages 77–93, April 2003
How to Cite
Tang, G.-Q., Hardin, S. C., Dewey, R. and Huber, S. C. (2003), A novel C-terminal proteolytic processing of cytosolic pyruvate kinase, its phosphorylation and degradation by the proteasome in developing soybean seeds. The Plant Journal, 34: 77–93. doi: 10.1046/j.1365-313X.2003.01711.x
- Issue published online: 28 MAR 2003
- Article first published online: 28 MAR 2003
- Received 30 September 2002; revised 17 December 2002; accepted 9 January 2003.
- pyruvate kinase;
- phosphorylation site;
- C-terminal truncation;
- soybean (Glycine max (L.) cv. Ransom)
Cytosolic pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 126.96.36.199) is an important glycolytic enzyme, but the post-translational regulation of this enzyme is poorly understood. Sequence analysis of the soybean seed enzyme suggested the potential for two phosphorylation sites: site-1 (FVRKGS220DLVN) and site-2 (VLTRGGS407TAKL). Sequence- and phosphorylation state-specific antipeptide antibodies established that cytosolic pyruvate kinase (PyrKinc) is phosphorylated at both sites in vivo. However, by SDS–PAGE, the phosphorylated polypeptides were found to be smaller (20–51 kDa) than the full length (55 kDa). Biochemical separations of seed proteins by size exclusion chromatography and sucrose-density gradient centrifugation revealed that the phosphorylated polypeptides were associated with 26S proteasomes. The 26S proteasome particle in developing seeds was determined to be of approximately 1900 kDa. In vitro, the 26S proteasome degraded associated PyrKinc polypeptides, and this was blocked by proteasome-specific inhibitors such as MG132 and NLVS. By immunoprecipitation, we found that some part of the phosphorylated PyrKinc was conjugated to ubiquitin and shifted to high molecular mass forms in vivo. Moreover, recombinant wild-type PyrKinc was ubiquitinated in vitro to a much greater extent than the S220A and S407A mutant proteins, suggesting a link between phosphorylation and ubiquitination. In addition, during seed development, a progressive accumulation of a C-terminally truncated polypeptide of approximately 51 kDa was observed that was in parallel with a loss of the full-length 55 kDa polypeptide. Interestingly, the C-terminal 51 kDa truncation showed not only pyruvate kinase activity but also activation by aspartate. Collectively, the results suggest that there are two pathways for PyrKinc modification at the post-translational level. One involves partial C-terminal truncation to generate a 51 kDa pyruvate kinase subunit which might have altered regulatory properties and the other involves phosphorylation and ubiquitin conjugation that targets the protein to the 26S proteasome for complete degradation.