Poster: Hormones

Abs # 564: The phosphorylation regulatory mechanism of LeACS2 turnover

Presenter:	Mori, Hitoshi , morihito@agr.nagoya-u.ac.jp
Authors	Mori, Hitoshi  (A)   Iwata, Mineko  (A)   Fukaya, Tomoko  (A)   Tatsuki, Miho  (A) (B)
Affiliations:	(A): Nagoya University (B): National Institute of Fruit Tree Science

ACC synthase (ACS) is a rate-limiting enzyme of ethylene biosynthesis that is regulated not only transcriptionally but also post-translationally. We found that LeACS2 is phosphorylated at Ser-460 at the C-terminal region. Phosphorylation/dephosphorylation of LeACS2 did not affect the enzyme activity. Western blot analyses with the anti-LeACS2 and anti-phosphorylated LeACS2 antibodies suggested that LeACS2 was phosphorylated immediately after translation of LeACS2. More LeACS2 protein accumulated by wounding with calyculin A or okadaic acid treatment than by wounding alone (control). In contrast, less LeACS2 protein accumulated by wounding with staurosporine or k252a treatment than in controls. These results suggested that the half-life of LeACS2 was controlled by phosphorylation. To determine the half-life of LeACS2, we performed pulse-chase experiments in the presence/absence of protein phosphatase/kinase inhibitors. The results indicated that the half-life of LeACS2 was 60 min (controls), whereas it was 100 min and 45 min when wounding with calyculin A and k252a, respectively. Based on these results, we propose the following regulatory mechanism: LeACS2 acts in the phosphorylated form in the cell. Phosphorylation prevents degradation of LeACS2 while dephosphorylation accelerates it. Furthermore, we identified the kinase that phosphorylates LeACS2. cDNA libraries of wounded tomato fruits were constructed using the ZAP II vector and infected E. coli BL21, in which LeACS2 can be expressed. cDNAs and LeACS2 were co-expressed with isopropyl thiogalactoside and clones that harbored kinases for LeACS2 were immuno-screened using the anti-phosphorylated LeACS2 antibody. A specific CDPK was cloned that was similar to NtCDPK2 that induced by elicitors and osmotic stress.