Identification of conserved tyrosine residues important for gibberellin sensitivity of Arabidopsis RGL2 protein.
DELLA proteins are regulators in the signaling pathway of gibberellin (GA), a plant growth regulator of diverse functions. GA typically induces the degradation of DELLA proteins to overcome their repressive roles in growth and development. We have previously evaluated the likely roles of Ser-Thr phosphorylation of DELLA proteins in GA signaling (Hussain et al., Plant J 44:88-99, 2005). Here we report that four DELLA proteins of Arabidopsis, namely GAI, RGL1, RGL2 and RGL3, expressed in tobacco BY2 cells, are degradable by GA. Both, proteasome inhibitor and protein tyrosine (Tyr) kinase inhibitors, strongly inhibit GA-induced DELLA degradation whereas phospho-Tyr phosphatase inhibitors have no effect, suggesting that Tyr phosphorylation is critical in GA-induced DELLA degradation. Mutation of eight conserved Tyr residues of RGL2 into alanine shows four mutant proteins (Y52A, Y89A, Y223A and Y435A) are resistant to GA-induced degradation. Substitution of these four critical Tyr residues into negatively charged glutamate (Y --> E) also resulted in stabilization of these mutants against GA treatment. However, further mutation of these four Tyrs into conservative phenylalanine (Y --> F) rendered the mutant proteins sensitive to GA like the wild-type RGL2. Since Y --> E mutations sometimes mimic phosphor-Tyr whereas Y --> F mutations render the protein unphosphorylatable at these Tyr sites, we conclude that these four conserved Tyrs, despite being critical for GA-sensitivity, are unlikely to be sites of Tyr phosphorylation but instead play important roles in maintaining the structure integrity of RGL2 for GA-sensitivity.
Hussain
Alamgir
A
Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore 138673.
Cao
Dongni
D
Peng
Jinrong
J
D017360:Arabidopsis / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005875:Gibberellins / Q000378:metabolism*; D009154:Mutation; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D014026:Nicotiana / Q000235:genetics; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D014443:Tyrosine / Q000737:chemistry
D029681:Arabidopsis Proteins; D004791:Enzyme Inhibitors; C109555:GAI protein, Arabidopsis; D005875:Gibberellins; C450824:RGL1 protein, Arabidopsis; C450823:RGL2 protein, Arabidopsis; C485889:RGL3 protein, Arabidopsis; D014157:Transcription Factors; D014443:Tyrosine