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A gibberellin‐regulated protein phosphorylated by a putative Ca 2+ ‐dependent protein kinase is G‐protein mediated in rice root
Author(s) -
KHAN M. M. K.,
YANG S.,
IWASAKI Y.,
FUJISAWA Y.,
FUKUDA H.,
KOMATSU S.
Publication year - 2005
Publication title -
plant, cell and environment
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.646
H-Index - 200
eISSN - 1365-3040
pISSN - 0140-7791
DOI - 10.1111/j.1365-3040.2005.01321.x
Subject(s) - protein kinase a , biology , biochemistry , map2k7 , mitogen activated protein kinase kinase , protein phosphorylation , kinase , microbiology and biotechnology , cyclin dependent kinase 2 , protein a/g , cgmp dependent protein kinase , gene , fusion protein , recombinant dna
The GA‐signal transduction pathways downstream to the G α protein in rice seedling root were investigated using in‐gel kinase assay and in vitro protein phosphorylation techniques with a G α protein defective mutant, d1. A 50‐kDa protein kinase was detected downstream to G α protein in the membrane fraction of rice seedling roots using an in‐gel kinase assay with histone III‐S as a substrate. The activity of a 50‐kDa protein kinase increased in the wild‐type rice by gibberellin (GA 3 ) treatment, but did not change in the d1 mutant. This protein kinase activity was inhibited by the Ca 2+ chelator ethyleneglycol‐bis‐(beta‐aminoethylether)‐ N , N , N 1 , N 1 ‐tetraacetic acid (EGTA), protein kinase inhibitors, staurosporine and H7, and calmodulin antagonist, trifluoperazine, suggesting that the 50‐kDa protein kinase is a putative plant Ca 2+ ‐dependent protein kinase (CDPK). The activity of the 50‐kDa putative CDPK reached its highest level at 3 h after GA 3 treatment and then gradually declined with time. In order to identify the endogenous substrate for 50‐kDa putative CDPK, two‐dimensional polyacrylamide gel electrophoresis followed by in vitro protein phosphorylation was carried out. The phosphorylation activity of an endogenous protein PP30, identified as an unknown protein having molecular weight 30 kDa and isoelectric point 5.8 was increased in the wild‐type rice by GA 3 treatment, compared with the d1 mutant. The addition of GA 3 treated membrane fraction, which predominantly represent a 50‐kDa putative CDPK further increased the phosphorylation of PP30. Almost similar to GA 3 treatment, phosphorylation activity of PP30 was also increased by the treatment with cholera toxin in the wild‐type rice but not in d1 mutant. These results suggest that the 50‐kDa putative CDPK and an unknown protein, PP30 promoted by GA 3 treatment are G‐protein mediated in rice seedling roots.