The Arabidopsis SOS2 protein kinase physically interacts with and is activated by the calcium-binding protein SOS3

Abstract

The Arabidopsis thaliana SOS2 and SOS3 genes are required for intracellular Na(+) and K(+) homeostasis and plant tolerance to high Na(+) and low K(+) environments. SOS3 is an EF hand type calcium-binding protein having sequence similarities with animal neuronal calcium sensors and the yeast calcineurin B. SOS2 is a serine/threonine protein kinase in the SNF1/AMPK family. We report here that SOS3 physically interacts with and activates SOS2 protein kinase. Genetically, sos2sos3 double mutant analysis indicates that SOS2 and SOS3 function in the same pathway. Biochemically, SOS2 interacts with SOS3 in the yeast two-hybrid system and in vitro binding assays. The interaction is mediated by the C-terminal regulatory domain of SOS2. SOS3 activates SOS2 protein kinase activity in a Ca(2+)-dependent manner. Therefore, SOS3 and SOS2 define a novel regulatory pathway important for the control of intracellular ion homeostasis and salt tolerance in plants.

Figure 1

Genetic interaction between sos2 and sos3. (A) Seedling phenotypes of wild-type (WT) plants and sos2-2, sos3-1, and sos2sos3 double mutants treated with various levels of NaCl. (B) Root growth measurement. Five-day-old seedlings were transferred from normal nutrient medium to media supplemented with different concentrations of NaCl, and root elongation after 7 days is presented. Error bars represent the standard deviation (n = 12). The pictures were taken 10 days after the NaCl treatments.

Figure 2

Amino acid alignment of SOS2 with SIP1, SIP2, SIP3, and SIP4. Amino acids identical in SOS2 and at least another two proteins are highlighted in black and conservative substitutions in gray.

Figure 3

Interaction between SOS3 and SOS2, SOS2 N-terminal (SOS2-NT) and C-terminal (SOS2-CT) domains, and SIP1, SIP2, SIP3, or SIP4 in the yeast two-hybrid assay. Yeast strains containing pAS-SOS3 and pACT-SOS2, pACT-SOS2-NT, pACT-SOS2-CT, pACT-SIP1-1, pACT-SIP2-1, pACT-SIP3-1, pACT-SIP4-1, respectively, were assayed for LacZ expression. pAS-p53 and pAS2 were used as negative controls in combination with pACT-SOS2. (Upper Right) Yeast grown on SC medium minus tryptophan and leucine to select for both the bait and prey proteins. (Lower Right) SC medium minus tryptophan, leucine, histidine, and plus 25 mM 3-amino-1,2,4-triazole (AT) to allow the growth of only positively interacting clones.

Figure 4

SOS2 interacts with SOS3 in vitro. (A) Interaction between SOS3 and SOS2 in a pull-down assay. [³H]leucine-labeled SOS3 was incubated with glutathione-Sepharose immobilized GST, GST fusion proteins of SOS2, or RB. Proteins bound to the Sepharose beads were pelleted, washed thoroughly, electrophoresed, and detected by fluorography. (B) Interaction between SOS3 and SOS2 in a gel blot overlay assay. GST, partially purified GST-RB, GST-SOS2, GST-SOS2(K40N), GST-SOS2(G197E), and protein size markers (M) were separated by SDS/PAGE. Two identical gels were run; one was stained with Coomassie blue (Left), and the other was electroblotted and probed with [³²P]labeled SOS3 (Right).

Figure 5

SOS2 kinase activity is activated by SOS3 in a Ca²⁺-dependent manner. (A) SOS2 phosphorylation of peptide substrates is activated by SOS3. Oligopeptides p1, p2, and p3 were incubated in the presence of [γ-³²P] ATP and Ca²⁺ in a kinase buffer with GST-SOS2, and with or without GST-SOS3. γ-³²P-incorporation was measured by scintillation counting. (B) SOS3 activation of SOS2 is Ca²⁺-dependent. Oligopeptide p3 was phosphorylated by GST-SOS2 in the presence of GST-SOS3, and with or without free Ca²⁺. Error bars represent the standard deviation (n = 3).

Figure 6

Proposed regulatory pathway for intracellular Na⁺ and K⁺ homeostasis and Na⁺ tolerance in plants.