Molecular Characterization of Functional Domains in the Protein Kinase SOS2 That Is Required for Plant Salt Tolerance

Abstract

The SOS3 (for SALT OVERLY SENSITIVE3) calcium binding protein and SOS2 protein kinase are required for sodium and potassium ion homeostasis and salt tolerance in Arabidopsis. We have shown previously that SOS3 interacts with and activates the SOS2 protein kinase. We report here the identification of a SOS3 binding motif in SOS2 that also serves as the kinase autoinhibitory domain. Yeast two-hybrid assays as well as in vitro binding assays revealed a 21-amino acid motif in the regulatory domain of SOS2 that is necessary and sufficient for interaction with SOS3. Database searches revealed a large family of SOS2-like protein kinases containing such a SOS3 binding motif. Using a yeast two-hybrid system, we show that these SOS2-like kinases interact with members of the SOS3 family of calcium binding proteins. Two-hybrid assays also revealed interaction between the N-terminal kinase domain and the C-terminal regulatory domain within SOS2, suggesting that the regulatory domain may inhibit kinase activity by blocking substrate access to the catalytic site. Removal of the regulatory domain of SOS2, including the SOS3 binding motif, resulted in constitutive activation of the protein kinase, indicating that the SOS3 binding motif can serve as a kinase autoinhibitory domain. Constitutively active SOS2 that is SOS3 independent also was produced by changing Thr(168) to Asp in the activation loop of the SOS2 kinase domain. Combining the Thr(168)-to-Asp mutation with the autoinhibitory domain deletion created a superactive SOS2 kinase. These results provide insights into regulation of the kinase activities of SOS2 and the SOS2 family of protein kinases.

Phosphoproteomic identification of targets of the Arabidopsis sucrose nonfermenting-like kinase SnRK2.8 reveals a connection to metabolic processes

“…OST1 is autophosphorylated at Ser-175 and this residue is essential for its activation and function and is located in the T-loop region (54). Phosphorylation sites of SnRK1 (AKIN10) and SnRK3 (SOS2) have also been found in the T-loop (20,55,56). SnRK2.8 is autophosphorylated at Thr-158, which corresponds to the Ser-175 in OST1.…”

Yokonolide B, a Novel Inhibitor of Auxin Action, Blocks Degradation of AUX/IAA Factors

“…Cells were washed two times with uracil dropout medium supplemented with 2% glucose to stop further transcription of the fusion protein and resuspended in the same medium containing YkB or MG132 and cultured at 28°C. Galactosidase activity was measured as previously described (30). ATP-dependent 20 S core unit activity of 26 S proteasome in Arabidopsis suspension T-87 cells was measured by peptide-hydrolysis using succinyl-Leu-Leu-Val-Tyr-4-methyl-coumaryl-7-amide as the substrate in the presence or absence of ATP and Mg 2ϩ , as previously described (31).…”

Ca2+-Dependent Protein Kinase11 and 24 Modulate the Activity of the Inward Rectifying K+ Channels inArabidopsisPollen Tubes

“…Besides MAPK cascades, the regulation of some other protein kinases has been reported to be achieved by transphosphorylation. Some residues in the activation loop of SOS2 (CIPK24) can be phosphorylated by unknown protein kinases (Guo et al, 2001;Gong et al, 2002). At least one CDPK from tobacco (CDPK2) requires transphosphorylation by unknown kinases to become fully activated (Romeis et al, 2000(Romeis et al, , 2001.…”