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The tissue-preferential distributed calcium sensors, SOS3 and SCaBP8, play important roles in SOS pathway to cope with saline conditions. Both SOS3 and SCaBP8 interact with and activate SOS2. However the regulatory mechanism for SOS2 activation and membrane recruitment by SCaBP8 differs from SOS3. SCaBP8 is phosphorylated by SOS2 at plasma membrane (PM) under salt stress. This phosphorylation anchors the SCaBP8-SOS2 complex on plasma membrane and activates PM Na+/H+ anti-porter, such as SOS1. Here, we describe that SOS2 has high binding affinity and catalytic efficiency to SCaBP8, suggesting that phosphorylation of SCaBP8 by SOS2 perhaps occurs rapidly in salt condition. SCaBP8 is also phosphorylated by PKS5 (SOS2-like Protein Kinase5) which negatively regulates PM H+-ATPase activity and functions in plant alkaline tolerance, providing a clue to roles of SCaBP8 in both salt and alkaline tolerance. SOS2 interacts with SOS3 and SCaBP8 with its FISL motif at C-terminus. However, luciferase activity complement assay indicates that SOS2 N-terminal is also essential for interacting with these proteins in plant.
Due to their sessile nature, plants have developed elaborate strategies to deal with a number of environmental challenges. One overwhelming constraint is high salinity in the soil, which inhibits plant growth and decreases the agricultural productivity. Efflux and/or sequestering of sodium ion to apoplastic space/vacuolar are well-known cellular mechanisms that plants protect them from saline stress.1 Recently identified SOS (salt overly sensitive) pathway plays critical roles in maintaining ion homeostasis in response to high salinity.2 Two calcium sensors, SOS3 and SCaBP8 (SOS3-like calcium binding protein8), perceive cytosolic calcium signature triggered by salt, interact with and activate a Thr/Ser protein kinase, SOS2 and recruit it to the plasma membrane. Then, the formed SOS3-SOS2 or SCaBP8-SOS2 complex activates a PM Na+/H+ anti-porter, SOS1.2–4 Moreover, SOS2 also regulates vascular Na+/H+ antiporter activity.5 Previously, we reported that SCaBP8 and SOS3 function distinctly in activation of SOS2.3 For instance, N-terminal myristoylation of SOS3 plays an important role in salt tolerance.6 However, there is no consensus myristoylated motif in SCaBP8. Instead, an N-terminal hydrophobic domain is sufficient to facilitate the association of SCaBP8 to plasma membrane.3 In addition, SCaBP8 is phosphorylated by SOS2 under salt stress and this phosphorylation stabilizes the interaction of SOS2 and SCaBP8.4 In this report, we describe that SCaBP8 possibly is rapidly phosphorylated by SOS2 under salt stress and also phosphorylated by another stress responsible protein kinase, implying additional roles of SCaBP8 in stress responses.
Previously, we have shown that activation of SOS2 requires SCaBP8 binding to SOS2 at FISL motif, which is similar to SOS3 in activating SOS2.3,4,7 SOS2 phosphorylates SCaBP8 at Ser-237 but not SOS3. To examine the affinity and catalytic efficiency of phosphorylation of SCaBP8 by SOS2, we determined the apparent kinetic parameters using SCaBP8 and P3 peptide (a synthetic peptide designed based on the recognition sequences of protein kinase C or SNF1/AMPK kinases, ALARAASAAALARRR) as substrates (Fig. 1). The apparent Km value and Vmax of SOS2 for SCaBP8 is determined to be 12.11 µM and 1.24 × 104 µM min−1. However, the apparent Km value is increased 10-fold and Vmax is reduced 100-fold when P3 peptide was used as substrate (data not shown). The data demonstrate that SOS2 prefers SCaBP8 as a substrate.
Lower apparent Km value (higher affinity) and higher Kcat/Km (high catalytic efficiency) of SOS2 for SCaBP8 compared to P3 may reflect their response under salt stress in planta.4,8 Upon salt stress, SCaBP8 is rapidly phoshphorylated by SOS2 and anchors SOS2 on plasma membrane for activating SOS1.4 One possibility to explain this hypothesis is synergistic effect between interaction and phosphorylation. The phosphorylated state of SCaBP8 stabilizes its interaction with SOS2 and that may be capable of activating SOS2 more efficiently, which is consistent with phenotype rescue of scabp8 by expressing SCaBP8S237D but only partially by expressing SCaBP8S237A.4
Soil alkalinity is often associated with increased soil salinity partly due to application of fertilizers and irrigation water. Our previously study showed that PKS5 function in alkaline stress response through directly phosphorylating PM H+-ATPase (AHA2) and repressing its activity, and SCaBP1 (SOS3-like calcium binding protein1) activates PKS5 in yeast.9 It is known that SOS2 also involves in regulating the PM proton pump activity. To test if other SOS2 like protein kinases (PKS) also phosphorylate SCaBP8, we find that PKS5 does (Fig. 2). More interestingly, Ser-237 likely is not the phosphorylation site for PKS5 as both SCaBP8S237A and SCaBP8S237D mutations are phosphorylated by PKS5. It is possibly that phosphorylations in multiple sites of SCaBP8 by PKSes are in response to various environmental changes. Due to scabp1 mutant did not show significant phenotypic change under external high pH treatment, it is reasonable to believe that other SCaBPs, like SCaBP8, together with SCaBP1 regulate PKS5 activity in response to alkaline condition.9,10
In yeast two-hybrid assay, the C-terminal regulatory domain of SOS2 interacts with its N-terminal kinase domain and this intremolecular interaction inhibits SOS2 kinase activity.2,5,11,12 The hypothesis is when SOS3 is evoked by salt stress, it interacts with SOS2 C-terminal at FISL motif and this in turn releases SOS2 kinase domain for substrate accessing, such as SOS1. FISL motif is necessary and essential for SOS2 interacting with SOS3 and SCaBP8 and serves as a kinase autoinhibitory domain.2–4,11 To extend our views of SOS3-SOS2 and SCaBP8-SOS2 interaction in vivo, we performed a luciferase complementation assay.13 C-terminal region of luciferase (cLUC) was fused to N-terminus of SOS3, SCaBP8 or SOS2. N-terminus region of luciferase (nLUC) was fused to the C-terminus of SOS3, SCaBP8 or SOS2. Different combinations of SOS2 and SOS3/SCaBP8 were cotransformed into Arabidopsis protoplasts. Luciferase activity was only observed when nLUC was fused to SOS2 C-terminus, suggesting that SOS2-nLUC was able to interact with cLUC-SOS3/SCaBP8. This interaction was abolished when FISL motif was removed from SOS2 and when cLUC was fused to SOS2 N-terminus (Fig. 3). These data suggest that N-terminal structure of SOS2 is also required for its interacting with SOS3 and SCaBP8 in plant.
Previously published online: www.landesbioscience.com/journals/psb/article/9641