A Kinase-Phosphatase Signaling Module with BSK8 and BSL2 Involved in Regulation of Sucrose-Phosphate Synthase

External supply of sucrose to carbon-starved Arabidopsis seedlings induced changes in phosphorylation
of Brassinosteroid
Signaling Kinase 8 (BSK8) at two different sites. Serine S20 lies within a phosphorylation hotspot at the N-terminal region of
the protein, while S213 is located within the kinase domain
of BSK8. Upon sucrose supply phosphorylation of BSK8S20 and BSK8S213 showed opposite behavior with increasing
phosphorylation of S213 and decreased phosphorylation of
S20 at 5 min after sucrose supply. Here we aim to systematically
analyze the effects of BSK8 mutations on downstream cellular regulatory
events and characterize molecular functions of BSK8 and its phosphorylation.
Comparative phosphoproteomic profiling of a bsk8 knockout
mutant and wild type revealed potential targets in sucrose metabolism.
Activity of sucrose-phosphate synthase (SPS) was decreased by phosphorylation
at S152, and SPS phosphorylation inversely correlated with
sucrose-induced BSK8 activity. Furthermore, BSK8 was found to interact
with BSL2, a Kelch-type phosphatase. On the basis of a combination
of kinase activity measurements, SPS activity assays, and phosphorylation
site mutations in BSK8 at S20 and S213, we conclude
that regulation of SPS by BSK8 occurs through activation of a phosphatase
that in turn may dephosphorylate SPS and thus activates the enzyme.