Higher plants have evolved numerous RLKs and RLCKs that modulate many different biological processes, including innate immunity, growth and development, abiotic stresses, and stomatal aperture. Phosphorylation of the RLK/RLCK complex is an essential step in the initiation of diverse biological and physiological processes. Among RLCKs in Arabidopsis, BIK1 is a serine/threonine kinase that possesses tyrosine kinase activity, and mass spectrometry, immunoblot, and genetic analyses have shown that it is autophosphorylated at multiple Ser/Thr/Tyr residues. BAK1, a co-receptor of BRI1, is able to phosphorylate BIK1 at both tyrosine and serine/threonine residues 22. BIK1 (Y150) blocks both tyrosine and serine/threonine kinase activity, whereas BIK1 (Y243 and Y250) are more specifically involved in tyrosine phosphorylation 22. In the present study, AtBIK1 showed strong autophosphorylation kinase activity on tyrosine and threonine residues, in contrast to BrBIK1, despite high amino acid similarity between these two proteins (Fig. 1). To identify downstream signaling components of BIK1 related to growth and defense signaling in A. thaliana, we used an Arabidopsis library for Y2H screening, and identified four proteins that interact with BrBIK1 but not AtBIK1(Fig. 2A); UDP-arabinopyranose mutase 2 (RGP2), Patellin-2 (PATL2), serine/threonine-protein phosphatase 7 (PP7), and sulfate transporter 4.1 (SULTR4.1). RGP2 is essential for proper cell wall and pollen development 13. PATL2 may be involved in membrane trafficking events associated with cell plate formation during cytokinesis 14. PP7 acts as a positive regulator of cryptochrome signaling involved in hypocotyl growth inhibition, cotyledon expansion under white and blue light conditions 15, and control of Arabidopsis stomatal aperture 16. SULTR4.1 is an H+/sulfate cotransporter that may play a role in the regulation of sulfate assimilation. Y2H results suggested that BrBIK1 interactors might be involved in plant growth and development processes, including sulfate assimilation, which indicates that non-phosphorylated BIK1 might not be involved in defense mechanisms, including PTI.
We also employed BiFC experiments to confirm interactions between BIK1 and RGP2, PP7, and PATL2 in N. benthamiana leaves. As a positive control we used SOS2, which encodes a novel Ser/Thr protein kinase that also functions in salt tolerance in Arabidopsis 22, and SOS3 that encodes a novel EF-hand Ca2+ sensor23. We confirmed that AtBIK1 does not interact with RGP2, PP7, and PATL2 (Fig. 2C), whereas BrBIK1 strongly interacts with these proteins (Fig. 2B). The results indicate that phosphorylation of BIK1 is not necessary for RGP2, PP7, PATL2, and BrBIK1 interactions, suggesting that BIK1 phosphorylation is essential for plant defense signaling, but not necessary for plant growth and development signaling.
Therefore, we performed amino acid sequence alignments and investigated known phosphorylation sites of AtBIK1 in A. thaliana. Protein phosphorylation is a key event in signal transduction pathways. When upstream signals are stimulated through receptor kinases, protein kinases are activated and phosphorylate their substrates on Ser/Thr and tyrosine residues, modulating their localization, conformation, and activity. In some cases, phosphorylated substrates become recognizable to other proteins, and such interactions transduce and propel the signal onward. Certain domains specifically recognize phosphorylated residues of proteins, regulating cell growth and differentiation 24. Analysis of recombinant RLCKs revealed distinct autophosphorylation activity at tyrosine and threonine residues in E. coli 25. Given that phosphorylation of RLCKs generally initiates and mediates signal transduction pathways, these two RLCKs might also be involved in sensing and responding to cellular needs in Arabidopsis 25.
In the present work, we focused on the status of BIK1 phosphorylation, which may be essential for interactions between BIK1 and downstream components to regulate diverse physiological processes. Therefore, we cloned BrBIK1 and AtBIK1 into the pDEST15B and pFlag recombinant expression vectors. The results of western blotting analysis with anti-pThr and anti-pY antibodies showed that GST-BrBIK1 and Flag-BrBIK1 did not undergo autophosphorylation at threonine and tyrosine residues. However, in contrast with BrBIK1, AtBIK1 exhibited strong autophosphorylation kinase activity at threonine and tyrosine residues (Fig. 3A and B). Thus, we performed site-directed mutagenesis (SDM) to manipulate autophosphorylation of AtBIK1 and BrBIK1 based on amino acid sequence alignment (Fig. 1), and generated three threonine phosphorylation site mutants in GST-AtBIK1 (T90S, T362P, and T368A). The three sites differ from those in BrBIK1. The results showed that GST-AtBIK1(T90S), GST-AtBIK1(T362P), GST-AtBIK1(T368A), and wild-type GST-AtBIK1 exhibited almost identical autophosphorylation kinase activities (Fig. 3B). We also used SDM to generate Flag-BrBIK1 mutants P362T and A368T. Interestingly, both mutations rescued autophosphorylation kinase activity at threonine residues to levels comparable with AtBIK1 in vitro (Fig. 3C). Thus, we tested protein interactions using BiFC experiments with BrBIK1 (P362T, A369T) and RGP2, PATL2, and PP7. Surprisingly, BrBIK1 (P362T, A369T) did not bind downstream components RGP2, PATL2, and PP7 (Fig. 4A and B). These results suggest that the phosphorylation status of BIK1 may play an important role in regulating between growth and defense in higher plants. The findings indicate that autophosphorylation of RLCKs such as BIK1 provides a convenient and powerful system for elucidating kinase specificity in vitro and in vivo.