An Autophosphorylation Site of the Protein Kinase SOS2 Is Important for Salt Tolerance in Arabidopsis

The protein kinase SOS2 （Salt Overly Sensitive 2） is essential for salt-stress signaling and tolerance in Arabidopsis. SOS2 is known to be activated by calcium-SOS3 and by phosphorylation at its activation loop. SOS2 is autophosphorylated in vitro, but the autophosphorylation site and its role in salt tolerance are not known. In this study, we identified an autophosphorylation site in SOS2 and analyzed its role in the responses of Arabidopsis to salt stress. Mass spectrometry analysis showed that Ser 228 of SOS2 is autophosphorylated. When this site was mutated to Ala, the autophosphorylation rate of SOS2 decreased. The substrate phosphorylation by the mutated SOS2 was also less than that by the wild-type SOS2. In contrast, changing Ser228 to Asp to mimic the autophosphorylation enhanced substrate phosphorylation by SOS2. Complementation tests in a sos2 mutant showed that the S228A but not the $228D mutation partially disrupted the function of SOS2 in salt tolerance. We also show that activation loop phosphorylation at Thr168 and autophosphorylation at Ser228 cannot substitute for each other, suggesting that both are required for salt tolerance. Our results indicate that Ser 228 of SOS2 is autophosphorylated and that this autophosphorylation is important for SOS2 function under salt stress.

Identification of Ser465 as a novel PINK1 autophosphorylation site

Background:PINK1(PTEN-induced putative kinase 1)gene is the causal gene for recessive familial type 6 of Parkinson’s disease(PARK6),which is an early-onset autosomal recessive inherited neurodegenerative disease.PINK1 has been reported to exert both autophosphorylation and phosphorylation activity,affecting cell damage under stress and other physiological responses.However,there has been no report on the identification of PINK1 autophosphorylation sites and their physiological functions.Methods:(1)We adopted mass spectrometry assay to identify the autophosphorylation site of PINK1,and autoradiography assay was further conducted to confirm this result.(2)Kinase activity assay was used to compare the kinase activity of both Ser465 mutant PINK1 and disease-causing mutant PINK1.(3)We use Pulse-chase analysis to measure whether Ser465 may affect PINK1 degradation.(4)Immunocytochemistry staining was used to study the PINK1 subcellular localization and Parkin transition in subcellular level.Result:In our study,we identified the 465th serine residue(Ser465)as one of the autophosphorylation sites in PINK1 protein.The inactivation of Ser465 can decrease the kinase activity of PINK1.Either dissipated or excessive Ser465 site phosphorylation of PINK1 can slow down its degradation.PINK1 autophosphorylation contributes to the transit of Parkin to mitochondria,and has no effect on its subcellular localization.PARK6 causal mutations,T313 M and R492X,display the same characteristics as Ser465A mutation PINK1 protein,such as decreasing PINK1 kinase activity and affecting its interaction with Parkin.Conclusion:Ser465 was identified as one of the autophosphorylation sites of PINK1,which affected PINK1 kinase activity.In addition,Ser465 is involved in the degradation of PINK1 and the transit of Parkin to mitochondria.T313 M and R492X,two novel PARK6 mutations on Thr313 and Arg492,were similar to Ser465 mutation,including decreasing PINK1 phosphorylation activity and Parkin subcellular localization.

Structural and biochemical basis of Arabidopsis FERONIA receptor kinase-mediated early signaling initiation

Accumulating evidence indicates that early and essential events for receptor-like kinase(RLK)function involve both autophosphorylation and substrate phosphorylation.However,the structural and biochemical basis for these events is largely unclear.Here,we used RLK FERONIA(FER)as a model and crystallized its core kinase domain(FER-KD)and two FER-KD mutants(K565R,S525A)in complexes with ATP/ADP and Mg^(2+) in the unphosphorylated state.Unphosphorylated FER-KD was found to adopt an unexpected active conformation in its crystal structure.Moreover,unphosphorylated FER-KD mutants with reduced(S525A)or no catalytic activity(K565R)also adopt similar active conformations.Biochemical studies revealed that FER-KD is a dual-specificity kinase,and its autophosphorylation is accomplished via an intermolecular mechanism.Further investigations confirmed that initiating substrate phosphorylation requires autophosphorylation of the activation segment on T696,S701,and Y704.This study reveals the structural and biochemical basis for the activation and regulatory mechanism of FER,providing a paradigm for the early steps in RLK signaling initiation.

Structural analysis of receptor-like kinase SOBIR1 reveals mechanisms that regulate its phosphorylation-dependent activation

Plant leucine-rich repeat(LRR)receptor-like kinases(RLKs)and LRR receptor-like proteins(RLPs)comprise a large family of cell surface receptors that play critical roles in signal perception and transduction.Both LRR-RLKs and LRR-RLPs rely on regulatory LRR-RLKs to initiate downstream signaling pathways.BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1/SOMATIC EMBRYOGENESIS RECEPTOR KINASE 3(BAK1/SERK3)and SUPPRESSOR OF BIR1-1(SOBIR1)are important and extensively studied regulatory LRR-RLKs with distinct functions.Although the regulatory mechanism of BAK1 activation has been studied in detail,the activation mechanism of SOBIR1 remains poorly understood.Here,the crystal structures of the catalytically inactive kinase domain of SOBIR1(SOBIR1-KD)from Arabidopsis thaliana were determined in complexes with AMP-PNP and Mg^(2+).The results show that SOBIR1-KD contains a uniquely long β3-αC loop and adopts an Src-like inactive conformation with an unusual architecture at the activation segment,which comprises three helices.Biochemical studies revealed that SOBIR1 is transphosphorylated by BAK1 following its autophosphorylation via an intermolecular mechanism,and the phosphorylation of Thr529 in the activation segment and the β3-αC loop are critical for SOBIR1 phosphorylation.Further functional analysis confirmed the importance of Thr529 and the β3-αC loop for the SOBIR1-induced cell death response in Nicotiana benthamiana.Taken together,these findings provide a structural basis for the regulatory mechanism of SOBIR1 and reveal the important elements and phosphorylation events in the special stepwise activation of SOBIR1-KD,the first such processes found in regulatory LRR-RLKs.