Receptor tyrosine kinases control many critical processes in metazoans,   but these enzymes appear to be absent in plants. Recently, two

   Arabidopsis receptor kinases-BRASSINOSTEROID INSENSITIVE 1 (BRI1) and

   BRI1-ASSOCIATED KINASE1 (BAK1), the receptor and coreceptor for

   brassinosteroids-were shown to autophosphorylate on tyrosines. However,

   the cellular roles for tyrosine phosphorylation in plants remain poorly

   understood. Here, we report that the BRI1 KINASE INHIBITOR 1 (BKI1) is

   tyrosine phosphorylated in response to brassinosteroid perception.

   Phosphorylation occurs within a reiterated [KR][KR] membrane

   targeting motif, releasing BKI1 into the cytosol and enabling formation

   of an active signaling complex. Our work reveals that tyrosine

   phosphorylation is a conserved mechanism controlling protein

   localization in all higher organisms

Belkhadir, Youssef

Chory, Joanne

Dabi, Tsegaye

Hothorn, Michael

Jaillais, Yvon

Meyerowitz, Elliot M.

Nimchuk, Zachary L.

English

PubMed

Caltech Authors - Main

Tyrosine phosphorylation controls brassinosteroid receptor activation   by triggering membrane release of its kinase inhibitor

Supplementary Material and methods 
Plant material and growth conditions. The wild type used in all experiments was 
Columbia (Col0) and plants were grown on either soil or Petri dishes containing 0.5X 
Linsmaier and Skoog salts medium in long days. The MAKR genes are defined as: 
MAKR1: At5g26230; MAKR2: At1g64080; MAKR3: At2g37380; MAKR4: At2g39370; 
MAKR5: At5g52870; MAKR6: At5g52900. We used bak1-3 (Chinchilla et al. 2007) and 
the null bri1 allele GABI_134E10.  
 
Constructs, generation of transgenic lines, and phenotype analysis. All the BKI1 
constructs (deletions and mutants included) are tagged with mCITRINE at their C-
terminus. mCITRINE-tagged lines are resistant to glufosinate (Basta), 6xHA-tagged lines 
to kanamycin and mCHERRY-tagged lines to hygromycin. Rosette radius phenotypes 
were quantified on 5 week-old plants. UBQ10prom was PCR amplified from 
pNIGEL(Geldner et al. 2009) (gift from N. Geldner), 35Sprom from pBJ36 (Gift from J. 
Long), BRI1prom (1.7 kb), BAK1prom (1.7 kb) and BKI1prom (2 kb) from Col0 genomic 
DNA and cloned into pDONR-P4P1R using gateway recombination (Invitrogen) (see 
supplementary table 1 for primers). BKI1, BKI1 deletion, BRI1, BAK1 and MAKR1 were 
PCR amplified from Col0 genomic DNA and recombined into pDONR221 (Invitrogen). 
mCITRINE (a pH-resistant YFP variant), mCHERRY (gift from R. Tsien) and 6xHA 
(pBK36, Gift from J. Long) were cloned into pDONR-P2RP3 (Invitrogen). Final 
destination vectors were obtained by using three fragments recombination system using 
the pB7m34GW, pH7m34GW and pK734GW destination vectors(Karimi et al. 2007). 
The vectors created are listed in supplementary table 2. BRI1 and BAK1 constructs were 
transformed into heterozygous bri1 (GABI_134E10) and homozygous bak1-3 
respectively and their transgenic expression fully rescued the bri1 and bak1 growth 
defect. For all constructs, more than 20 independent T1 lines were isolated and between 
3 to 6 representative mono-insertion lines were selected in T2. Confocal microscopy, 
phenotypic analysis and protein extraction were performed on segregating T2 and 
omozygous T3 lines.  h
 
 
 
Supplementary References: 
 
 
Chinchilla, D., Zipfel, C., Robatzek, S., Kemmerling, B., Nürnberger, T., Jones, J.D.G., Felix, G., 
and Boller, T. 2007. A flagellin-induced complex of the receptor FLS2 and BAK1 initiates 
plant defence. Nature 448(7152): 497-500. 
Deprez, C., Lloubes, R., Gavioli, M., Marion, D., Guerlesquin, F., and Blanchard, L. 2005. 
Solution structure of the E.coli TolA C-terminal domain reveals conformational changes 
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visualization using persistence of vision ray-tracing. Journal of Applied Crystallography 
36: 944-947. 
Geldner, N., Dénervaud-Tendon, V., Hyman, D.L., Mayer, U., Stierhof, Y.-D., and Chory, J. 2009. 
Rapid, combinatorial analysis of membrane compartments in intact plants with a 
multicolor marker set. Plant J 59(1): 169-178. 
Karimi, M., Bleys, A., Vanderhaeghen, R., and Hilson, P. 2007. Building blocks for plant gene 
assembly. Plant Physiol 145(4): 1183-1191. 
Kuglstatter, A., Villasenor, A.G., Shaw, D., Lee, S.W., Tsing, S., Niu, L., Song, K.W., Barnett, 
J.W., and Browner, M.F. 2007. Cutting Edge: IL-1 receptor-associated kinase 4 
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Receptor tyrosine kinases control many critical processes in metazoans, but these enzymes appear to be absent in plants. Recently, two Arabidopsis receptor kinases—BRASSINOSTEROID INSENSITIVE 1 (BRI1) and BRI1-ASSOCIATED KINASE1 (BAK1), the receptor and coreceptor for brassinosteroids—were shown to autophosphorylate on tyrosines. However, the cellular roles for tyrosine phosphorylation in plants remain poorly understood. Here, we report that the BRI1 KINASE INHIBITOR 1 (BKI1) is tyrosine phosphorylated in response to brassinosteroid perception. Phosphorylation occurs within a reiterated [KR][KR] membrane targeting motif, releasing BKI1 into the cytosol and enabling formation of an active signaling complex. Our work reveals that tyrosine phosphorylation is a conserved mechanism controlling protein localization in all higher organisms.</jats:p

Yvon Jaillais

Michael Hothorn

Youssef Belkhadir

Tsegaye Dabi

Zachary L. Nimchuk

Elliot M. Meyerowitz

Joanne Chory

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Genes & Development

Tyrosine phosphorylation controls brassinosteroid receptor activation by triggering membrane release of its kinase inhibitor

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https://authors.library.caltech.edu/22865/1/Jaillais2011p12985Genes_Dev.pdf

Tyrosine phosphorylation controls brassinosteroid receptor activation by triggering membrane release of its kinase inhibitor

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