Interaction of calmodulin, a sorting nexin and kinase-associated protein phosphatase with the Brassica oleracea S locus receptor kinase

Abstract

Recognition of self-pollen during the self-incompatibility response in Brassica oleracea is mediated by the binding of a secreted peptide (the S locus cysteine-rich protein) to the S locus receptor kinase (SRK), a member of the plant receptor kinase (PRK) superfamily. Here, we describe the characterization of three proteins that interact with the cytosolic kinase domain of SRK. A B. oleracea homolog of Arabidopsis kinase-associated protein phosphatase was shown to interact with and dephosphorylate SRK and was itself phosphorylated by SRK. Yeast (Saccharomyces cerevisiae) two-hybrid screens identified two additional interactors, calmodulin and a sorting nexin, both of which have been implicated in receptor kinase down-regulation in animals. A calmodulin-binding site was identified in sub-domain VIa of the SRK kinase domain. The binding site is conserved and functional in several other members of the PRK family. The sorting nexin also interacted with diverse members of the PRK family, suggesting that all three of the interacting proteins described here may play a general role in signal transduction by this family of proteins.

Figure 1.

Interaction of the Brassica KAPP and SRK proteins. A, Alignment of the FHA domains from the Brassica (Bo KAPP) and Arabidopsis (At KAPP) KAPP proteins and yeast (Saccharomyces cerevisiae) RAD53 (Sc RAD53). Arrowheads indicate residues required for binding of Arabidopsis KAPP to PRKs (Li et al., 1999). B, Kinase interaction (KI) domain of Brassica KAPP and the C-terminal end of ARC1 (containing the Arm repeats) interact with the wild type but not with a kinase-inactive form of the SRK₂₉ kinase domain in the yeast two-hybrid assay. Different numbers of yeast strain PJ69-4A cells expressing the prey and bait indicated above and below the panel, respectively, were inoculated by spotting onto selective medium lacking His and adenine and grown at 30°C for 5 d. C, Phosphorylation of Brassica KAPP by SRK. Glutathione S-transferase (GST) fusion proteins with Brassica KAPP or with either wild-type or mutant forms of the SRK₂₉ kinase domain were mixed in phosphorylation reactions as indicated. The KAPP and SRK fusion proteins are indicated by arrowheads. Proteins were detected by autoradiography (top) or by immunoblotting with an anti-GST antibody (bottom). Note that the active form of the SRK₂₉ kinase domain was detected as a diffuse band (indicated by a bar). Phosphatase treatment showed that this was due to the presence of differentially phosphorylated isoforms (data not shown). D, Dephosphorylation of SRK by KAPP. Radiolabeled, phosphorylated GST::SRK₂₉kin was incubated in the presence or absence of GST::KAPP, and the proteins were separated on an SDS-PAGE gel. Radiolabeled GST::SRK₂₉kin was detected by autoradiography (top) and total GST::SRK₂₉kin protein was detected with an anti-GST antibody (bottom).

Figure 2.

Calmodulin binds to the kinase domain of SRK in vitro. A, Binding of GST or the GST protein fused with either wild-type or mutant forms of the SRK₂₉ kinase domain to calmodulin-Sepharose beads. GST-containing proteins were detected with an anti-GST antibody. B, Binding of the integral SRK protein to calmodulin-Sepharose beads. A recombinant hexa-His epitope-tagged, kinase-inactive form of SRK (^mSRK₃His; Giranton et al., 2000) was used for this experiment. SRK protein was detected with the monoclonal antibody MAb85-36-71 (Giranton et al., 2000). C, Binding of Brassica calmodulin to glutathione-Sepharose beads carrying either GST or GST protein fused to either wild-type or mutant forms of the SRK₂₉ kinase domain. Eluted proteins were detected by silver staining. Note that calmodulin alters its conformation in the presence of calcium resulting in a change in mobility. CAM, Calmodulin with no bound Ca²⁺; CAM/Ca, calmodulin with bound Ca²⁺; C, purified fusion protein; UB, unbound fraction; W1 and W40, washes; E1 to E4, eluted fractions. C and UB, One-quarter of the amount of protein loaded on and unbound to the column, respectively.

Figure 3.

Calmodulin binds to sub-domain VIa of the kinase domain of SRK in vitro. A, Amino acid sequence of the HEL1 region of sub-domain VIa of SRK₃. The wheel projection shows the amphiphilic nature of HEL1. The sequence of the control peptide PEP1 is also shown. B, Binding of GST::HEL1 and GST::PEP1 fusion proteins to calmodulin-Sepharose beads. GST-containing proteins were detected with an anti-GST antibody. C, purified GST fusion protein; UB, unbound fraction; W1 and W40, first and last washes; E1 to E4, eluted fractions.

Figure 4.

Calmodulin binds to the kinase domains of SFR1, RLK4, and CLV1, but not BRI1, in vitro. A, Analysis of the binding of the kinase domains of SFR1, RLK4, CLV1, and BRI1 to calmodulin-Sepharose. The kinase domains were expressed as fusion proteins with either GST or MBP. Both a wild-type (GST::SFR1kin) and a kinase-inactive mutant form (GST::SFR1kin^K555R) of the SFR1 kinase domain were tested. GST- and MBP-containing proteins were detected with anti-GST and anti-MBP antibodies, respectively. B, Abundance of calmodulin transcripts in different Brassica organs. Calmodulin transcripts were detected with a probe corresponding to the entire coding sequence. Lower, Ethidium bromide-stained rRNA. The positions of RNA size markers are shown at right in kilobase pairs. R, Root; C, cotyledon; L, leaf; Se, sepal; P, petal; A, anther; O, ovary; St, stigma.

Figure 5.

Sequence analysis of Brassica SNX1 and the sorting nexin family in Arabidopsis. A, Alignment of Brassica SNX1 (Bo SNX1) with Arabidopsis (At SNX1) and human (Hs SNX1) SNX1. Residues conserved in at least two proteins are shaded. The phox homology (PX) and coiled-coil domains of Brassica SNX1 are overlined with a continuous and a dotted line, respectively. B, Neighbor-joining tree based on an alignment of the PX domains of Brassica SNX1 and of the Arabidopsis, human, and yeast sorting nexin families. The tree was constructed using the PAM250 amino acid substitution matrix. Numbers next to the branches are bootstrap values expressed as percentage confidence level and based on 1,000 repeats. The PX domain of human PI-3 kinase was used to root the tree. The PX domain polypeptide sequences compared were Brassica SNX1, Arabidopsis SNX1 (At SNX1; accession no. At5g06140), SNX2a (At SNX2a; At5g58440), and SNX2b (At SNX2b; At5g07120), several human (Hs SNX1, Q13596; Hs SNX2, O60749; Hs SNX3, O60493; Hs SNX4, O95219; Hs SNX5, Q9Y5X3; Hs SNX6, Q9UNH7; Hs SNX7, Q9UNH6; Hs SNX8, Q9Y5X2; Hs SNX9, Q9Y5X1; Hs SNX10, Q9Y5X0; Hs SNX11, Q9Y5W9; Hs SNX12, Q9UMY4; Hs SNX15, Q9NRS6), and yeast (Sc YJL036W, S56808; Sc Vps17p, NP_014775; Sc Mvp1p, P40959; Sc Grd19p, Q08826; Sc Vps5p, Q92331; Sc YJL113C, CAA98681; Sc YDR425W, S69707; Sc YHR105W, S48947; Sc Vam7p, P32912) sorting nexins and human PI-3 kinase (Hs PI-3 kinase, JC5500). The domain architecture of the sorting nexins is shown at right. Gray boxes, PX domains; white circles, coiled-coil domains.

Figure 6.

Brassica SNX1 binds to the kinase domains of SRK, SFR1, and CLV1 in vitro. A, Elution of SNX1 from affinity resins loaded with GST or MBP proteins alone or GST or MBP fused with the kinase domains of SRK₂₉, SFR1, or CLV1. Both wild-type and kinase-inactive mutant forms of the SRK₂₉, SFR1, and CLV1 kinase domains were tested. B, Interaction between the SRK kinase domain and the PX domain (amino acids 1–157) of Brassica SNX1. Hexa-His-tagged SNX1 was detected with an anti-HIS antibody. In some cases, multiple bands were detected presumably due to the presence of either breakdown products or incompletely synthesized recombinant SNX1. E1 and E2, Eluted fractions. SRK29kin, GST::SRK29kin; SRK₂₉kin^K561R, GST::SRK₂₉kin^K561R; SFR1kin, GST::SFR1kin; SFR1kin^K555R, GST::SFR1kin^K555R; CLV1kin MBP::CLV1kin; CLV1kin^K720E, MBP::CLV1kin^K720E. C, RT-PCR analysis of SNX1 expression. Calmodulin (CAM) and actin cDNA were also amplified as controls. R, Root; C, cotyledon; L, leaf; Se, sepal; P, petal; A, anther; O, ovary; St, stigma; –, no cDNA control.