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Plant Physiology 133:919-929 (2003) © 2003 American Society of Plant Biologists Interaction of Calmodulin, a Sorting Nexin and Kinase-Associated Protein Phosphatase with the Brassica oleracea S Locus Receptor KinaseReproduction et Développement des Plantes, Unité Mixte de Recherche 5667, Centre National de la Recherche Scientifique-Institut National de la Recherche Agronomique-Ecole Normale Supérieure de Lyon-Université Claude Bernard Lyon, Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, 69364 Lyon cedex 07, France
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.
Plant genomes encode large numbers of receptors with Ser/Thr protein kinase activity. These plant receptor kinases (PRKs) are related to, but phylogenetically distinct from, the receptor Tyr kinase (RTK) and receptor Ser/Thr kinase (RSK) families in animals, suggesting that the three families have evolved independently from a common ancestor (Shiu and Bleecker, 2001  ). The independent evolution of these gene families is reflected in the fact that, to date, none of the proteins that have been shown to interact with PRKs are closely homologous to RTK- and RSK-interacting proteins. However, despite the many differences between plant and animal receptor kinase systems, there are indications that they function in a similar manner with, for example, unrelated interacting proteins carrying out analogous roles in the two kingdoms (Cock et al., 2002).
One of the best characterized PRKs is the S locus receptor kinase (SRK). SRK is located in the plasma membrane of the stigmatic papillar cells and is the female component of the self-incompatibility (SI) response in Brassica spp. (Stein et al., 1991
SRK is phosphorylated after an incompatible pollination (Cabrillac et al., 2001
The signal transduction pathway downstream of SRK has been partially characterized. ARC1 (arm repeat containing 1; a complex protein with multiple domains including a Leu zipper, a U box, and a C-terminal Arm repeat domain; accession no. AJ427335) interacts with the SRK kinase domain and is required for efficient rejection of self-pollen (Gu et al., 1998
There are also indications that negative control of SRK signaling may play a role in SI signaling, both in the basal state, before pollination, and after SRK activation. SRK interacts with two thioredoxin h-like proteins (THL1 and THL2; Bower et al., 1996
SRK also interacts, in vitro, with Arabidopsis kinase-associated protein phosphatase (KAPP; accession no. AJ427336; Braun et al., 1997
Down-regulation and internalization by endocytosis are essential for correct regulation of receptor kinases in animals, and a wide range of mechanisms have been described, including inhibitors acting on receptors in their basal state; regulation by dephosphorylation, by phosphorylation, or by calmodulin binding; Cbl-mediated receptor ubiquitylation; and sorting nexin-mediated targeting to endosomes (San José et al., 1992 We show here that SRK interacts with and is dephosphorylated by the Brassica homolog of KAPP, indicating that this protein plays a role in SRK down-regulation. SRK was also shown to interact with calmodulin and a sorting nexin, both of which are proteins that have been implicated in receptor kinase down-regulation in animals. KAPP, calmodulin, and the sorting nexin also interact with additional, diverse PRKs. The possible roles of these three proteins in PRK down-regulation and endocytosis are discussed.
SRK Interacts with a Brassica oleracea Homolog of KAPP
The demonstration that the Arabidopsis KAPP protein interacts with SRK in vitro (Braun et al., 1997
We used the yeast two-hybrid system to test whether Brassica KAPP interacts with the kinase domain of SRK. The KI domain of Brassica KAPP was expressed as a fusion protein with the activation domain of Gal4 (Gal4AD::KI) in yeast cells that also expressed the kinase domain of SRK29 fused to the DNA-binding domain of Gal4 (Gal4DB::SRK29kin). As a positive control for this experiment, we tested whether the kinase domain of SRK interacted with ARC1 in this system. For this, we cloned the Arm-repeat region of a B. oleracea homolog of ARC1 (residues 362–661) and expressed it as a fusion protein with Gal4 (Gal4AD::ARC) in the same kinase domain-expressing yeast strain. ARC1 has been shown to interact with SRK in closely related B. napus (Gu et al., 1998 To test whether KAPP could be phosphorylated by SRK, a fusion protein consisting of GST fused to Brassica KAPP lacking its predicted N-terminal signal anchor (residues 1–47) was expressed in Escherichia coli, and the purified protein (GST::KAPP) was added to a phosphorylation reaction in the presence of either the wild type or a mutant form of the SRK29 kinase domain that had also been expressed in E. coli as protein fusions with GST (GST::SRK29kin and GST::SRK29kinK561R). KAPP was phosphorylated by the wild-type SRK29 kinase but not by the kinase-negative mutant form (Fig. 1C). A phosphorylation assay confirmed that the wild-type kinase domain construct, GST::SRK29kin, was capable of autophosphorylation in vitro, whereas the mutant form, GST::SRK29kinK561R, was enzymatically inactive (data not shown). Furthermore, Figure 1D shows that when GST::KAPP was added to radiolabeled, phosphorylated GST::SRK29kin in vitro, the latter was dephosphorylated, indicating that SRK is a substrate for the phosphatase activity of KAPP.
The above experiments showed that the kinase domain of SRK29 was able to interact specifically with KAPP and ARC1 in the two-hybrid assay. Based on this result, we initiated a screen to identify novel interacting partners using the two-hybrid system. Several two-hybrid screens were performed, using both wild-type (Gal4DB::SRK29kin) and mutant (Gal4DB::SRK29kinK561R) forms of the SRK kinase domain and the kinase domain of SFR1 (Gal4DB::SFR1kin), a receptor closely related to SRK that is also expressed in stigmas (Pastuglia et al., 2002
The calmodulin cDNAs identified in the three screens corresponded to two different genes (designated Calmodulin 1, accession no. AJ427337; and Calmodulin 2, accession no. AJ427338) that differed significantly in their 3' non-translated regions. However, the coding regions of these two calmodulin genes are 93.2% identical, and they encode identical proteins. The deduced amino acid sequence of the Brassica calmodulins is 98.0% identical to that of a previously described B. napus calmodulin (accession no. AF150059).
Calmodulin-Sepharose affinity binding was carried out to confirm the interaction between calmodulin and the kinase domain of SRK. Figure 2A shows that fusion proteins consisting of GST plus either the wild type or a mutant form of the SRK29 kinase domain bound to calmodulin-Sepharose beads in the presence of 5 mM CaCl2 and were eluted from the beads when the 5 mM CaCl2 was replaced by 5 mM EGTA. When GST alone was loaded on the column, some protein bound to the beads, but this was removed by washing with 5 mM CaCl2 and was not specifically eluted by 5 mM EGTA.
Similarly, when a kinase-negative, hexa-His epitope-tagged form of the integral SRK3 protein (mSRK3His) expressed in insect cells was subjected to calmodulin-Sepharose affinity binding, the recombinant protein bound to the calmodulin-Sepharose beads and was eluted in the presence of 5 mM EGTA (Fig. 2B). The interaction between the kinase domain of SRK and calmodulin was also confirmed using an alternative affinity-binding approach. GST alone or fused to either the wild type or the mutant form of the SRK29 kinase domain was bound to glutathione-Sepharose beads. Brassica calmodulin 2 tagged with a hexa-His epitope was then expressed in insect cells, affinity purified on nickel-nitrilotriacetic acid agarose beads, and applied to beads carrying the SRK fusion proteins in the presence of calcium. After washing, calmodulin was specifically eluted from the beads in the presence of 5 mM EGTA. Figure 2C shows that calmodulin bound to beads carrying both wild-type and mutant kinase domain protein fusions but not to beads carrying GST alone.
Calmodulin has been shown to interact with its target proteins by binding to small amphiphilic
Figure 3B shows that, when the HEL1 peptide was fused to GST (GST::HEL1), the fusion protein bound to calmodulin-Sepharose beads and was eluted in the presence of 5 mM EGTA, indicating that HEL1 interacts specifically with calmodulin. GST fused to a non-helical, nonamphiphilic peptide of the same size as, and of similar hydrophilicity to, HEL1 (PEP1 in Fig. 3A) did not bind to calmodulin-Sepharose beads (Fig. 3B).
The fact that calmodulin interacted with the kinase domains of both SRK and SFR1 in the yeast two-hybrid assay (Table I) indicated that this interaction was not specific to SRK and suggested that calmodulin may interact with other receptor kinases. To test this, we expressed both wild-type and mutant forms of the SFR1 kinase domain and wild-type kinase domains of three additional members of the PRK superfamily, RLK4 (receptor-like kinase 4), CLV1, and BRI1 (brassinosteroid insensitive 1) as fusion proteins with either GST or the maltose-binding protein (MBP). RLK4 is a member of the S gene family of receptor kinases but is distantly related to SRK (approximately 25% amino acid identity depending on the allele of SRK); CLV1 and BRI1 are members of the family of PRKs that contain Leu-rich repeats in their extracellular domains. All five of the recombinant proteins were then purified and subjected to a calmodulin affinity-binding assay. Figure 4A shows that all the proteins tested, apart from GST::BRI1kin, bound to the calmodulin-Sepharose beads in the presence of 5 mM CaCl2 and were eluted after addition of 5 mM EGTA. Analysis of the sub-domain VIa regions of these proteins showed that all except BRI1 were predicted to include amphiphilic helices. Therefore, the presence of a predicted amphiphilic helix in kinase sub-domain VIa was correlated with the ability to bind calmodulin.
Interestingly, we consistently observed that MBP::RLK4kin was eluted from the calmodulin-Sepharose beads in two peaks, indicating the presence of more than one calmodulin-binding site in the RLK4 kinase domain, the sites having different binding affinities. The expression pattern of calmodulin was consistent with it interacting with multiple PRKs expressed in different parts of the plant because transcripts were detected in a wide range of organs (Fig. 4B).
We were interested in determining whether calmodulin binding modified SRK autophosphorylation because HEL1 is located close to the activation loop in the kinase domain of SRK. In a kinase assay carried out in vitro, addition of calmodulin in the presence of 5 mM CaCl2 did not prevent autophosphorylation of GST::SRK29kin, and calmodulin was not phosphorylated by the kinase domain fusion protein (data not shown). The same result was obtained when the experiment was repeated with full-length SRK protein that had been expressed in insect cells (data not shown). This result suggests that calmodulin binding has no direct effect on SRK kinase activity. Again, calmodulin was not phosphorylated in these reactions, indicating that it is not a substrate of SRK kinase activity.
Sorting nexins have been implicated in cargo trafficking through the endosomal system in mammals and yeast, and several members of this family interact with animal receptor kinases (Kurten et al., 1996
Sequence analysis showed that Brassica SNX1 was most similar to an SNX1 homolog from Arabidopsis but also shared extensive similarity with human (Homo sapiens) SNX1 (Fig. 5A). Human SNX1 contains a PX domain plus three predicted coiled-coil domains in the C-terminal part of the protein. Comparison of the PX domains of sorting nexins has allowed the identification of three highly conserved motifs, two of which (RRY/FSD/EFxxLxxxL and RR/KxxLxxY/F where x is any amino acid) are predicted to form a basic pocket involved in phosphoinositol binding and a third, Pro-rich motif (PPxPxK), which may be involved in protein-protein interactions (Sato et al., 2001
Both the human and yeast genomes contain moderately large families of sorting nexins (Teasdale et al., 2001 To confirm the interaction between SRK and Brassica SNX1, GST alone or GST fused to wild-type or kinase-negative forms of the SRK29 kinase domain were bound to glutathione-Sepharose beads and then incubated with hexa-His-tagged SNX1. Kinase/SNX1 complexes were then eluted from the resin by addition of glutathione. Figure 6A shows that SNX1 was eluted from the resin only if kinase domain fusion proteins were present as bait. No SNX1 was detected in the glutathione elution fractions if the bait used was GST protein alone. Deletion analysis demonstrated that the PX domain of Brassica SNX1 was sufficient to mediate the interaction with SRK (Fig. 6B).
Using the same approach, we were also able to demonstrate interactions between SNX1 and two other PRK kinase domains, from SFR1 and CLV1 (Fig. 6A). SNX1 interacted with both wild-type and kinase-negative forms of the SRK kinase domain, but this was not the case for SFR1 and CLV1 where only the wild-type or only the kinase negative forms interacted, respectively. It is not clear why such differences were observed, but the differences were clearly reproducible. The interaction of SNX1 with the kinase domains of three diverse receptors suggests that this protein may play a role in multiple PRK signal transduction pathways. This hypothesis is supported by the fact that SNX1 transcripts were detected in a wide range of Brassica organs (Fig. 6C).
In this study, we have investigated interactions between several cytoplasmic stigma proteins and the kinase domain of SRK. We show that a stigma-expressed Brassica homolog of KAPP interacts with, and is phosphorylated by, the kinase domain of SRK in vitro. KAPP was also shown to dephosphorylate SRK. These results, together with the observation that SRK is phosphorylated in vivo approximately 1 h after an incompatible pollination (Cabrillac et al., 2001 ), suggest that KAPP may play a role in SRK down-regulation, as has been proposed for CLV1 and FLS2 (Williams et al., 1997; Stone et al., 1998; Gomez-Gomez et al., 2001). KAPP may not be the only protein phosphatase involved in the SI response because a breakdown of SI has been observed in pistils after treatment with okadaic acid or microcystin LR (Rundle et al., 1993; Scutt et al., 1993). Note, however, that okadaic acid and microcystin LR are inhibitors of type 1 and type 2A phosphatases and would not be expected to inhibit type 2C phosphatases such as KAPP.
Four previously unidentified interacting proteins were recovered by a two-hybrid screen carried out to search for new SRK-interacting proteins. One of these interactors, calmodulin, was of interest because it is known to be a component of many signal transduction pathways in both plants and animals (Sanders et al., 1999
Calcium fluxes have been measured in both the cytoplasm and in the cell wall of stigmatic papillar cells after both compatible and incompatible pollination (Dearnaley et al., 1997 In addition to SRK, calmodulin interacted in a calcium-dependent manner with the kinase domains of three distantly related PRKs (SFR1, RLK4, and CLV1) but not with the kinase domain of BRI1. This suggests that calmodulin may interact with a broad range of PRKs.
A short region from within sub-domain VIa of the SRK kinase domain resembled a calmodulin-binding site based on its predicted amphiphilic
The interaction observed between SRK and Brassica SNX1 is of particular interest because sorting nexins have been implicated in the down-regulation of animal receptor kinases. Several members of the sorting nexin family have been shown to interact with RTKs or RSKs both in vitro and in vivo (Kurten et al., 1996
In yeast, the SNX1 homolog Vps5p/Grd2p is part of the retromer complex that is essential for retrograde transport from the endosome to the Golgi (Seaman et al., 1998
Apart from the PX domain, no other sequences are widely conserved in sorting nexins (Teasdale et al., 2001
In contrast to previous studies, which have indicated that plant and animal receptor kinases interact with structurally distinct sets of cytoplasmic proteins (Bower et al., 1996
Cloning the Brassica oleracea Homolog of KAPP
An internal fragment of the Brassica KAPP coding sequence was amplified from a stigma cDNA library in the yeast (Saccharomyces cerevisiae) two-hybrid vector, pAD-Gal4 2.1 (see below), using two oligonucleotides, Kap1 (5'-GGAGGGATCCCAAGTTGGCTGTTCCTGGAAGTCAT-3') and Kap2 (5'-CGCTGGATCCGGAAGTTTCCTGCCTCCTCGACGCAT-3'), based on the Arabidopsis KAPP gene sequence. Overlapping fragments corresponding to the 5' and 3' ends of the KAPP cDNA were then amplified using the two above oligonucleotides and two additional Brassica KAPP oligonucleotides, Kap3 (5'-TGGGCTTCAATTTGCTGTTCACTCC-3') and Kap4 (5'-GGGATCTGAGGCCACACCAACT-3'), in combination with vector oligonucleotides from a second stigma cDNA library (Giranton et al., 1995
Total RNA was extracted, using the method described by Cock et al. (1997
For two-hybrid assays, yeast strains PJ69-4A (James et al., 1996
Gene fusions of the wild-type and mutant kinase domains of SRK29 and SFR1 with GST were constructed (Mazzurco et al., 2001
Recombinant baculovirus containing the hexa-His-tagged Brassica calmodulin 2 was obtained with the Bac-to-Bac baculovirus expression system (Invitrogen, Carlsbad, CA). Hexa-His-tagged wild-type and kinase-negative forms of the mSRK3 (SRK3His and SRK3His) and Brassica calmodulin 2 were expressed in insect cells as described (Giranton et al., 2000
Purified GST::kinase fusion proteins (2 µg in 300 µL) were incubated in 50 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM dithiothreitol (DTT), 5 mM CaCl2, and antiprotease cocktail (Boehringer Ingelheim GmbH, Ingelheim am Rhein, Germany) with 100 µL of calmodulin-agarose resin (Sigma, St. Louis) for 1 h at 4°C in Eppendorf tubes with gentle agitation. The resin was washed with 40 column volumes of the binding buffer, and proteins eluted in 50 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM DTT, 5 mM EGTA, and antiprotease cocktail were detected by immunoblotting using an anti-GST antibody (Pharmacia Corporation). Before carrying out the calmodulin-binding assay, mSRK3His expressed in insect cells was solubilized in 62.5 mM Tris-HCl (pH 6.8), 2.5% (w/v) SDS, 0.13 M DTT, and 20% (w/v) glycerol by heating at 100°C for 10 min and desalted twice on Bio-Gel P6-DG columns (Bio-Rad, Hercules, CA). For kinase domain affinity-binding assays, 1 µg of GST::SRK29kin or GST::SRK29kinK561R was bound to glutathione-Sepharose beads (100 µL) and then incubated with 2 µg of Brassica calmodulin. Washing and elution were as above, and proteins were detected on silver-stained SDS-PAGE gels. Binding to SNX1 was detected by incubating E. coli extracts containing equivalent amounts of soluble, recombinant GST- or MBP-kinase domain fusion proteins in affinity lysis buffer (50 mM HEPES [pH 7.4], 150 mM NaCl, 10 mM EDTA, 1 mM DTT, and antiprotease cocktail) with 25 µL of glutathione-Sepharose (Amersham Pharmacia Biotech Inc., Piscataway, NJ) or amylose-Sepharose resin (New England BioLabs, Beverly, MA), respectively. The resin was washed once with affinity wash buffer (50 mM HEPES [pH 7.4], 150 mM NaCl, 10 mM EDTA, and 1 mM DTT) and then incubated with E. coli extracts containing recombinant hexa-His-nexin fusion proteins in affinity lysis buffer. After washing with 120 column volumes of affinity wash buffer, fusion proteins and their interacting partners were eluted twice with 25 µL of either 10 mM glutathione, 10 mM Tris (pH 8), or affinity wash buffer plus 10 mM maltose. All experiments were repeated at least once.
Phosphorylation assays were carried out using either the procedure described by Williams et al. (1997
Sequence data analysis and construction of multiple sequence alignments were carried out using Lasergene sequence analysis software (DNASTAR, London). Neighbor joining trees were constructed from multiple alignments using ClustalW (Thompson et al., 1994
Total RNA was isolated from a range of different organs of B. oleracea line P57Si using the method described by Cock et al. (1997
Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes, subject to the requisite permission from any third party owners of all or parts of the material. Obtaining any permissions will be the responsibility of the requestor.
We thank Shahinez Madi, Claire Rollin, Anne-Marie Thierry, Hervé Leyral, and Claudia Bardoux for technical assistance; Masao Watanabe for the SRK29 cDNA clone; and Daphne Goring, Erin Morris, John Walker, Steve Clark, and Steve Clouse for various PRK domain expression constructs. We are grateful to Gwyneth Ingram for her comments on the manuscript. Received March 19, 2003; returned for revision April 15, 2003; accepted June 23, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.023846.
1 Present address: Swann Building, Institute of Cell and Molecular Biology, King's Buildings, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JR, UK.
2 Present address: Unité Mixte de Recherche 7139, Centre National de la Recherche Scientifique-Goëmar-Université Paris 6, Station Biologique, Place Georges Teissier, BP 74, 29682 Roscoff Cedex, France * Corresponding author; e-mail cock{at}sb-roscoff.fr; fax 33–2–9829–2324.
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ABSTRACT
RESULTS
receptor II (Wang et al., 1996
-galactosidase enzyme activity (data not shown). In contrast, neither the KAPP nor the ARC1 fusion proteins interacted with an enzymatically inactive, mutant form of the SRK29 kinase domain (Gal4DB::SRK29kinK561R; 
