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In this work, we used a combination of coimmunoprecipitation based on the GFP variant CFP as a protein tag and LC/MALDI-TOF/MS
to identify proteins associated with the membrane receptor SERK1. Based on our data with other membrane proteins from Arabidopsis, this technique appears to be of general use. Compared with other techniques to identify interacting proteins, such as in
vitro pulldown assays and yeast two-hybrid screens, the main advantage is that the immunoprecipitation combined with LC/MALDI-TOF/MS
allows protein complex isolation under native conditions, therefore allowing functional studies in which the activity or posttranslational
modification of one or several proteins in the complex can be examined (Drewes and Bouwmeester, 2003). Another advantage is that the CFP tag allows one to directly follow all subcellular localizations of the fusion proteins.
This allows predictions concerning the nature of the proteins recovered, especially in the case of membrane receptors undergoing
recycling (Russinova et al., 2004). Other tags have been used, such as tandem affinity purification tags for plant protein complex isolation in a transient
expression system or in stably transformed transgenic lines, using the cauliflower mosaic virus 35S promoter (Rohila et al.,
2004; Rubio et al., 2005). Our procedure differs in several respects: we used an endogenous promoter-based construct and only used CFP as a protein
tag, thereby minimizing potential biological problems arising from the use of strong constitutive promoters. Also, we used
integral membrane protein as a bait, rather than the soluble proteins used previously.
SERK1 and BR Signaling
A highly interesting finding was the identification of two members of the BR signaling pathway, the main BR receptor BRI1
(Li and Chory, 1997) and its coreceptor BAK1 (SERK3) (Li et al., 2002; Nam and Li, 2002), as components of the SERK1 complex. This finding implies that SERK1 is a previously unrecognized element of BR signaling.
Recently, we showed that the SERK3 receptor may have a role in the internalization of BRI1 after heterodimerization and that
BRI1 can also form homodimers in the plasma membrane (Russinova et al., 2004). Further biochemical evidence comes from the finding here that BRI1 and SERK1 as well as SERK1 and SERK3 can interact in
the plasma membrane of protoplasts. FRET studies between SERK1-CFP and SERK1-YFP have shown that only 15% of SERK1 exists
as homodimers on the plasma membrane of protoplast cells (Shah et al., 2001a). Our results showed that both SERK1 and SERK3 heterodimerize with the BRI1 receptor, suggesting that both coreceptors can
have comparable activity in terms of BRI1 signaling and/or internalization. Another piece of evidence is that a bak1 null allele shows less severe phenotypes than bri1 loss-of-function alleles, indicating that partially redundant activity could be attributed to different SERK family members
(Li et al., 2002; Nam and Li, 2002). Genetic analysis of the serk1-1 bri1-119 double mutant suggests that SERK1–BRI1 interactions indeed affect BRI1-mediated signaling in planta, but to a lesser extent
than SERK3–BRI1 interactions. Notably, none of the proposed downstream components of BRI1, such as BES1, BZR1, or BIN2 (Li
and Nam, 2002; Wang et al., 2002b; Yin et al., 2002), was detected in the SERK1 complex.
It is unlikely that all proteins (as identified by MALDI-TOF/MS) listed in Table 1 are present in a complex together with SERK1 at the same time. Especially because proteins such as receptors and CDC48A (Rancour
et al., 2002, 2004) are known to shuttle between monomeric and multimeric forms and can change their interaction properties upon activation
and relocalization, multiple forms of the same complex are to be expected. An indication of the presence of different complexes
is the localization of SERK1 in the plasma membrane as well as in internalized membrane compartments (Russinova et al., 2004; Kwaaitaal et al., 2005).
It was proposed that BRI1 forms heterodimers with SERK3 to initiate BR signaling upon BR binding (Li et al., 2002; Nam and Li, 2002). This was recently confirmed in an elegant demonstration of the in vivo phosphorylation properties of the BRI1 receptor
(Wang et al., 2005a). In that work, it was clearly shown that the interaction between the BAK1 (SERK3) proteins and BRI1 is brassinolide-dependent.
Because we did not apply exogenous ligands before isolation of the SERK1 complex, it is likely that only a small fraction
of the complexes we isolated are actively signaling via the SERK1 protein. This notion seems to be supported by the fact that
most of the BRI1 receptors were unassociated with SERK1 in seedlings. Although our results do not allow us to precisely predict
the composition, it is likely that SERK1, SERK3, and BRI1 receptors can form tetrameric complexes analogous to the functional
complex of transforming growth factor-ß (TGF-ß) receptors. Each type of TGF-ß receptor is also present as a ligand-independent
dimer on the cell surface. TGF-ß binding to the TßRII homodimers promotes the formation of (TßRI)2/(TßRII)2 heterodimers, in which TßRI is phosphorylated by the constitutively active TßRII and becomes activated to propagate the TGF-ß
signal. Combinatorial interactions in the tetrameric receptor complex allow differential ligand binding or differential signaling
in response to the same ligand. One receptor combination often binds different ligands, and patterns of ligand and receptor
expression dictate which receptor–ligand combination is activated (Feng and Derynck, 1997). This so-called tetrameric model was also recently proposed for BRI1 and BAK1 (SERK3) (Wang et al., 2005b).
Other SERK1 Complex Components
The presence of the PP2C phosphatase KAPP, 14-3-3ν, and CDC48A in the SERK1 complex confirms previously obtained results using
yeast two-hybrid screening and in vitro interaction
studies (Rienties et al., 2005). Collectively, these proteins appear to be involved in dephosphorylation, protein interaction and membrane interaction,
and protein degradation, respectively. It is likely that they represent receptor maintenance or trafficking functions. Previously,
it was shown that SERK1 could interact with 14-3-3λ (Rienties et al., 2005). The results presented here suggest that the receptor preferentially interacts with 14-3-3ν in seedlings and perhaps with
14-3-3λ in siliques, given the origin of the Arabidopsis cDNA library from young silique tissue that was used for yeast two-hybrid screening. The kinase domain of SERK1 is able to
transphosphorylate and bind in vitro to CDC48A, 14-3-3λ, and KAPP (Rienties et al., 2005). In vitro, the interactions are all phosphorylation-dependent. In vivo, it was shown that SERK1 interacts with KAPP only
in intracellular vesicles, and it was proposed to play a role in receptor internalization as well as in dephosphorylation
(Shah et al., 2002). For two other LRR-RLKs, CLV1 (Williams et al., 1997) and FLS2 (Gomez-Gomez et al., 2001), it has been shown that KAPP functions as a negative regulator, and it may have the same role in controlling signaling through
the SERK1 receptor.
CDC48 protein assembles mainly in hexameric forms (Rancour et al., 2002). In Arabidopsis suspension-cultured cells, soluble (cytosolic) CDC48A was found in a high molecular mass protein of 640 kD. Recently, it
was proposed that CDC48A interacts with PUX1 as a monomer and may function in regulating plant growth (Rancour et al., 2004) and also in the plant endoplasmic reticulum–associated protein degradation system (Muller et al., 2005).
Plant 14-3-3 proteins have also been found associated with G-box transcription factors (Lu et al., 1992). Five Arabidopsis 14-3-3s, including 14-3-3λ, have been shown to interact with other transcription factors (Pan et al., 1999), and current models propose that the interaction with members of the 14-3-3 family is client-driven (Paul et al., 2005).
Two transcription factors were found to be associated with the SERK1 complex: AGL15 and a putative CO-like B-box zinc finger
protein. Most likely, these two proteins do not interact directly with the receptor but require 14-3-3 proteins as adaptor
proteins. It has been shown that 14-3-3 proteins can also promote the cytoplasmic localization or, conversely, the nuclear
localization, of transcription factors (reviewed in Muslin and Xing, 2000). One of the other MADS box transcription factors, AGL24, was shown to interact directly with the kinase domain of the Arabidopsis meristematic receptor-like kinase and to be phosphorylated by the kinase domain of the receptor in vitro (Fujita et al.,
2003). AGL15 was shown to accumulate in nuclei but also to be present in the cytoplasm (Perry et al., 1996). SERK1 and AGL15 are highly expressed during embryogenic cell formation in culture and during early embryogenesis. As found
for SERK1 (Hecht et al., 2001), AGL15 promotes somatic embryo production from the shoot apical meristem in liquid culture when ectopically overexpressed
(Perry et al., 1996; Harding et al., 2003). Interestingly, AGL15-overexpressing tissues also had increased expression of SERK1 (Harding et al., 2003). It was shown that AGL15 could bind directly to the promoter regions of different targets (Wang et al., 2002a). Those authors described how after chromatin immunoprecipitation, they obtained DNA fragments containing cis-regulatory elements targeted by AGL15, which may contribute to the regulation of the SERK gene by directly binding to its promoter. These data and our findings suggest that SERK1 and AGL15 can be involved in the
same signaling pathway.
The other putative transcription factor found in the SERK1 complex belongs to a large family of CO-like zinc finger transcription
factors, in which the zinc finger region regulates protein–protein interactions, as found for several animal transcription
factors (reviewed in Griffiths et al., 2003). Recently, it was shown that CO accumulation is regulated by photoreceptors in photoperiodic flowering (Valverde et al.,
2004).
In conclusion, we propose that in Arabidopsis seedlings, signaling mediated by the SERK1 receptor combines elements of the BR pathway with a short signal transduction
chain, in which the plasma membrane receptor is in a complex with its cognate transcriptional regulators, such as AGL15.
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