 and well-characterized members of the receptor kinase family are the brassinosteroid receptor BRI1 (72), the flagellin receptor FLS2 (73), and CLAVATA1, a receptor for the peptide CLAVATA3 (74). Other members of the receptor kinase family are also involved in the perception of peptide hormones (75) or other growth-promoting substances (76). Receptor kinases are known to be phosphorylated in a stimulus-dependent manner, as discovered in various comparative phosphoproteomics experiments (37,42,77,78). Under the assumption that transient stimulus-dependent phosphorylation indicates functional roles under the respec-tive conditions, SIRK1 was identified as potentially involved in sucrose-induced signaling processes (43). The present study aimed at gaining functional insights into the role of SIRK1 in sucrose-induced responses.
Use of Kinase Mutants for the Identification of Putative Substrates-In mammalian systems, systematic studies for the identification of kinase substrates have been carried out using ATP-analog-sensitive kinase mutants (79,80). This approach was also followed in QIKS (the quantitative identification of kinase substrates), which was used to identify cytoplasmic substrates of the MAP-kinase MEK1 by incubating protein extracts of mek1 cells with recombinant mutated MEK1, which takes an ATP analog as a substrate (81). Other proteomic approaches in studying protein kinase functions include the use of specific kinase inhibitors (82) or kinaseselective enrichment methods (83). However, in our case, these latter strategies were not readily applicable because of insufficient knowledge of specific kinase inhibitors or binding proteins for SIRK1.
Therefore, we used an unbiased approach of phosphopeptide profiling to identify possible target proteins of receptor kinase SIRK1. It was assumed that in a sirk1 expression mutant, the same stimulus (in our case, sucrose) would result in reduced protein phosphorylation of SIRK1 substrate proteins. This approach also assumed that other kinases with similar functions would not completely take over SIRK1 functions and phosphorylation patterns. For mammalian receptor tyrosine kinases, it was described that different kinases can actually phosphorylate the same target proteins but differ in their quantitative degree of phosphorylation (84). A large-scale and systematic comparative analysis of protein phosphorylation patterns in wild-type and kinase-or phosphatase-deletion mutants of yeast (85) revealed specific phosphorylation patterns for 78% of the kinases analyzed. However, a high degree of robustness and interconnectivity in signaling pathways was concluded from lacking or mild growth phenotypes for most of the kinase/phosphatase deletions. Thus, although the identification of kinase substrates merely based on the absence of identified phosphopeptides is likely to be very difficult, the characterization of putative kinase targets based on the quantitation of phosphorylation levels was found to be quite successful. Thus, enrichment of the compartment of interest-such as, for instance, membrane proteins-will improve the chance of finding relevant proteins.
SIRK1 as a Membrane-bound Kinase Directly Regulates Transport Processes-Here, we present evidence in support of a functional interaction of a transmembrane receptor kinase with at least one member of the plasma membrane aquaporins (PIP2F), and possibly other transporters such as the sucrose exporter SWEET11 (14). The molecular interaction of SIRK1 with aquaporins could be proven at different levels and with independent experiments: firstly, several plasma membrane intrinsic proteins were identified as interaction partners in pull-downs of GFP-tagged SIRK1 from microsomal preparations. Secondly, a PIP2F-specific peptide served as a phosphorylation target in a kinase-substrate relationship with SIRK1. Finally, physiological analysis of water uptake properties in mutants of different expression levels of SIRK1 revealed that SIRK1 directly regulates aquaporins in a sucrosedependent manner via phosphorylation.
SIRK1 was initially identified with rapid transient changes in protein phosphorylation in a sucrose-starvation/resupply experiment (43), and it was assumed that the early time point with maximum phosphorylation change at 3 min after sucrose resupply placed SIRK1 rather upstream in a signaling cascade. However, for protein phosphorylation and for signaling in general, 3 min is already quite long. Phosphorylation and heteromerization of receptor kinases can occur within seconds, as shown for stimulus-induced FLS2 phosphorylation and its interaction with the co-receptor BAK1 (86). Therefore, it cannot be excluded that SIRK1 activation and the response induced by SIRK1 are secondary effects of the altered osmolarity of the medium when 30 mM sucrose is resupplied to sucrose-starved seedlings.
The primary osmo-sensor in plants was identified as a histidine-receptor kinase, and connections to the MAP-kinase signaling cascade were postulated (24). Prolonged exposure of Arabidopsis plants to mild osmotic stress induced by 25 mM mannitol leads to global adjustments of protein levels also affecting mitochondrial respiration and photosynthetic proteins (27). Thus, the plant may have more than one mechanism of response to external osmotic changes, and possibly even substance-specific responses. Receptor-like protein kinases were suggested as a protein family with high potential in the context of understanding and preventing drought stress (87). Our finding that the receptor-like kinase SIRK1 is involved in the regulation of sucrose-specific osmotic responses supports this concept. Sucrose specificity of the response is suggested, as SIRK1-dependent protoplast swelling occurred after sucrose treatment, but not in sirk1 mutant lines. No difference was observed if mannitol was used at the same osmolarity.
Proposed Model of SIRK1 Function-In general, we cannot exclude the possibility that SIRK1 functions within a larger protein complex. Particularly, the receptor kinase AT3G02880, identified as a sucrose-dependent interaction partner and phosphorylation target of SIRK1, is a candidate for the formation of a receptor complex with SIRK1. Therefore, targets identified using the sirk1 T-DNA insertional mutant, as well as identified SIRK1 interaction partners, might in fact have to be referred to as targets and partners of a larger SIRK1 receptor complex.
In its active state, SIRK1 complex interacts with other transport proteins, particularly aquaporins (Fig. 7). Therefore, it is likely that some aquaporins are direct substrates to SIRK1, without other protein kinases being involved. There are other, recent indications for phosphorylation-dependent direct short signaling pathways at the plasma membrane involving receptor kinases (88) that mainly affected transporters-for instance, the plasma membrane H ϩ -ATPase. Furthermore, SIRK1 can also interact with other receptor kinases in a sucrose-dependent manner (see Table I), which suggests that SIRK1 forms heteromers under sucrose supply. The heteromerization of receptor kinases has been shown for various other plant receptor kinases (86,89,90). Stimulation and heteromerization can induce endocytosis, as shown for BRI1 and FLS2 (91,92). Therefore, SIRK1 also could be targeted to endocytotic vesicles after sucrose stimulation. This hypothesis is supported by increased interaction with clathrin protein after sucrose supply and observations of increased SIRK1 endocytosis within 15 min after sucrose treatment (see Table  I and Fig. 6).
An interesting finding in this study was the interaction of SIRK1 with remorin proteins and indications that remorins also are phosphorylation targets of SIRK1. Remorins are considered as marker proteins for sterol-rich membrane microdomains (69,70), also known as ordered lipid phases within the membrane. Stimulus-dependent phosphorylation of remorins was also found upon elicitor treatment of plant cell cultures (37), as well as in other phosphoproteomic studies (41,93). A possible function of the interaction of SIRK1 with remorins could be in the recruitment of SIRK1 and its target proteins to the same ordered lipid phase, or the formation of a functional SIRK1 protein complex. Such rearrangements of signaling proteins, particularly of receptor kinases, between the lipidordered phase and the disordered phase (94) were demonstrated in mammalian systems for the EGF receptor, which is recruited to the membrane-ordered phase before inactivation and endocytosis (95). Also, the phosphorylation-dependent phase transition and subsequent assembly of signaling complexes in the Nck-signaling pathway (96) suggests that such stimulus-dependent recruitment of proteins to particular lipid and protein environments might represent a general regulatory principle in all organisms. In plants, phosphorylation-dependent recruitment of protein-protein interaction partners specifically to sterol-rich membrane microdomains has recently been demonstrated for the anion channel SLAH3 and its phosphorylating kinase CPK21 (97). We propose similar mechanisms for SIRK1 and its associated proteins in interactions with clathrins for internalization.
Besides transport proteins and other receptor-like kinases, the candidate targets to SIRK1 included a number of soluble proteins, and even proteins of internal membranes, such as the Golgi or tonoplast. The identification of SnRK2.5 suggests a link to further cytosolic signaling cascades. Members of the SnRK family were previously associated with signaling in relation to metabolic stresses (98,99), and in particular the SnRK2 kinases were suggested to function in intracellular osmotic responses (26). There are indications that phosphorylation is required for the activation of SnRK2.5, but the precise sites of activation are not yet identified (25). Two members of the soluble BSK kinase family were identified as interaction partners to SIRK1 but did not show up as differentially phosphorylated. BSK1 and BSK2 were initially characterized as members of the brassinosteroid signaling pathway induced by receptor kinase BRI1 (68). BSKs are activated by