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Challenges in understanding RLK function Kevin Lease*, Erika lnghamt and John C Walker+

Plants use receptor-like kinases (RLKs) to transduce

extracellular signals into the cell. Recent advancements in RLK

research include the cloning of the BRASSlNOSTEROlD INSENSITIVE 7 and CLAVATA 7 genes, revealing RLK roles in

development. Our understanding of RLK function has also

been broadened by transgenic approaches in the study of the

RLKs pollen receptor kinase 1, and wail associated kinase 1.

These results extend the observations that RLKs function in

developmental processes and plant defense responses.

Additionally, expression based studies suggest roles for other

newly reported RLKs in development and light responses.

Taken together, the studies confirm the importance of RLKs in

diverse plant processes, yet major challenges remain. These

include identifying ligands that activate RLKs and

characterizing downstream pathways. These challenges can

be conquered by coordinated efforts from investigators using

molecular, genetic, and biochemical approaches.

Addresses 308 Tucker Hall, University of Missouri-Columbia, Columbia MO 65202, USA *e-mail: c684855@showme,missouri,edu ic675904@showme.missouri.edu .ijcw@biosci.mbp.missouri.edu

Current Opinion in Plant Biology 1998, 1:388-392

http://biomednet.com/elecref/1369526600100388

0 Current Biology Ltd ISSN 1369-5266

Abbreviations BR brassinolide BRI brassinosteroid insensitive CLV clavata KAPP kinase associated protein phosphatase RLK receptor-like kinase RPK receptor protein kinase SA salicylic acid SRK self-incompatibility locus receptor protein kinase WAK wall associated receptor kinase

Introduction Multicellular organisms need to carry out many processes in a co-ordinated fashion, that is to sense and respond to both external and internal signals in an intricate and pre- cise way. Multi-step signal transduction creates the necessary complexity for refined regulation of a cell’s response to its environment. A commo6way cells relay molecular messages is reversible protein phosphorylation; protein kinases add phosphates to their target protein(s), protein phosphatases remove them. Cells can begin this process with receptor protein kinases, using phosphoryla- tion status to transduce external messages into the cell [ 11.

Candidate receptor protein kinas-s have been found in plants [Z]. These plant receptor-like kinases (RLKs) have not been definitively shown to be receptors: none has a

known ligand. RLKs, however, possess all of the following structural characteristics to be receptors. They have an amino-terminal signal peptide followed by an ‘extracellu- lar’ domain which varies from protein to protein. The variability of the proteins suggests the presence of several different ligands in plants and it it likely that a specific receptor binds to a specific ligand. The extracellular domain lies next to a hydrophobic region predicted to be membrane spanning. This is followed by a carboxy-termi- nal protein kinase domain which has serine/threonine protein kinase activity [3].

Numerous RLKs have been cloned but only a few have been further investigated. Function for RLKs has been shown in diverse biological processes such as development [4”], disease resistance [S] and self incompatibility [6]. Expression studies have also implicated RLKs in embryo- genesis, pollen function and light responses. Although their biological roles are diverse, they may share common signaling elements. RLKs likely function in an analogous way to known receptor kinases, being in multi-step path- ways which lead to changes in gene regulation. Little is known about downstream components, however, or which genes are regulated by different RLKs. Many important insights into how plants perceive and respond to their environment will be gained by characterizing RLKs and their pathways.

Functional roles for RLKs BRASSINOSTEROID INSENSITIVE 1 Examination of RLK mutants has provided the greatest insights into their physiological importance. Mutant analy- ses, natural variation, and reverse genetics have suggested diverse functions for RLKs. An example of the power of genetics for elucidation of RLK function was the identifi- cation of BRASSINOSTEROID INSENSITIVE 1 (BRIl). Three different screens for Ambicz’opsis mutants insensitive to the plant steroid hormone brassinolide (BR) defined the bm’l locus [7,8,9”]. Li and Chary positionally cloned BRZl and revealed that it was an RLK [lo]. ‘This cloning proved insightful because there was nothing previously known about how plants might transduce BR signals.

The hormone insensitivity trait of hail had previously led to the expectation that the gene would encode a BR receptor or signaling component. In animals, steroid hor- mones act by diffusing across the plasma membrane and binding to transcription factors in the cytoplasm or nucle- us, resulting in a change in gene expression [ll]. Thus, it was hypothesized that BRIl would be a l&and activated transcription factor. That BRIl turned out to be a pre- dicted transmembrane protein kinase was unexpected. Research conducted on mammals had previously suggest- ed that some steroid hormones may act at the cell surface,

Challenges in understanding RLK function Lease, lngham and Walker 389

in addition to acting within the cell, but the membrane bound receptors are unknown. ‘I’his finding in plants has now stimulated research to test whether transmembrane protein kinases similar to BRI 1 function in animal steroid hormone signaling.

Is BRIl really a receptor for BR? It will be important to test whether Bril can physically bind BR. If it cannot bind BR alone, it may be that BRll associates with BR in concert with another protein. Identification of other factors required for BR signal transduction is an important goal. Further genetic screens for loci conditioning RR insensi- tivity may not be the best approach as extensive mutant screens by three laboratories have yielded only alleles of l/ri;l. Screening for modifiers of bril may uncover new genes that regulate this pathway [12]. This involves a genetic screen in which plants homozygous for a mutation at one locus are mutagenized to create secondary lesions that could enhance or suppress the phenotype caused by the original mutation. Protein-protein interaction based studies may also be useful to identify components and pro- vide new clues for how RR signals are transduced.

In addition to BRIl, another RI,K involved in develop- ment is CLAVA’I’Al (CLVl) [4 1. To date, CLVl is the best characterized of all the RLKs. CLVl has been proposed to maintain the proper balance of cells in shoot meristems: undifferentiated cells versus cells dedicated to organ for- mation. The clvl phenotype has enlarged shoot and floral meristems which typically cause fasciated stems, extra flowers, and excess floral organs [ 13). The current model of CLVl signaling proposes that CIXl acts like classical RPKs. ‘I-hat is, CIXl recognizes an extracellular ligand, multimerizes and transautophosphorylates. IJpon phos- phorylation of the cytoplasmic domain other proteins would bind in a phosphorylation dependent manner to transduce this signal to other downstream components. Ultimately, the signal will result in a change in gene expression and a cellular response. There is both bio- chemical and genetic data supporting this model. CLVl has been demonstrated to be an active protein kinase that autophosphorylates on multiple serines [14’,15]. Genetic evidence suggests that CLVl functions as a homomulti- mer, based on dominant interference seen when some mutant alleles are heteroallelic with wild-type [16].

‘l’herc is also evidence for other components of the CLVl ‘pathway.’ Results from two complementary approaches suggest a role for a type ZC protein phosphatase, designated KAPI’ (kinase associated protein phosphatase) in CLVl sig- naling [ 14’,15]. Protein phosphatases are defined biochemically by their substrate preferences, inhibitor sen- sitivities, and requirements for divalent cations. KAPP, like other type 8.2 protein phosphatases, is insensitive to okada- ic acid, and requires divalent cations for activity. Previously, KAPP has been shown to bind to a subset of RLKs in a phosphorylation dependent manner. The physiological

Figure 1

c/vi-9 A+V

a

”

d Qb -

WILD-TYPE bril WILD-TYPE c/v 1 SILIQIJE SILIOUE

Current Op~mon I” Plant Bdogy

Importance of the activation loop for RLK functibn. Top, a schematic diagram of an RLK. Domains: EXT, extracellular; TM, transmembrane; PK, protein kinase. Middle, the BRIl activation loop amino acid sequence is shown with missense mutant alleles of bril and clvl. Conserved residues are in boxes. Bottom, phenotypic effects of mutations in the activation loop with bril (left), and c/v7 (right).

function of KAPP binding to an RLK, however, was unclear. Williams eta/ demonstrated that KAPP binds CLVl in vitro. Furthermore, they showed that a clv phenotype could be obtained by overexpression of KAPP in a wild-type back- ground. Independently, Stone et aL. demonstrated in vitro KAPP and CLVl interaction, as well showing co-immuno- precipitation from plant extracts. In a distinct approach, complementary to the overexpression experiment of Williams et al., Stone and co-workers were able to suppress the clv phenotype by reducing the amount of KAPP expres- sion in a homozygous c/v2 mutant background. Results from these two reports convincingly demonstrate that KAPP functions in the CLVl signal transduction pathway, and sug- gest that KAPP is a negative regulator of CLVl. Genetic interactions have also implicated CLAVATA3 (CLV3) in CLVl signaling. Double mutant combinations place CLVl and CLV3 in the same pathway because recessive hypomor- phic alleles of C/UZ become dominant when placed in a &13 mutant background [ 161. This suggests CLV3 may encode a ligand for the CLVl receptor, or act downstream in the path- way. The genetic and biochemical interaction of CLVl with other components, KAPP and CLV3, provides a foundation for future work which will elaborate the molecular mecha- nisms of RLK biology.

hlutant alleles from clvl and other RLKs have now been sequenced revealing that many mutations map to the

390 Cell signailing and gene regulation

protein kinase domain. Many protein kinase domains con- tain a small structural feature known as the activation loop and are positively regulated by phosphorylation on a key residue in this loop [ 171. Several mutations found in (XV1 and BRIl map to the activation loop, which has important implications for RLK potential activation mechanisms (see Figure 1). Of the eight reported missense RLK mutations that map to the protein kinase domain, five fall within the activation loop [4”,9”,18]. The importance of this loop for function becomes apparent as the activation loop, which only represents about 15% of the total protein kinase domain, contains about 60% of the missense mutations which fall in the protein kinase domain. It will be impor- tant to test biochemically whether phosphorylation of the activation loop is required for proper RLK function.

Classic genetic studies such as those with bril and r/v1 have been instrumental in uncovering the functional roles for RLKs, but reverse genetic approaches have also yield- ed clues about some RLKs. For example, the role of PRKl, a receptor-like kinase has been inferred from trans- genie PtWniu ir$ata lines expressing an antisense copy of PRKZ [19]. Previous work had shown high levels of endogenous PRKZ gene expression in the male gameto- phyte of wild-type plants, and some antisense lines showed pollen abortion. This indicates PRKl is required for pollen viability. The PRKZ transgene was not inherited at the expected frequency, however, when the antisense transgenic plant was used as either the male or the female parent. ‘I’his prompted investigation of PRKZ expression in female tissues [ZO]. RT-PCR experiments detected a low level of PRKI mRNA in ovules of wild-type plants. Antisense lines, which are expected to have reduced expression of PRKZ, showed embryo abortion. This sup- ports the possibility that PRKl may also function in the female contribution to the embryo and that PRKl is required for both pollen and ovule viability.

Beyond suggesting functions for RLKs, transgenic approaches are also a potentially powerful means of testing RI,K mechanisms. In particular, transgenic experiments with the cell wall associated receptor-like kinase (WAKl) of Ar&id@sic have provided clues about how they function [Zl’]. WAKl is a pathogen-related protein and its expres- sion is stimulated by salicylic acid (SA). Wild-type plants produce SA in response to pathogen attack. Application of high amounts of SA to wild-type plants, however, is lethal. WAKE antisense and dominant negative transgenic plants do not survive treatment with low amour& of SA analogs. Conversely, ectopic expression of the protein kinase domain or full length protein confers resistance to high lev- els of SA. Thus WAKl seems to be necessary for plants to survive SA induced defense responses.

Although reverse genetics experiments were successful with WAKl, there have been examples with another RLK when they did not answer the questions raised by researchers. ‘I’he mechanism of a self-incompatibility locus

receptor protein kinase (SRK) action was tested by trans- genie approaches in two different labs. ‘I-he question addressed was whether SRK functions by multimerization and transactivation. In both cases, a self-incompatible strain became self-compatible because of silencing of the endogenous gene through cosuppression rather than through interference by the transgene encoded protein [Z&23]. The epigenetic phenomena of cosuppression has both frustrated and intrigued researchers trying to make inferences from transgenic plants. Since cosuppression thwarted two labs’ attempts to test SRK mechanisms, other approaches will have to be tried. Biochemical exper- iments, such as estimation of the SRK complex size and determination of the SRK phosphorylation mechanism, would address the mechanism of activation originally investigated with a genetic strategy.

Expression based studies Genetic analysis has provided information about the bio- logical relevance of RLKs. Another useful approach has been to look at the location, inducibility, and timing of gene expression to provide correlative data. These studies can then be followed by physiological, genetic and biochemical characterization to fully understand the role of the RLK.

Pollination is one process where expression profiles impli- cate RLK involvement. Two pollen specific RLKs were cloned from tomato, LeRPKl and LeRPK2 [24]. ‘I’hese proteins are expressed late in pollen development and are plasma membrane associated. Although both proteins have kinase activity, they have amino acid differences in invari- ant and highly conserved residues within the subdomains. Additionally, steady-state LeRPK2 mRNA levels increase after pollination and the protein can be partially dephos- phorylated by style extracts. Although the specific function of these RLKs remains to be discovered, their dif- ferent phosphoylation states suggest they have different roles following pollination.

The function of another RLK, the receptor protein kinase 1 (RI’Kl) from Arabidvpsis [ZS], was also inferred from its expression profile. RPKI is expressed in all tissues, but expression increases after treatment with ABA, dehydra- tion, cold and high salt treatment. Thus, RPKl may be involved in an ABA induced stress pathway, although this has not been clearly demonstrated.

Expression of the carrot somatic cmbryogenesis receptor- like kinase (S’ERK) and the light-repressible receptor kinase (L.RRPK) of Arahidopsis have been recently described. SERK expression begins during the transition from somatic to embryogenic competent cells in carrot cell cultures, suggesting that it is involved in this developmen- tal switch [26]. LRRPK is expressed in blue-light growth conditions in all organs, but not in red-light grown plants [27]. Any light applied to plants reduced the level of steady-state mRNA. ‘I’hese results suggest that LRRPK may be involved in responding to different light qualities.

Challenges in understanding RLK function Lease, lngham and Walker 391

New RLKs continue to be isolated and their function inferred from their mRNA expression. What exact roles they play and what their signaling mechanisms are remain to be investigated by other means involving genetic and biochemical screens as well as Z%Z vitro assays.

Future directions and concluding remarks A combination of classical and reverse genetic approaches have been powerful tools used to decipher the physiologi- cal functions and signal transduction downstream of RLKs. It is useful to look to the future and examine the areas that will move the field forwards with the greatest celerity. Scanning the Arabidopsis genome project database reveals a large number of RLK genes that have been sequenced but not described in the literature. Although it may seem tempting to put these new RLKs through the battery of typical molecular genetic experiments, the RLK field will move farther faster if focus is first placed upon a few RLKs which are already partially characterized. The choice of which RLK to study will principally depend on the biolog- ical questions addressed by the researcher; however, elucidation of the signal transduction cascades employed by RLKs most amenable to genetic or biochemical charac- terization will likely serve as a blueprint for studies on less tractable, but still biologically interesting RLKs.

It will be important to identify additional interacting pro- teins for these RLKs, both biochemically and genetically. Protein-protein interaction based experiments will likely continue to be successful at identifying regulatory compo- nents. Furthermore, characterization of phosphorylation events in plant extracts by immunoprecipitation and pro- tein microsequencing may be a useful means of discovering RLK substrates. Identification of genes func- tioning in the pathways of ‘present-day’ RLKs will accelerate as large scale insertional mutageneses will satu- rate the genome, facilitating cloning. DNA chips will allow for assaying genome wide differential gene expres- sion in RLK mutant versus wild-type backgrounds. Finally, the sequencing of the Arabidopsis thaliana genome will soon be complete: we will know what genes RLKs could potentially draw upon to transduce their signals. This will settle controversies about whether plants con- tain well established signal transduction components found in animals and may also identify candidate signal transduction genes by virtue of their containing phospho- protein recognitioii domains.

The focused efforts of a critical mass of investigators will facilitate the unraveling of RLK molecular mechanisms. In this way, the field can move forward from continually rebuilding the basic foundation of understanding for each RLK and push towards a high level characterization of RLK signaling and biology. A great deal of synergy is antic- ipated among RLK investigators, so that hopefully, in ten years, several receptors will be characterized in great detail rather than a small amount known about each of more than several hundred RLKs.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:

?? of special interest ??* of outstanding interest

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meristem size in Arabidopsis. Cell 1997, 89575-585. CLVl is necessarv for maintainina the orooer balance between differentiated and undifferentiated cellsin apical a;d floral meristems. The finding that CLVl encodes an RLK established a critical role for RLKs in cell-cell communication during development This is an important insight for understanding the molecular basis of development and provides a biological context for further investigation of RLK signaling mechanisms and components.

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Along with the Williams et al. paper below, this study shows that KAPP physiologically interacts with an RLK, CLVl As previous data was entirely in vitro binding studies, it was a major breakthrough to demonstrate KAPP’s function in planta. These studies also suggest that KAPP is a negative regulator of RLK signaling.

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Lee HS, Chung YY, Das C, Karunanandaa B, Vanwent JL, Mariana, C, Kao, TH: Embryo sac development is affected in Petunia Mata plants transformed with an antisense gene encoding the extracellular domain of receptor kinase PRKI. Sex Plant Repro 1997, IO:341 -350.

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