- © 1999 American Society of Plant Physiologists
The balance that multicellular organisms must strike between cell proliferation and differentiation is the very focus of developmental biology. From slime molds to humans, developmental programs must direct both the temporal and spatial expression of specific genes despite the exacting exigencies of a DNA replication regime designed to provide each new cell with an identical genome. Unchecked cell proliferation or, equivalently, the failure of cells to differentiate properly, has been hypothesized for decades to account for tumorigenesis and cancer. In more recent years, the discovery of oncogenes and their protein products has deepened our understanding of the molecular biology not only of oncogenesis, but also of programmed cell differentiation, proliferation, and death (apoptosis).
Regardless of whether one chooses to study cell growth or apoptosis, the obvious fact of multicellular life is that cells must coordinate their activities with one another. It should not be so surprising, therefore, that some of the transforming proteins described, beginning in the early 1980s, as the products of oncogenes have turned out to be key factors in cell–cell communication that, through mutation, have gone awry. The c-erbB family of mammalian proto-oncogenes, capable of undergoing specific mutations so as to transform cells in culture, provides just one example. Indeed, a number of tumors, such as gliomas, engender mutations in one or more members of the c-erbB gene family. But it is the normal biology of proto-oncogenes and the cell regulatory factors that they encode that stands as one of the most hotly pursued areas of biomedical research in recent years. And as the signal transduction pathways in plant cells are currently being elaborated, a familiarity with themes that have arisen in animal systems becomes ever more pertinent to plant biologists.
To pursue the example further, therefore, the normal c-erbB products are now recognized as cell receptors that bind a number of ligands, the best studied of which is epidermal growth factor (EGF). The binding of EGF to its receptor thus represents a primary message that subsequently triggers a cascade of signaling events within the targeted cell. Specifically, the binding of EGF to the extracellular domain of the EGF receptor (ErbB-1) activates the intrinsic tyrosine kinase activity of the cytoplasmic domain, resulting in the autophosphorylation and homodimerization of ErbB-1 (or heterodimerization of differing ErbB variants). In this way ErbB-1 is typical of a wide variety of receptor kinases.
In response to ErbB-1 receptor activation, a number of other proteins, many of which have long been regarded as proto-oncogene products, are subsequently recruited to effect a net response (e.g., cell proliferation). In this respect the Ras superfamily of proteins represents a particularly important and early “switch,” alternating between GDP-bound (“off”) and GTP-bound (“on”) forms. Its inherent GTPase activity ensures that the given Ras protein does not remain active for extended periods of time in the absence of receptor activation, whereas GDP-displacement factors (Grb2 and SOS) are otherwise activated to supply Ras with GTP (see, e.g., Tari et al., 1999).
Upon activation, Ras proteins propagate a phosphorylation cascade that involves multiple regulatory proteins, the most dynamic participants being protein kinases and phosphatases. A simplified textbook scheme that holds for EGF and many other cell signals is: receptor kinase→Ras→Raf→MEK→ MAPK. (Each arrow denotes the activation of the subsequent kinase by the preceding kinase.) The broad-specificity, mitogen-activated protein kinase (MAPK) in turn phosphorylates a variety of transcription factors, protein kinases and phosphatases, which culminates either in cell division or inhibition of cell division, depending on the particular signal and cell type.
Plants, like animals, must also balance the processes of cell proliferation and differentiation, and they manifest many parallels to signal transduction in animals. Ethylene, for instance, one of the primary hormones to regulate growth and development in plants, induces the sequential phosphorylation of several proteins that have counterparts in animal cells. The ethylene receptor itself cannot be directly placed amid the spectrum of animal cell receptor kinases that includes EGF receptors; however, other of the cellular responses to ethylene seem to depend on a number of orthologs of animal signal transducing proteins. In Arabidopsis, for example, kinases of the Raf, MEK, and MAPK types have all been directly implicated in ethylene signaling, albeit as negative regulators (for review, see Solano and Ecker, 1998).
Nevertheless, at the cell surface, where extracellular messages must be received and where the phosphorylation cascades that result in cell division or differentiation must be initiated, the mechanisms whereby binding of ligand is realized in the plant cell cytoplasm have yet to be elucidated. Not only are the protein–protein interactions involved in early signal relay uncertain, but the handful of plant cell receptors implicated in plant development must be subsumed under the single heading of “receptor-like kinases” (RLKs). This somewhat noncommittal moniker reflects the fact that it has been very difficult to experimentally verify ligands for plant receptor kinases, and even the kinase activity of many RLKs (as well as of established receptors, such as the ethylene receptor) has to be assumed on the basis of sequence similarities to nonplant receptor kinases (Hua et al., 1998). Nevertheless, sequence comparisons have been helpful in assigning extracellular, membrane-spanning, and intracellular kinase domains to all plant RLKs (for review, see Lease et al., 1998).
One of the more intriguing RLKs is encoded by the Arabidopsis gene CLAVATA1 (CLV1) (Clark et al., 1997). In addition to addressing mechanisms of signal transduction per se, a full understanding of CLV1 will certainly elucidate themes central to plant development and organogenesis, inasmuch as clv1 mutants manifest extra floral organs, fasciated stems, disrupted phyllotaxis, and enlarged shoot and flower meristems. The accumulation of undifferentiated cells (up to a 1000-fold increase) in clv1 meristematic tissue is particularly significant because it represents a perturbation of the normal developmental balance whereby the commitment of certain cells to primordial development is precisely counter-balanced by the proliferation of cells that replenish the meristem. Exactly how meristematic cells sense the requirement to divide in order to maintain this equilibrium is unknown.
On pages 393–405 of this issue, Trotochaud et al. present data that establish a biochemical understanding of how CLV1 functions as an RLK and give a glimpse into the early signaling events that may underlie control of meristem integrity. Signal cascades by definition require that proteins interact, and Trotochaud et al. successfully reveal at least three of the relay partners engaged by CLV1. Specifically, they demonstrate that CLV1, which has a monomeric molecular mass of approximately 105 kD, shows up predictably in either of two distinct complexes that have respective molecular masses of 185 and 450 kD. Both complexes are labile to reducing conditions, which indicates that their construction is mediated by the formation of intermolecular disulfide bridges. Correspondingly, the authors point out that most RLKs include cysteine residues in conserved motifs, so that the specific complexes formed with CLV1 may turn out to be mechanistically representative of multiple signal transduction pathways that involve RLKs. Further mechanistic insight comes from the authors' suggestion that the 185-kD species is intermediate to the formation of the larger complex of 450 kD.
Using an antibody specific for CLV1, the authors have previously established the interaction of the CLV1 protein with KAPP, a kinase-associated protein phosphatase that had been isolated together with an RLK of unknown function (Stone et al., 1994, 1998). The interaction of CLV1 with KAPP is, furthermore, indicative of a phosphorylation cascade to the extent that it is dependent on the prior autophosphorylation of CLV1. Because specific reductions of KAPP expression in vivo can suppress the clv1-conferred phenotype, whereas KAPP overexpression mimics clv1-conferred phenotypes (Williams et al., 1997), KAPP can be regarded as a negative regulator of CVL1 signaling. Having produced antibodies to KAPP, the authors now confirm that KAPP is also present in the 450-kD complex, although it is not present in the 185-kD complex.
In view of the molecular weights of CLV1 (105 kD) and KAPP (65 kD), the authors confront the problem of further elucidating the constituency of the 450-kD complex, which genetic experiments define as the active complex (see below). Recalling the simplified scheme worked out for animal systems and outlined above (receptor kinase→ Ras→ Raf→ MEK→MAPK), an especially intriguing question is whether a Ras-related protein could associate with CLV1 and provide the cytoplasmic “switch” for signal transduction. There is indeed a subfamily of monomeric GTPases, known as Rho in animal cells and as Rop in plant cells (Rho-related GTPases from plants), that participates in signal transduction pathways that elicit reorganization of actin-based cytoskeletal elements (see, e.g., Ridley and Hall, 1992). Significantly, Trotochaud et al. demonstrate that a 25-kD protein, consistent with the size of Rop, exists in the 450-kD CLV1 complex and reacts strongly with anti-Rop antisera.
The detected interaction of Rop with an RLK such as CLV1 (i.e., CLV1:Rop) is an important step in identifying an early “switch” in plant signal transduction. The identification of the ligand that binds to and activates CLV1 so as to initiate a phosphorylation cascade (i.e., CLV1→Rop) would represent yet a further advance in elucidating the mechanistic underpinnings of RLK activities in plants. To verify their 450-kD complex as a biologically meaningful entity, therefore, the authors have exploited a number of Arabidopsis clv mutants. The clv1-10 mutation, for instance, results in a protein that exhibits no kinase activity, and is therefore presumably unable to interact with KAPP; correspondingly, clv1-10 extracts contain no detectable complex in the 450-kD range, whereas a 185-kD complex is readily formed.
Mutations of the CLV3 gene are also instructive with regard to formation of the 450-kD complex. clv3 mutants are similar in phenotype to clv1 mutants, and an epistatic relationship has been established for the two loci such that the CLV1 and CLV3 proteins have been assumed to interact or to be active at a common position in a signaling pathway (see, e.g., Clark, 1997). Trotochaud et al. now show that clv3 mutant extracts do not produce the 450-kD complex, despite the fact that such mutants express the wild-type CLV1 protein. (The 185-kD complex is nevertheless abundant in clv3 extracts, which the authors offer as evidence that the larger 450-kD complex cannot merely be an artifactual aggregation involving CLV1.) Their intriguing hypothesis is that the CLV3 protein may in fact be the activating ligand of the CLV1 receptor kinase. This possibility is attractive in that intermolecular associations of protein kinases are known to be instigated in animal systems by the binding of ligands to cell receptors. As the authors point out, the cloning of CLV3 will help to elucidate the signal transduction pathway that is promoted by CLV1 to regulate meristematic function.
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