Intercellular communication is critical to the development and normal functioning of higher eukaryotes; breakdowns in this communication can result in developmental defects or contribute to the onset of cancer. Therefore, a thorough understanding of the molecular mechanisms of these cellular interactions is obviously essential for the improved treatment of these often devastating human maladies.
The dissection of signal transduction pathways has been greatly assisted by multiple experimental approaches including classical and molecular genetic studies with Caenorhabditis elegans and Drosophila and biochemical studies in mammalian cell culture systems (Fantl et al., 1993). The underlying biochemical basis of intercellular communication seems to be common to all animals, since relation (Cantley et al., 1991; Egan and Weinberg, 1993). The activation of the signal transduction pathways ultimately results in new patterns of gene expression that affect either cell proliferation, cell differentiation, or other cellular activities, such as, for example, interferon-stimulated anti-viral responses (Darnell et al., 1994).
The role of PTKs in regulating biological processes is most apparent when the PTKs are altered due to mutations. PTKs originally gained prominence as the product of viral and tumor oncogenes whose expression led to unrestricted growth and as the receptors for several growth factors that regulate cell division (Yarden and Ullrich, 1988; Ullrich and Schlessinger, 1990). The homology between these oncoprotein and growth factor receptors indicated that the oncogenes were mutated forms of normal cellular genes required for proper growth regulation. For example, the v-erbB viral oncogene was found to encode a truncated version of the epidermal growth factor (EGF) receptor; the kinase domain of the truncated receptor is no longer subject to its normal regulation and therefore constitutively signals the cell to divide (Yarden and Ullrich, 1988). Subsequently, many developmentally important genes from both invertebrates and vertebrates have been shown to encode receptor PTKs. Many of these genes, such as the C. elegans let-23 gene, the Drosophila sevenless gene and the mouse W/kit locus, have been shown to regulate cell fate choice based on cellular interactions (Pawson and Bernstein, 1990; Kayne, 1994).
A novel role for nonreceptor PTKs, in cytokine signalling, became clear in just the past year. Binding of cytokines, lymphokines, and some growth factors to their non-catalytic receptors activates members of the Janus kinase (JAK) group of nonreceptor PTKs (reviewed in Ihle et al., 1994, Darnell et al., 1994). These kinases phosphorylate the STAT (signal transducer and activators of transcription) proteins which directly participate in transcriptional regulation. The recent characterization of the JAK family members and the novel signal transduction pathway in which they participate illustrates our incomplete understanding of the mechanisms of intercellular communication. To further investigate the potential functions of PTKs in multicellular organisms, I therefore initiated an analysis of PTKs in the experimentally manipulable model organism C. elegans.
To study PTKs in C. elegans, I first conducted a molecular screen for receptor PTK genes and thereby cloned two genes, kin-15 and kin-16 (Morgan and Greenwald, 1993). These tandem genes, apparently the result of a gene duplication event, appear to encode PTKs based on their homology to known members. The predicted gene products are structurally similar and have several features characteristic of receptor PTKs, most notably a potential signal sequence and membrane spanning domain. However, both KIN-15 and KIN-16 have unusually short (<50 amino acids) extracellular domains which lack any cysteine residues. No other receptor PTK has a similar structure, except perhaps the murine Ltk protein with a predicted extracellular domain as short as 108 amino acids (Bernards and de la Monte, 1990). The analysis of human cDNAs, however, indicates Ltk possesses a more typical extracellular domain of over 300 amino acids (Toyoshima et al., 1993). In any case, KIN-15 and KIN-16 have the shortest extracellular domains described so far.
The unusual structure of KIN-15 and KIN-16 renders the typical mechanism of receptor PTK regulation, through the binsted several alternative mechanisms for the regulation of kinase activity (Morgan and Greenwald, 1993). First, KIN-15 and KIN-16 could function in signal transduction by interacting with a second subunit that binds an extracellular signal molecule, as seen with some receptor PTKs (the insulin receptor and Drosophila Sevenless protein, for example; although in these cases both subunits are cleaved from a common precursor; Simon et al., 1989), and some non-receptor PTKs including members of both the Src-like and JAK groups (Veillette and Davidson, 1992; Ihle et al., 1994). Second, intracellular events, such as phosphorylation/dephoshorylation could control KIN-15/KIN-16 activity. Phosphorylation by a second protein kinase could either permit the binding of substrate proteins by creating SH2-binding sites or squelch kinase activity as seen with the EGF receptor and Src-like proteins (Yarden and Ullrich, 1988; Cooper, 1990; Koch et al, 1991). Finally, because KIN-15/KIN-16's short extracellular domains and C-terminal regions devoid of potential autophosphorylation sites superficially resemble some oncogenic PTKs, such as the v-erbB, v-fms, and v-kit products (Yarden and Ullrich, 1988), KIN-15 and KIN-16 may be constitutively active. If so, then the restricted expression of kin-15 and kin-16 (see below) may be a way to regulate kinase activity, and ectopic expression of KIN-15/KIN-16 would then be expected to have deleterious consequences.
Each of these models makes different predictions about which regions of KIN-15 and KIN-16 are functional. For example, amino terminal sequences (the extracellular, transmembrane, and/or juxtamembrane regions) are likely to be essential for function if these PTKs interact with an extracellular ligand binding protein, but not if KIN-15 and KIN-16 are regulated intracellularly or are constitutively active. One objective of the proposed research is to compare the evolutionary conservation of kin-15 and kin-16 homologues from related organisms and perform site-directed mutation studies to help distinguish between these possible models (see Research Design and Methods).
To investigate the possible organismal functions of kin-15 and kin-16, we initially examined their pattern of gene expression during development using lacZ fusion gene constructs (Morgan and Greenwald, 1993). These studies indicated that kin-15 and kin-16 may be needed for the proper development or functioning of the hypodermal syncytium called hyp7. This multinucleate epidermal cell is formed just prior to hatching by the fusion of 23 mononucleate cells; as larval development proceeds, hyp7 grows by further cell fusions (with 110 more cells) until it covers most of the adult worm (Sulston and Horvitz, 1977). Based on the fusion gene studies kin-16, and probably kin-15, is expressed in hyp7 shortly after hatching and throughout larval development (and into the adult) as the syncytium grows by cell fusion. This expression patterns suggests that KIN-15 and KIN-16 may regulate the fusion of cells with hyp7 or, alternatively, may play a functional role in hyp7, such as maintaining nuclei in a mitotically quiescent state following cell fusion.
While the expression studies indicate a site of gene action, they
still leave the question of gene function in the organism unanswered;
therefore we have also been searching for kin-15 and
kin-16 gene mutations to examine the phenotypic effects of
gene disruption. To initiate a reverse genetic approach,
kin-15 and kin-16 was mapped using chromosomal
deficiencies to a small genetic interval containing several
previously isolated mutations (let-240 and
let-31) were complemented by transgenic plasmids harboring
kin-15 and/or kin-16. We have therefore continued our
screen for kin-15 and kin-16 mutations by testing other
candidates and by screening for transposon-induced mutations.
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