stumps: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - stumps

Synonyms - downstream of FGFR (dof), heartbroken

Cytological map position - 88D

Function - signal transduction protein

Keywords - mesoderm, trachea, Ras pathway. FGF signaling

Symbol - stumps

FlyBase ID: FBgn0020299

Genetic map position -

Classification - novel protein

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | Entrez Gene
BIOLOGICAL OVERVIEW

Drosophila possesses two FGF receptors that are encoded by the heartless and breathless genes. Heartless is essential for early migration and patterning of the embryonic mesoderm; Breathless is required for proper branching of the tracheal system. A new gene, stumps, has been identified that participates in the signaling pathways of both FGF receptors. stumps has been cloned and although it appears to be a novel protein, it possesses several sequences characteristic of a signal transduction protein (Vincent, 1998). Mutations in stumps are associated with defects in the migration and later specification of mesodermal and tracheal cells. Genetic interaction and epistasis experiments indicate that stumps acts downstream of the two FGF receptors, but either upstream of, or parallel to, Ras1. Furthermore, stumps is involved in both the Heartless- and Breathless-dependent activation of Mapk. It has been concluded that stumps may contribute to the specificity of developmental responses elicited by FGF receptor signaling (Michelson, 1998, and Vincent, 1998).

Mutations have been described in the Drosophila heartless gene that eliminate development of the single somatic muscle and the subset of pericardial cells that express Even-skipped (Eve) in the dorsal region of the embryonic mesoderm. An independent complementation group, later named stumps, with a mesodermal Eve phenotype identical to that of heartless was obtained in the same genetic screen (Gisselbrecht, 1996). In a second laboratory, stumps was discovered in a search for mutations affecting morphogenetic movements in the Drosophila embryo (Vincent, 1998). stumps was originally cloned in a search for genes expressed downstream of twist and snail (Casal, 1996). As occurs for heartless, the development of other dorsal mesodermal derivatives is severely reduced in the stumps mutant, including the cardial cells of the heart, most dorsal somatic muscles and pericardial cells, in addition to those expressing Eve, and the midgut visceral mesoderm. On the basis of these and other phenotypic similarities to heartless (htl), this new gene was named stumps (Michelson, 1998).

The earliest phenotype observed in heartless mutant embryos is a lack of dorsolateral migration of the invaginated mesoderm. This abnormality accounts for the later absence of dorsal mesodermal derivatives, since these structures require induction by Decapentaplegic (Dpp) which is supplied by the dorsal ectoderm. Since the phenotype of later stage stumps embryos is very similar to that of heartless, the effect of a stumps mutation on earlier stages of mesoderm development was examined. As revealed by transverse sections of stage 10 Twist (Twi)-stained embryos, there is a severe defect in the dorsolateral spreading of stumps mutant mesodermal cells. Thus, as is the case for htl, the heart, dorsal somatic muscles and visceral mesoderm fail to develop properly when stumps function is reduced because mispositioned mesodermal progenitors are not exposed to Dpp (Michelson, 1998).

Ras1 is a key signal transducer acting downstream of all (receptor tyryosine kinases) Rtks, including Htl. Since htl and stumps mutants have similar mesodermal phenotypes and genetic interaction studies suggest a functional relationship between the products of these genes, it became interesting to see whether stumps could also be related to Ras1 function. It is known that targeted mesodermal expression of a constitutively activated form of Ras1 can partially rescue the htl mutant phenotype. This conclusion was reached by examining both the activated Ras1-induced migration of Twi-expressing cells and the recovery of dorsally restricted Eve-positive muscle and cardiac progenitors in htl embryos. Using these same assays, it has been found that activated Ras1 is capable of partially rescuing the strong stumps (hbrYY202) mutant. The above results suggest that stumps acts either upstream of Ras1 or on a parallel pathway involved in either initiating or transducing the Htl signal. It was next asked where stumps functions in relation to the receptor by determining if a constitutively activated form of Htl can rescue the stumps phenotype. When expressed in the mesoderm of wild-type embryos, activated Htl induces the formation of additional Eve founder cells but has no effect on mesoderm migration. In a htl mutant background, activated Htl partially corrects the mesoderm migration defect and contributes to the specification of significant numbers of Eve progenitors. Quantitation of the latter effect reveals that activated Htl is significantly more efficient at rescuing loss of htl function than is activated Ras1. In contrast, the influence of activated Htl is completely blocked by a homozygous stumps mutation. These results, as well as the dominant suppression of activated Htl by stumps, argue that stumps acts either downstream of, or parallel to, this mesodermal Fgf receptor (Michelson, 1998).

Rolled MapK is another important component of the Rtk signalling cascade. A monoclonal antibody specific for the dual phosphorylated, activated form of MapK (diphospho-MapK) has recently been shown to be highly effective for monitoring the activity of Rtk pathways during Drosophila development. Using this reagent, high levels of activated MapK were localized to the leading edge of the migrating mesoderm, with much lower levels present at more ventral positions. Activation of MapK is very weakly enhanced in the ventral mesoderm by twi-GAL4-induced expression of a constitutive form of Htl, although the normal gradient of diphospho-MapK expression does not appear to be significantly altered by this manipulation. Activated MapK is completely absent from the early mesoderm of htl mutants, confirming that this mesodermal expression of diphospho-MapK is entirely Htl-dependent. Moreover, no activated MapK is detectable at comparable stages in the mesoderm of stumps mutant embryos. Activated Htl expressed in a null htl mutant generates a low, uniform level of diphospho-MapK throughout the mesoderm. In addition, reduction of stumps function is capable of completely blocking MapK activation by constitutive Htl. These results suggest that stumps acts upstream of MapK in the Htl signal transduction pathway, a hypothesis that is consistent with the findings of the above genetic epistasis experiments (Michelson, 1998).

A second Drosophila Fgf receptor is encoded by the breathless (btl) gene. Btl activity is required for the migration of tracheal cells to form primary branches, and for the subsequent induction of secondary and terminal tracheal cell fates. Mutations in btl are associated with a marked inhibition of tracheal branching. Given the involvement of stumps in the Htl Fgf receptor signaling pathway, an examination was carried out to see if stumps might also function with Btl in the tracheal system. Reduction of stumps function is indeed associated with significant defects in tracheal development. In hbrYY202 mutant embryos, numerous primary and secondary tracheal branches are missing and the extension of those that do form is frequently stalled. These results imply that stumps is necessary both for tracheal cell migration and for the acquisition of secondary tracheal cell fates. One stumps allele, hbrems7, exhibits a very similar tracheal phenotype to another, hbrYY202, while a third allele, hbrems6, has a more severe reduction in tracheal branching. Consistent with findings for the mesoderm, both hbrYY202 and hbrems6 are hypomorphic with respect to their effects on tracheal development, since more severe phenotypes occur when either allele is in trans to a deficiency. Interestingly, although hbrems6 has the strongest tracheal phenotype, its mesodermal defects are the least severe of the three stumps alleles. As is the case with heartless and stumps, breathless and stumps exhibit strong genetic interactions. Thus, stumps is capable of dominantly enhancing a hypomorphic btl allele and btl can dominantly enhance the stumps tracheal phenotype. Together, these genetic interaction experiments suggest that stumps participates in both the Htl and Btl signaling pathways (Michelson, 1998).

stumps completely blocks the effects of activated Htl in the mesoderm. The potential requirement for stumps in mediating the effects of Btl hyperactivation was also investigated. Ectopic ectodermal expression of Bnl, the Btl ligand, leads to widespread Btl activation, which causes a strong inhibition of primary tracheal branching, accompanied by the induction of disorganized networks of secondary and terminal tracheal branches. A homozygous stumps mutation strongly suppresses this effect of ectopic Bnl - the formation of long primary branches is recovered and the additional fine, higher order branches are markedly reduced in number. Thus, as with activated Htl in the mesoderm, a hypomorphic stumps mutation is capable of at least partially suppressing the effect of Btl hyperactivation (Michelson, 1998).

Expression of activated MapK can be used to follow Rtk involvement in tracheal development. Specific tracheal cell fates are established initially under the influence of Egfr, whose activity is reflected in the expression of activated MapK in the tracheal placodes at stage 10. By stage 11, Btl-dependent expression of diphospho-MapK occurs in the tracheal pits prior to the onset of tracheal branch migration. In either btl or bnl mutant embryos, the Egfr-dependent expression of activated MapK in the tracheal placodes is not affected, while the later expression of activated MapK in the tracheal pits is largely but not completely eliminated. With reduced stumps function, the Egfr-dependent diphospho-MapK pattern at stage 10 is normal, while Btl-dependent expression at stage 11 is very weakly but significantly reduced. The extent to which tracheal pit MapK expression is affected in the bnl and stumps mutants appears to be commensurate with the relative severities of their tracheal migration defects (Michelson, 1998).

Having established that stumps is not involved in Egfr-dependent MapK activation in the tracheal placodes, it was next asked if stumps is required for any other Egfr-mediated process in embryogenesis. Egfr has many critical embryonic functions, as reflected in the multiple sites of Egfr-dependent expression of diphospho-MapK. However, none of these sites of diphospho-MapK expression is affected in stumps mutant embryos, including the head folds, cephalic furrow, dorsal folds and ventral ectoderm at stage 8, the ventral midline at stage 11 and the segmental epidermal pattern at stages 12/13. Moreover, Egfr-dependent patterning of the ventral ectoderm occurs normally in stumps mutant embryos, as determined from the wild-type cuticle pattern and normal expression of Fasciclin III in the ventral epidermis of the three thoracic segments. Finally, it was found that a constitutively activated form of Egfr is able to partially rescue mesodermal Eve expression equally well in both htl and stumps mutant embryos, an effect that is due to the capacity of Egfr to activate the Ras/MapK cascade (which also functions downstream of Htl) in a stumps-independent manner. This is in contrast to the ability of the same stumps mutation to completely block the mesodermal effects of constitutively activated Htl. That is, a mutation in stumps interferes with mesodermal Htl but not Egfr signaling. Thus, by multiple criteria, stumps functions in the Htl and Btl but not in the Egfr signaling pathways. It is concluded that stumps may contribute to the specificity of developmental responses elicited by FGF receptor signaling (Michelson, 1998).


GENE STRUCTURE

Two classes of cDNAs were isolated, consistent with Northern blot analysis, when the original short cDNA fragments were used to screen an embryonic cDNA library. Restriction mapping and sequencing shows that the two classes of cDNAs represent two different transcripts (I and II) which share 3555 bp of identical 3' sequence but differ in their 5' ends. When probes corresponding to the unique regions of the two transcripts were used for in situ hybridization experiments, it was found that both transcripts coexpress in most tissues, except for the anterior midgut primordium, which expresses only transcript II. Transcript II contains a full open reading frame, including an AUG in a context closely matching the consensus translation start site, which is preceded by in-frame stop codons. Since this transcript can rescue the defects in stumps mutants, its product is considered as the canonical Stumps protein. Comparison of the structure of the two cDNA clones with genomic DNA shows that the 3' sequences share between the two transcripts are encoded by one exon while their 5' ends are encoded by one further exon for transcript II and at least two further exons for transcript I (Vincent, 1998).


PROTEIN STRUCTURE

Amino Acids - 1009

Structural Domains

The open reading framed of transcript II encodes a protein of 1009 amino acids. There is no recognizable leader peptide, indicating that the protein is neither secreted nor a typical transmembrane protein. Database searches fail to identify any other proteins with significant overall homology to Stumps. However, two regions with similarities to structural motifs in known proteins have been found. Amino acids 366-440 show homology to two ankyrin repeats, and the sequence in the region of amino acids 703-741 shows some similarity to myosins in a region predicted to fold into a coiled coil. Another characteristic of the protein is a cluster of 15 positively charged residues at the C terminus between amino acids 970 and 1006. While the protein has no structural domains typical of molecules involved in signal transduction, the environments of the tyrosines at positions 97, 914, and 930 (YQNT, YLNT, and YQNQ, respectively) suggest that if phosphorylated, these tyrosines might act as binding sites for the SH2 domain of the Drosophila Grb2 homolog, Drk. In addition, the context of the tyrosine at position 486, YMEM, might represent a binding site for the SH2 domain of the regulatory subunit of phosphatidyl 3-kinase, while the sequence downstream of the tyrosine at position 844, YMVP, could represent a binding site for RasGAP. There is also a potential binding site for the protein tyrosine phosphatase SH-PTP2 (coded for by the corkscrew gene in Drosophila) around the tyrosine at position 515, LNYISVET, although it is noted that the threonine at position 520 is less than optimal (Vincent, 1998).

EVOLUTIONARY HOMOLOGS

Mice deficient in the B cell adaptor for phosphoinositide 3-kinase (BCAP) have reduced numbers of mature B lymphocytes, which show defects in cell survival and proliferation. The NF-kappa B (Rel) pathway is impaired in BCAP-deficient mature B cells, and NF-kappa B target genes, indispensable for cell survival and division, are not induced in response to B cell receptor (BCR) stimulation. Among the NF-kappa B (Rel) family, expression of c-Rel is specifically reduced in BCAP-deficient B cells. Retrovirus-mediated reintroduction of c-Rel restores the pool size of immunoglobulin (Ig)M(lo)IgD(hi) mature B cells in the spleen as well as proliferative responses to BCR stimulation. These results indicate BCAP is essential in the maintenance of mature B cells through functional coupling with c-Rel (Yamazaki, 2004).

B-cell activation mediated through the antigen receptor is dependent on activation of protein tyrosine kinases (PTKs) such as Lyn and Syk and subsequent phosphorylation of various signaling proteins. This study reports on the identification and characterization of the B-cell scaffold protein with ankyrin repeats (BANK), a novel substrate of tyrosine kinases. BANK is expressed in B cells and is tyrosine phosphorylated upon B-cell antigen receptor (BCR) stimulation; the phosphorylation is mediated predominantly by Syk. Overexpression of BANK in B cells leads to enhancement of BCR-induced calcium mobilization. It was found that both Lyn and inositol 1,4,5-trisphosphate receptor [IP(3)R] associate with the distinct regions of BANK and that BANK promotes Lyn-mediated tyrosine phosphorylation of IP(3)R. Given that IP(3)R channel activity is up-regulated by its tyrosine phosphorylation, BANK appears to be a novel scaffold protein regulating BCR-induced calcium mobilization by connecting PTKs to IP(3)R. Because BANK expression is confined to functional BCR-expressing B cells, BANK-mediated calcium mobilization may be specific to foreign antigen-induced immune responses rather than to signaling required for B-cell development (Yokoyama, 2003)


stumps: Regulation | Developmental Biology | Effects of Mutation | References

date revised: 3 March 99

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