BIOLOGICAL OVERVIEW

The proper alignment or polarity of the fly along its dorsal-ventral axis is the result of cooperative functioning among a number of genes collectively termed the Dorsal group. tube is one component of this group. Dorsal group proteins are supplied maternally; that is, they are present in the egg prior to fertilization. Their functioning leads to the activation of Dorsal protein, a transcription factor responsible for structuring dorsoventral polarity. The cascade of events begins with signals from the Toll receptor. These are transmitted via Tube and Pelle to Dorsal. Dorsal activation must also be accompanied by the destruction of Cactus, which otherwise would bind Dorsal, in effect imprisoning it in the cytoplasm and thereby rendering it inactive. Once freed (activated), Dorsal migrates from cytoplasm to nucleus where it then functions to activate and repress genes involved in dorsoventral polarity, including decapentaplegic, twist and snail.

Tube accomplishes two critical tasks. The first involves its interaction with Pelle, leading to the destruction of Cactus. Signal-dependent degradation of Cactus does not require the presence of Dorsal, indicating that Cactus degradation is a direct response to Pelle signaling. But Cactus degradation requires both Tube and Pelle (Belvin, 1995). So what gets Tube into the act?

Tube is membrane associated in the absence of Toll. Upon activation of Toll, Tube activates Pelle, a serine-threonine kinase. The only known phosphorylation target of Pelle is Tube (Grosshans, 1994). Each of these interactions leads to the destruction of Cactus, which in the case of I kappa B, the vertebrate homolog of Cactus, is ultimately carried out by ubiquitin, the cell's machinery for protein degradation.

The second function of Tube involves an interaction with Dorsal. Tube accompanies Dorsal on its migration into the nucleus (having acted with Pelle to destroy Cactus's cytoplasmic hold on Dorsal). Expression of tube does not by itself affect the localization of Dorsal. Toll's part is essential. Activation of Toll enhances nuclear localization of DL and increases dl transcriptional activation. When tube is coexpressed with Toll, dl transcriptional activity is further enhanced. In addition, coexpression of tube and Toll resulting in the nuclear localization of Dorsal, results in the nuclear localization of Tube as well.

Quite possibly Tube is a chaperone for Dorsal, accompanying it into the nucleus. In addition, the effect of Tube on dorsal transcription suggests that Tube might be a transcription factor acting to regulate dorsal transcription (Norris, 1995). Thus Tube has a cytoplasmic role in the activation of Pelle and the destruction of Cactus, and a subsequent role in association with Dorsal in Dorsal's migration to the nucleus, where it might serve to activate dorsal transcription.

GENE STRUCTURE

Bases in 5' UTR - 192

Bases in 3' UTR - 431

PROTEIN STRUCTURE

Amino Acids - 462

Structural Domains

Deletion analysis, together with an evolutionary comparison of tube genes in Drosophila species has defined two domains for the Tube protein. The amino-terminal domain is well conserved and is sufficient to rescue tube null embryos. The C-terminal half of Tube protein contains five copies of an eight-residue motif and shares no significant sequence similarity with known proteins (Letsou, 1991 and Letsou, 1993).

The interaction between the death domains (DDs) of Tube and the protein kinase Pelle is an important component of the Toll pathway. Published crystallographic data suggests that the Pelle-Tube DD interface is plastic and implies that in addition to the two predominant Pelle-Tube interfaces, a third interaction is possible. The NMR solution structure of the isolated death domain of Pelle is presented along with a study of the interaction between the DDs of Pelle and Tube. The data suggests the solution structure of the isolated Pelle DD is similar to that of Pelle DD in complex with Tube. Additionally, they suggest that the plasticity observed in the crystal structure may not be relevant in the functioning death domain complex (Moncrieffe, 2005).

The crystal structure suggests that the Pelle­Tube DD complex can exist as a tetramer comprised of Pelle and Tube heterodimers arranged in a linear sequence P1:T1:P2:T2 and whether this persists in solution needs to be determined as it may have implications for the function of the DD complex. To address these issues, the structure of the isolated Pelle-DD in solution and the interaction between the DDs of Pelle and Tube have been solved using nuclear magnetic resonance (NMR) spectroscopy. The results suggest that in the beta-helical regions, the structure of Pelle-DD is similar to that of Pelle-DD in complex with the DD of Tube, and of the two types of Pelle-Tube dimer interfaces observed in the crystal structure (P1:T1 and P2:T2) only one is likely to persist in solution (Moncrieffe, 2005).

The NMR data presented confirm the extensive nature of the Pelle-Tube death domain interface because all twenty seven residues that constitute the core interaction surface on Pelle-DD show significant chemical shift differences between the ¹H-¹⁵N HSQC spectrum of Pelle-DD and the ¹H-¹⁵N HSQC-TROSY spectrum of the Pelle-Tube DD complex. The NMR data also suggest that the Tube1:Pelle2 interaction observed in the crystals, may persist in solution. This implies that the death domains of Pelle and Tube are capable of forming a tetramer and this is corroborated by the sedimentation velocity data which reveals that the concentration of the tetrameric complex is very small (0.4%) relative to that of the dimer. The dominant species formed by the interaction of Pelle-DD and Tube-DD is a dimer and whether the very small amount of the tetrameric species plays a role in signaling is unknown; for example, a mutant of Pelle bearing a mutation in the Tube1:Pelle2 interface (D50K) fails to express protein. It is conceivable that the residues in Pelle that are part of the Tube1:Pelle2 interface are sites of interaction for other members of the signaling complex. A recent report argues against that partner being dMyD88 since this component is thought to bind predominantly to Tube. A more likely scenario however, given that dMyD88 appears to bind predominantly to Tube and not Pelle, is that in the functioning complex, P2 is occupied by dMyD88. Thus, the residues in Tube-DD that contribute to the T1:P2 interface are likely to be the scaffold that recruits dMyD88 (Moncrieffe, 2005).

In summary, the structure of the isolated Pelle DD is similar to that of the Pelle DD in complex with the DD of Tube. While the death domains of Pelle and Tube may form a tetramer, consistent with the crystallographic observation the concentration of this species is extremely low. Consequently, inferences regarding the “plasticity” of the Pelle-Tube DD complex in vivo should be reevaluated (Moncrieffe, 2005).

date revised: 25 August 98

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