(699a) MAPK Substrate Competition Integrates Patterning Signals in the Drosophila Embryo

Anteroposterior (AP) patterning of the Drosophila embryo depends on three inductive signals [1]: The head and thorax are specified by the anterior gradient of Bicoid (Bcd), a homeobox transcription factor; abdomen formation is directed by the reciprocal gradient of Nanos (Nos), a translational repressor; and the non-segmented termini are patterned by the localized activation of the Extracellular Signal Regulated/Mitogen Activated Protein Kinase (ERK/MAPK) pathway. MAPK signaling is induced by a uniformly expressed receptor tyrosine kinase Torso (Tor), which is activated by its ligand produced at the poles of the embryo.

Activated Tor promotes the expression of tailless (tll) and huckebein (hkb), zygotic gap genes required for the development of the terminal structures. This depends on MAPK-mediated phosphorylation of the transcriptional repressors Capicua (Cic) and Groucho (Gro), both of which are initially distributed uniformly throughout the embryo [2, 3]. Phosphorylation of Cic and Gro relieves their repression of tll and hkb and enables expression of these genes at both poles of the embryo [2, 3]. We found that downregulation of Cic is antagonized by Bcd, the anterior patterning morphogen. Here, we provide evidence suggesting that Bcd, a direct substrate of MAPK [4], decreases the availability of MAPK for other substrates, such as Cic. Based on the quantitative analysis of MAPK signaling in different genetic backgrounds, we propose that MAPK substrate competition coordinates the actions of the anterior and terminal patterning systems.

The spatial pattern of MAPK activation can be visualized using an antibody that recognizes the double phosphorylated form of ERK (dpERK) [5]. We found that this pattern is strongly asymmetric at the two poles: the anterior levels of dpERK are significantly higher than the posterior levels [6]. Since the early Drosophila embryo is highly polarized, multiple factors can potentially contribute to the observed asymmetry. These include the differences in the anterior and posterior levels of the extracellular ligand that activates Tor and/or in the intracellular localization of maternal determinants.

We hypothesized that the higher levels of dpERK at the anterior pole reflect the presence of an anteriorly localized maternal factor. One candidate for such a factor is Bcd, which is localized to the anterior of the embryo and is one of the phosphorylation targets of MAPK. To test whether Bcd can affect the AP asymmetry of the dpERK pattern, we examined this pattern in embryos with different levels of Bcd. We found that the anterior levels of dpERK are significantly decreased in embryos from bcd null or heterozygous mothers, and increased in embryos with two extra copies of bcd. Importantly, the posterior levels of dpERK are unaffected in these embryos, which reflect the anterior localization of Bcd. Thus, changing the level of Bcd, a substrate of MAPK, influences the level of MAPK phosphorylation.

Previous studies had shown that overexpressing MAPK substrates in a heterologous cell culture system leads to a higher level of MAPK phosphorylation. Furthermore, experiments with purified components revealed that MAPK substrates can directly compete with the MAPK phosphatases for binding to the common docking domain of MAPK [7]. Thus, the level of MAPK substrates can increase the level of MAPK phosphorylation by outcompeting the MAPK phosphatase. Correspondingly, a similar effect can be responsible for the observed dose-dependent control of MAPK phosphorylation levels by Bcd: The total concentration of MAPK substrates at the anterior pole is higher than at the posterior, due to the anterior localization of Bcd, resulting in a stronger interference with MAPK dephosphorylation in this region of the embryo. This can account for the higher level of dpERK at the anterior pole.

The fact that the anterior level of dpERK is sensitive to the dose of Bcd is consistent with the proposed competition model. According to the same model, Bcd can make the phosphorylated MAPK less available for its other substrates. To explore this possibility, we examined the distribution of Cic, which is downregulated at the poles as a result of its direct phosphorylation by MAPK. Statistical analysis revealed that, similar to the wild type pattern of dpERK, the wild type pattern of Cic is asymmetrical, with the levels of Cic significantly higher at the anterior pole. Since the distribution of Cic is uniform in the absence of MAPK signaling, the wild type pattern of Cic downregulation suggests that, although the level of MAPK phosphorylation is higher at the anterior pole, its activity directed towards Cic is actually lower.

We emphasize that this observation argues against the possibility that the AP asymmetry of the wild type MAPK phosphorylation pattern is generated only by the asymmetry in the extracellular activation of Tor. If this were true, then higher levels of MAPK phosphorylation at the anterior pole would lead to a higher level of Cic downregulation, which is contrary to what we observe. Based on this, we argue that the AP asymmetry of the wild type MAPK signaling pattern is generated mainly by the intracellular asymmetries of the early embryo. Upon quantifying the spatial pattern of Cic downregulation in embryos with varying levels of Bcd, we established that this asymmetry is increased in embryos with an extra copy of bcd and reduced in embryos with progressively lowered levels of bcd. These effects are consistent with the model, where anteriorly localized Bcd acts as a competitive inhibitor of MAPK-mediated Cic downregulation.

Competitive binding effects have been shown to influence the operation of signaling networks in mathematical models and in vitro. Our results support the existence of these effects in vivo and demonstrate how MAPK signaling can be regulated by the levels of MAPK substrates. Competitive interactions can provide a general control strategy in signaling networks where enzymes, such as MAPK, interact with their multiple regulators and targets.

References

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[3] Jimenez, G., Guichet, A., Ephrussi, A., and Casanova, J. (2000). Relief of gene repression by Torso RTK signaling: role of capicua in Drosophila terminal and dorsoventral patterning. Genes & Development 14, 224-231.

[4] Janody, F., Sturny, R., Catala, F., Desplan, C., and Dostatni, N. (2000). Phosphorylation of bicoid on MAP-kinase sites: contribution to its interaction with the torso pathway. Development 127(2):279-89.

[5] Coppey, M., Boettiger, A.N., Berezhkovskii, A.M., and Shvartsman, S.Y. (2008). Nuclear trapping shapes the terminal gradient in the Drosophila embryo. Curr. Biol. 18, 915-919.

[6] Kim, Y., Coppey, M., Grossman, R., Ajuria L, Jimenez, G., Paroush, Z., and Shvartsman, SY. (2010). MAPK substrate competition integrates patterning signals in the Drosophila embryo. Curr. Biol. 20, 446-451.

[7] Bardwell, A.J., Abdollahi, M., and Bardwell, L. (2003). Docking sites on mitogen-activated protein kinase (MAPK) kinases, MAPK phosphatases and the Elk-1 transcription factor compete for MAPK binding and are crucial for enzymic activity. Biochem J 370, 1077-1085.