The ETS domain protein Pointed-P2 is a target of MAP kinase in the Sevenless signal transduction pathway

Abstract

The fate of the R7 photoreceptor cell in the developing eye of Drosophila is controlled by the Sevenless (Sev) receptor tyrosine kinase. Sev activates a highly conserved signal transduction cascade involving the proteins Ras1 and Raf and the Rolled/mitogen-activated protein (Rl/MAP) kinase. Here we show that the ETS domain protein encoded by the P2 transcript of the pointed (pnt) gene is a nuclear target of this signalling cascade which acts downstream of Rl/MAP kinase. The PntP2 protein is phosphorylated by Rl/MAP kinase in vitro at a single site and this site is required for its function in vivo. Furthermore, we present genetic and biochemical data suggesting that MAP kinase controls neural development through phosphorylation of two antagonizing transcription factors of the ETS family, Yan and PntP2.

Molecular characterization of the zebrafish PEA3 ETS-domain transcription factor

“…ETSdomain proteins have been implicated in regulating several key developmental processes. For example, Pnt regulates both eye (Brunner et al, 1994;O'Neill et al, 1994) and tracheal (Samakovlis et al, 1996) development in Drosophila whereas in mammals, Ets-1 (Bories et al, 1995;Muthusamy et al, 1995) and PU.1 (Scott et al, 1994;McKercher et al, 1996) have been implicated in regulating di erent aspects of haematopoiesis.…”

The PNT domain from Drosophila pointed‐P2 contains a dynamic N‐terminal helix preceded by a disordered phosphoacceptor sequence

“…However, the two MAP kinases are highly related, sharing 82% (89%) sequence identity (similarity), exhibit comparable docking interactions, 40 and function in homologous signaling cascades. 9,18,20 Furthermore, Ets1 is also phosphorylated in vitro by ERK2 at the corresponding Thr38 and Ser41, and in vivo tests have confirmed that both these residues contribute to Ras-enhanced transactivation by ETS1. 4,17 Thus, the potential roles of Thr145 and Thr154 in the control of gene expression by Pointed-P2 remain to be evaluated.…”

“…[18][19][20][21] However, the molecular mechanisms underlying this process have not been defined. By analogy to ETS1, 4,17 it is plausible that phosphorylation of Pointed-P2 leads to enhanced recruitment of Nejire, the Drosophila ortholog of the mammalian coactivator CBP.…”

“…Mutational studies have demonstrated that phosphorylation of Thr151 is critically required for the in vivo function of Pointed-P2, [18][19][20] whereas the biological roles, if any, of these additional nonconsensus sites have not been examined. It is certainly possible that these modifications are an artifact resulting from using a large scale in vitro kinase system to produce milligram quantities of modified PntP2 142-252 for NMR spectroscopic studies, and/or owing to differences between mammalian ERK2 and Drosophila Addition of EKR2 leads to a significant reduction in the relative signal intensities of specific amides in the phosphoacceptor region, as well as in helices H0 and H5 of PntP2 142-252 (blue histogram bars).…”

“…4,17 Pointed-P2, the Drosophila ortholog of ETS1, plays a similar role in the well-characterized EGF and Sevenless receptor tyrosine kinase/Ras-mediated signal transduction pathway to control fly eye development. [18][19][20][21] Monomeric Pointed-P2 is a transcriptional activator and antagonist to Yan, an ETS family transcriptional repressor that polymerizes via head-to-tail self-association of its PNT/SAM domain. 7 Both control the expression of a common set of target genes, including a crucial regulator called Mae.…”