Spatial signal repression as an additional role of Sprouty2 protein variants

Abstract

Sprouty2 (Spry2) is a prominent member of a protein family with crucial functions in the modulation of signal transduction. One of its main actions is the repression of mitogen-activated protein kinase (MAPK) pathway in response to growth factor-induced signalling. A common single nucleotide polymorphism within the Spry2 gene creates two protein variants where a proline adjacent to the serine rich domain is converted to an additional serine. Both protein variants perform similar functions although their efficiency in fulfilling these tasks varies. In this report, we used biochemical fractionation methods as well as confocal microscopy to analyse quantitative and qualitative differences in the distribution of Spry2 variants. We found that Spry2 proteins localize not solely to the plasma membrane, but also to other membrane engulfed compartments like for example the Golgi apparatus. In these less dense organelles, predominantly slower migrating forms reside indicating that posttranslational modification contributes to the distribution profile of Spry2. However there is no significant difference in the distribution of the two variants. Additionally, we found that Spry2 could be found exclusively in membrane fractions irrespective of the mitogen availability and the phosphorylation status. Considering the interference of extracellular signal-regulated kinase (ERK) activation in the cytoplasm, both Spry2 variants inhibited the levels of phosphorylated ERK (pERK) significantly to a similar extent. In contrast, the induction profiles of pERK levels were completely different in the nuclei. Again, both Spry2 variants diminished the levels of pERK. While the proline variant lowered the activation throughout the observation period, the serine variant failed to interfere with immediate accumulation of nuclear pERK levels, but the signal duration was shortened. Since the extent of the pERK inhibition in the nuclei was drastically more pronounced than in the cytoplasm, we conclude that Spry2 - in addition to its known functions as a repressor of general ERK phosphorylation - functions as a spatial repressor of nucleic ERK activation. Accordingly, a dominant negative version of Spry2 was only able to enhance the pERK levels of serum-deprived cells in the cytosol, while in the nucleus the intensity of the pERK signal in response to serum addition was significantly increased.

Keywords: Localisation; MAPK; Nuclear ERK; Phosphorylation variants; Spatial signal transduction; Sprouty2.