Validation of commercial ERK antibodies against the ERK orthologue of the scleractinian coral Stylophora pistillata

Introduction

Mitogen activated protein kinases (MAPKs) are highly conserved proteins involved in signalling pathways and control key cellular processes such as proliferation, differentiation, migration, survival and apoptosis (Dhillon et al., 2007). The MAPK gene family encompasses three major subfamilies: the extracellular signal-regulated kinase (ERK), p38/HOG and c-Jun N-terminal kinase (JNK) groups. The ERK family is the most studied in mammals (Boulton et al., 1990; Dhillon et al., 2007) because it is involved in meiosis, mitosis and post mitotic functions in differentiated cells, as well as in the oxidative stress response and wound healing (Castellano et al., 2014; Johnson & Lapadat, 2002; Matsubayashi et al., 2004; Runchel et al., 2011). The ERK gene family is evolutionnarily conserved and is found in all eukaryotes, including yeasts, plants, vertebrates and invertebrates (Chen et al., 2001; Widmann et al., 1999). Although recent molecular studies have shown the existence of ERK genes in different coral species (Mayfield et al., 2010; Siboni et al., 2012; van de Water et al., 2015), ERK activity and specific functions are not yet clearly defined. ERK activation occurs through phosphorylation of the Threonine and Tyrosine residues of an ERK-specific TEY motif by the upstream kinases of ERK, the mitogen-activated protein kinase kinase (MAPKK or MEK). ERK phosphorylation on these residues is classically considered the most appropriate readout for the activity of the ERK signalling pathway. However, it has never been monitored in corals. Overall, MAPK activities in corals have only been investigated once, in a study focusing on the JNK subfamily (Courtial et al., 2017).

In this work, we used the scleractinian coral Stylophora pistillata, a very abundant species in most tropical reefs (Veron & Stafford-Smith, 2000). We applied the same protocol as in Courtial et al. (2017) to demonstrate the efficiency of antibodies directed against the mammalian phosphorylated forms of ERK (pERK) and total ERK to detect the ERK orthologs in S. pistillata (Table 1). According to the manufacturer’s instructions, the antibody used in this study and directed against the Thr202/Tyr204 di-phosphorylated active ERK (Thermo Scientific Pierce; MA5-15174) showed reactivity with fruit fly, human, mink, mouse, non-human primate, pig, rat and zebrafish. The immunogen used to generate this rabbit IgG monoclonal antibody was a synthetic phosphopeptide corresponding to residues surrounding the phospho-Thr202/Tyr204 of the human p44/ERK1 MAP kinase. This antibody is not cross-reactive with the corresponding phosphorylated residues of either JNK/SAPK or p38. The ERK1/ERK2 antibody (Thermo Scientific Pierce; MA5-15605) used in the study previously showed reactivity with human and mouse samples. The immunogen used to generate this mouse IgG2b monoclonal antibody was a purified recombinant fragment of human MAPK.

Methods

Results and discussion

In order to confirm the presence of an ERK ortholog in corals, the human protein sequence of ERK1 (NP_001035145) was compared to the transcriptome database of Stylophora pistillata using the BLAST software (Altschul et al., 1990; Karako-Lampert et al., 2014). An open reading frame was retrieved from the best hit sequence with a predicted molecular weight of 42 kDa (Spi_isotig05348). This sequence (hereafter referred to as Spi-ERK for S. pistillata ERK) is the only one that shows an homology as high as 81%, 80% and 78% with the protein sequences of the cnidarians Nematostella vectensis ERK (Nv-ERK; XP_001629498.1), Hydra vulgaris ERK (Hv-ERK; XP_002154499.3) and the human MAPK3/ERK1 (Hs-ERK1), respectively (Figure 1) (Krishna et al., 2013; Putnam et al., 2007). These sequences all contain both the conserved kinase domains (Hanks & Hunter, 1995) and the TEY motif of the catalytic domain, which is unique for ERK orthologs (Davis, 2000; Figure 1). An interesting point to note is that a unique sequence showing these features is present in N. vectensis and H. vulgaris genomes, as well as in the S. pistillata transcriptome database. This result suggests that a single ortholog of ERK is present in these cnidarians, consistently with previous work where only one ERK ortholog was found (Castellano et al., 2014; Russo et al., 2004) but as opposed to the two genes encoding ERKs in most mammalian genomes (Ip & Davis, 1998). Furthermore, based on the high level of sequence conservation between distant species (Hanks & Hunter, 1995), antibodies directed against portions of the ERK human proteins may recognize ERKs from other species. Accordingly, we detected a single immune-reactive band with the total-ERK antibody by western blot on S. pistillata extracts (Figure 2A and Supplementary Figure S1). Spi-ERK should retain the mechanism of activation by phosphorylation of the Threonine and the Tyrosine residues of the ERK-specific TEY motif. Hence, the MA5-15174 antibody directed against the phosphorylated Thr202 and the Tyr204 (i.e. the phosphorylated TEY motif) should detect a phosphorylated TEY motif of Spi-ERK (phospho-ERK). This is consistent with what we observed, as we detected a unique immune-reactive band of approximately 40 kDa with both antibodies (Figure 2A).

Figure 1. Sequence alignment of MAPK orthologs.

The ERK orthologs of Stylphora pistillata (Spi-ERK), Nematostella vectensis (Nv-ERK), Hydra vulgaris (Hv-ERK), and the human ERK1 (Hs-ERK1) protein sequences are shown. The ERK-specific TEY motif is highlighted in red. The eleven conserved kinase domains are underlined.

Figure 2. Detection of ERK activity in corals.

(A) Fluorescent immunoblot revealing activated (pERK) and total forms of ERK (ERK) present in Stylphora pistillata nubbins. Molecular weight standards in kilo Daltons (kDa) are indicated on the left side of the figure. (B) Immunoblot performed with ERK and pERK antibodies on protein extracts from coral nubbins incubated in the absence (Control) or presence of the MEK inhibitor U0126. Densitometric analysis of activated ERK intensities is presented on the right of the figure. The amido black total protein staining of the western blot membrane is shown as a loading control. The medians and standard deviations of three independent experiments are presented (***, p<0.01, t-test).

Interestingly, the fluorescent immunoblot technique showed that the bands detected with the phosphorylated- and the total-ERK antibodies mostly co-migrate, suggesting that the same protein is detected (Figure 2A). The slight electrophoretic migration shift of the band detected with the anti-phosphorylated ERK antibody would be consistent with the phosphorylation of the threonine and tyrosine residues of the TEY motif as previously described (Aoki et al., 2011). These results suggest that ERK and its phosphorylated form are correctly recognized by the antibodies.

RNAi interference techniques are not available in coral, and the confirmation that the immune reactive bands observed here specifically correspond to ERK could not be obtained through this method. In order to test the specificity of the antibodies, we therefore used U0126, a very potent and selective inhibitor of MEK (Bain et al., 2007). The limited thickness of the animal tissue covering the skeleton and the very large surface of contact of both ectoderm and endoderm with the seawater render S. pistillata suitable for treatment with drugs directly diluted in the seawater as we previously showed (Courtial et al., 2017). U0126 was previously shown to efficiently block MEK activity in a wide variety of organisms, including cnidarians (Hasse et al., 2014; Picco et al., 2007; Röttinger et al., 2004). When the inhibitor was added to the seawater, the intensity of the band detected by the anti-total ERK did not vary, while the intensity of the band detected with the anti-phosphorylated ERK antibody was significantly reduced (Figure 2B and Supplementary Figure S1). Altogether, our results strongly suggest that the proteins detected with the two antibodies were ERK and pERK.

To confirm that Spi-ERK activity can dynamically respond to changes in experimental conditions, we performed an induction experiment by modifying culture conditions of the corals. Courtial et al. (2017) showed that thermal and UVR stresses induced the formation of reactive oxygen species which are known to trigger ERK phosphorylation (McCubrey et al., 2006). ERK phosphorylation was enhanced in corals exposed to UVR, high temperature or a combination of both (Figure 3 and Supplementary Figure S2). These results confirm that the antibodies characterized herein can be used to monitor ERK activity in corals.

Finally, to assess the performance of these antibodies, we compared the signal obtained on S. pistillata and human fibroblasts protein extracts (Figure 4 and Supplementary Figure S3). We loaded on the same gel 10µg of fibroblast total protein extract and different amounts of S. pistillata extracts (ranging from 80 to 10 µg). A signal comparable to the one obtained with the fibroblast extract was observed using 40 µg of coral proteins for both antibodies. This suggests that the affinity of the antibodies towards the coral proteins may be lower than for their human counterparts.

Figure 3. Induction of Spi-ERK phosphorylation by thermal and UV stresses.

Immunoblot performed with ERK and pERK antibodies on protein extracts from coral nubbins incubated for 30 minutes in control (Cont.), thermal stress (T), UV stress (UV) or a combination of thermal and UV stresses (UV + T) conditions. The amido black total protein staining of the western blot membrane is shown as a loading control.

Figure 4. Relative sensitivities of ERK antibodies toward the human and coral proteins.

Immunoblot performed with anti-ERK and anti-phospho-ERK on total protein extracts of human fibroblasts (BJ) and Stylphora pistillata. The amount of protein loaded in each lane is indicated on the Supplementary Figure S4.

Conclusion

This work showed that MA5-15174 and MA5-15605 are two specific antibodies that can be used to quantitatively assess Stylophora pistillata ERK phosphorylation/activity in different experimental or environmental conditions. We demonstrated the specificity of these antibodies and their good affinity towards their coral targets. It therefore provides the coral research community with a potent tool for the analysis of the activity of a signalling pathway involved in a wide variety of biological processes.

Data availability

Supplementary Figure S1. Uncropped blot images for Figure 2 and supplementary replicates. (A) Biological replicates of fluorescent immunoblots performed in control conditions (Ct) are shown (Replicates 1 and 2). The portions of the images used in the main text are outlined. (B) Biological replicates of immunoblots performed on protein extracts from coral nubbins incubated in the absence (Control) or presence of the MEK inhibitor U0126 (UO) (Replicates 1 to 5). The portions of the images used in the main text are outlined. doi, 10.5256/f1000research.11365.d159188 (Courtial et al., 2017a).

Supplementary Figure S2. Uncropped blot images for Figure 3 and supplementary replicates. The portions of the images used in the main text are outlined. doi, 10.5256/f1000research.11365.d166821 (Courtial et al., 2017b).

Supplementary Figure S3. Uncropped blot images for Figure 4 and supplementary replicates. The portions of the images used in the main text are outlined. doi, 10.5256/f1000research.11365.d166825 (Courtial et al., 2017c).