Techniques update: Crossreactivity of anti-dual-phosphorylated antibodies with actin

Abstract

Mitogen-activated protein kinases (MAPKs) and stress-activated protein kinases (SAPKs) are components of protein kinase cascades that permit eukaryotic cells to respond to many extracellular signals[1xMarshall, C.J. Curr. Opin. Genet. Dev. 1994; 4: 82–89Crossref | PubMed | Scopus (798)See all References[1]. To monitor the activity of these signalling pathways under physiological conditions, several companies have recently developed specific antibodies directed against phosphopeptides corresponding to the TXY motif essential for MAPK and SAPK activation. These products specifically recognize single- or dual-phosphorylated forms of ERK MAPKs and the SAPKs Jun N-terminal kinase (JNK) and P38 kinase.These antibodies have been used to analyse processes ranging from the effects of growth hormone stimulation in different cell types[2xLove, D.W. et al. Endocrinology. 1998; 139: 1965–1971Crossref | PubMedSee all References[2]to the role of MAPKs in macrophage migration induced by integrins[3xWei, J. et al. J. Biol. Chem. 1998; 273: 5903–5907Crossref | PubMed | Scopus (62)See all References[3]. They have also been applied to biochemical studies, such as the elucidation of the nonprocessive mechanism of activation of MAPKs by MEK[4xBurack, W.R. and Sturgill, T.W. Biochemistry. 1997; 36: 5929–5933Crossref | PubMed | Scopus (128)See all References[4]. Two recent publications have made a new, even more exciting, use of these tools. Shilo and his colleagues have studied in situ the activation of MAPK during Drosophila development[5xGabay, L. et al. Science. 1997; 277: 1103–1106Crossref | PubMed | Scopus (297)See all References, 6xGabay, L. et al. Development. 1997; 124: 3535–3541PubMedSee all References], and the work was highlighted in this journal[7xHafen, E. Trends Cell Biol. 1997; 7: 436See all References[7].Using a similar approach, we examined the pattern of activation of the P38 kinase during Drosophila embryogenesis. In this study, we used the Anti-Active P38 antibody developed by Promega against a phosphopeptide containing the TGY motif of the mouse P38 kinase. The epitope recognized by this antibody is well conserved in the two recently isolated Drosophila homologues of the P38 kinase (VPQESEMVATRWYRAPE—DMPK2A; RPTENEMVATRWYRAPE—DMPK2B)[8xHan, S.J. et al. J. Biol. Chem. 1998; 273: 369–374Crossref | PubMed | Scopus (78)See all References, 9xHan, Z.Q.S. et al. Mol. Cell. Biol. 1998; 18: 3527–3539Crossref | PubMedSee all References].An Anti-Active P38 signal was detected from very early stages of Drosophila development, decorating all cell membranes from cellular blastoderm stages (Fig. 1Fig. 1a). At late stages, Anti-Active signal appeared associated mainly with cuticle structures (denticle belts; Fig. 1Fig. 1b) and the neuropile in the central nervous system (CNS; Fig. 1Fig. 1c). These patterns of activation correlated closely with the distribution of actin during development, and the remarkable similarity between the actin and active P38 expression led us to re-analyse the specificity of the antibody. We found that Anti-Active P38 associated with actin filaments polymerized in vitro from Drosophila embryo extracts (E. Martin-Blanco and A. Martinez-Arias, unpublished). More definitively, we found by western analysis that the antibody recognized rabbit skeletal actin as well as the dual-phosphorylated form of P38 (Fig. 1Fig. 1d). Immunoadsorption of the antibody eliminated this crossreactivity but maintained the specific binding to dual-phosphorylated P38 (Fig. 1Fig. 1e). The association of the signal with actin at early stages and in the embryo cuticle was eliminated (E. Martin-Blanco and A. Martinez-Arias, unpublished), whereas specific staining in the CNS (as in Fig. 1Fig. 1c) and several other conspicuous locations was reproducibly detected (a detailed analysis will be described elsewhere).Fig. 1Old, contaminated antibody detected phosphorylation of the stress-activated kinase P38 (arrowheads) during embryonic development of Drosophila. (a) In the cellular blastoderm, Anti-Active P38 staining associates with cell membranes. (b) In a stage 15 embryo, denticle structures accumulate Anti-Active P38 signal. (c) In a stage 16 embryo, Anti-Active P38 signal occurs in commissures and connectives of the embryonic central nervous system (CNS). In most cases, these signals correlated with actin accumulation. The anti-actin component of Promega Anti-Active P38 antibody was eliminated by adsorption to rabbit skeletal actin [compare (d) and (e)], but the antibody still retains its ability to recognize dual-phosphorylated P38 (e). Using new, clean batches, P38 phosphorylation is still detected in the Drosophila CNS [data not shown, but similar to (c)].View Large Image | Download PowerPoint SlideProduction and sale of the antibody by Promega were halted, all customers were informed, technical support has been provided and new uncontaminated batches have now been supplied. This type of in vivo analysis is one approach that can be used to verify antibody specificity. Users of this or similar antibodies should nevertheless take extreme care in the interpretation of their results and investigate carefully if any ambiguity arises.