Hunting for Hippo Proteins

With highly specific ABfinity recombinant monoclonal antibodies

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The Hippo signaling pathway is an evolutionarily conserved pathway that has been shown to play a critical role in controlling organ size through the regulation of both cell proliferation and apoptosis. Dysregulation of the Hippo pathway results in aberrant cell growth and neoplasia. Given its involvement in these vital cell processes, it is not surprising that mutations in key Hippo pathway proteins are linked to a variety of cancers [1,2].

At the cellular level, the Hippo pathway integrates signals through several mechanisms, including G protein–coupled receptor (GPCR) signaling and the apicobasal polarity fundamental to epithelial cell function [3]. Kinase cascade and nuclear transcription modules form the backbone of the Hippo pathway. The kinase cascade includes serine/threonine kinases such as mammalian STE20–like protein kinases (MST1/2) and large tumor suppressors (LATS1/2), along with adaptor proteins including Salvador homolog 1 (SAV1), MOB kinase activator 1A (MOB1A), and MOB kinase activator 1B (MOB1B). The kinase cascade functions to restrict the activity of two transcriptional coactivators—Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ)—which are further modified by several Hippo regulators.

Antibodies are essential to the study of pathway proteins. However, for many components of the Hippo pathway, specific antibodies are either unavailable or poorly characterized. To address this need, Thermo Fisher Scientific has made a concerted effort to develop highly specific, application-tested antibodies directed against key proteins in this pathway.

Antibodies for kinase cascade proteins

In the kinase arm of the Hippo pathway, MST1/2-mediated phosphorylation of SAV1 and MOB1A/B leads to the recruitment, phosphorylation, and subsequent activation of LATS1/2. In conjunction with MOB1, the activated LATS1/2 kinases phosphorylate YAP/TAZ. These phosphorylation events result in the cytoplasmic sequestration and degradation of YAP/TAZ, mediated by 14-3-3 proteins. Figures 1A and 1B demonstrate the specificity of Invitrogen anti-MST1/2 and anti-SAV1 antibodies in western blot and immunofluorescence/immunocytochemistry (IF/ICC) applications using siRNA-mediated knockdown. In response to nocodazole treatment, LATS2 translocates from the cytoplasm into the nucleus, then binds to and activates p53, inducing LATS2 expression [4]. Figure 1C shows the specificity of the Invitrogen anti- LATS2 antibody in IF/ICC using this nocodazole-induced upregulation of LATS2 expression.

Antibodies for transcription factors and Hippo regulators

In the nuclear transcription module of the Hippo pathway, several DNA-binding proteins regulate the transcription of genes encoding Hippo pathway proteins and also undergo modification themselves. When not phosphorylated, YAP and TAZ translocate to the nucleus and serve as transcriptional coactivators for Hippo pathway genes. Their most significant interacting partners are the TEA-domain family member transcription factors (TEAD1–4) [5]. Figure 2A shows the specificity of the Invitrogen anti-TEAD4 antibody in western blot analysis using siRNA-mediated knockdown. Another transcription regulator, VGLL4, selectively binds to TEAD and interferes with the YAP–TEAD interaction. The specificity of the Invitrogen anti-VGLL4 antibody has been demonstrated with IF/ICC using siRNA-mediated knockdown (Figure 2B).

Angiomotin proteins (AMOT), which play a central role in tight junction maintenance, are also important negative regulators of YAP/TAZ, either by binding YAP/TAZ or by promoting their inhibitory phosphorylation. Phosphorylated AMOT (ser176) is downregulated during serum starvation, a characteristic that was used to show the specificity of the Invitrogen anti-AMOTpS176 antibody in IF/ICC (Figure 2C). In contrast to AMOT, 53BP2 (ASPP2) is a positive regulator of YAP, facilitating the dephosphorylation of YAP/TAZ. Specificity of the Invitrogen anti-53BP2 antibody has been demonstrated in IF/ICC using siRNA-mediated knockdown (Figure 2B).

WWC1 (KIBRA), a mechanical regulator that associates with tight junctions and other cell polarity complexes, can induce the phosphorylation of LATS1/2 [6]. The specificity of the Invitrogen anti-WWC1 antibody is shown in IF/ICC using siRNA-mediated knockdown (Figure 2B). The Hippo kinase cascade can also be initiated by TAO kinases, which phosphorylate MST1/2 [7]. TAO kinases, including TAO3 and JIK, are differentially expressed in androgen-dependent and -independent cell lines, a feature that was used to show the specificity of the Invitrogen anti-JIK antibody (Figure 2D).

References

1. Aylon Y, Michael D, Shmueli A et al. (2006) Genes Dev 20:2687–2700. 
2. Barron DA, Kagey JD (2014) Clin Transl Med 3:25. 
3. Royer C, Koch S, Qin X et al. (2014) PLoS One 9:e111384. 
4. Park JH, Shin JE, Park HW (2018) Mol Cells 41:83–92. 
5. Zhou Y, Huang T, Cheng AS et al. (2016) Int J Mol Sci 17:E138. 
6. Xiao L, Chen Y, Ji M et al. (2011) J Biol Chem 286:7788–7796. 
7. Boggiano JC, Vanderzalm PJ, Fehon RG (2011) Dev Cell 21:888–895.