Immunohistochemical assessment of protein phosphorylation state: the dream and the reality

Abstract

The development of phosphorylation state-specific antibodies (PSSAs) in the 1980s, and their subsequent proliferation promised to enable in situ analysis of the activation states of complex intracellular signaling networks. The extent to which this promise has been fulfilled is the topic of this review. I review some applications of PSSAs primarily in the assessment of solid tumor signaling pathway activation status. PSSAs have received considerable attention for their potential to reveal cell type-specific activation status, provide added prognostic information, aid in the prediction of response to therapy, and most recently, demonstrate the efficacy of kinase-targeted chemotherapies. However, despite some successes, many studies have failed to demonstrate added value of PSSAs over general antibody immunohistochemistry. Moreover, there is still a large degree of uncertainty about the interpretation of complex and heterogeneous staining patterns in tissue samples and their relationship to the actual phosphorylation states in vivo. The next phase of translational research in applications of PSSAs will entail the hard work of antibody validation, gathering of detailed information about epitope-specific lability, and implementation of methods for standardization.

Fig. 1

Continuous growth of publications with immunohistochemical applications of PSSAs. A Google Scholar Advanced search (full text search) using the Boolean search: {(“phosphospecific” OR “phospho-specific” OR “phosphorylation-specific” OR “phosphorylation state-specific”) AND “immunohistochemistry”} revealed a continuous increase in the annual number of publications including these key phrases in the full text content. The apparent slight decline in 2007 is likely due to the restricted full text access of many journals in the first year after publication

Fig. 2

An example of a panel of phosphorylation state-specific antibodies applied to a human glioblastoma multiforme. a Overexpression of total EGFR is apparent [(compared to little or no signal in adjacent nonneoplastic brain, (not shown)]. In addition, strong tyrosine phosphorylation of the receptor was found at Tyr1173 (b) but only focal weak phosphorylation at Tyr1068 (c). Downstream intracellular kinases ERK (d) and mTOR (e) show strong cytoplasmic and phosphorylation in a subset of tumor cells, whereas the transcription factor CREB (f) is strongly phosphorylated in the nuclei of most tumor cells. Phospho-Histone H3 (g) is a marker of all cells in mitosis. Phosphorylation of the actin-associated proteins Ezrin/Radixin/Moesin, potential mediators of tumor invasiveness, is strong in all cells (h). Finally, a generic anti-phospho-tyrosine antibody reveals the general preservation of tyrosine phosphorylation in this tumor section

Fig. 3

Ex Vivo phosphoepitope lability: preliminary comparison of phospho-NF-kappaB (Ser276) and phospho-CREB (Ser133). To model the potential post-surgical loss of phosphoepitopes, tumor xenografts were grown in nude mice, harvested, and fixed either immediately (a, c), or 4 h later (b, d)