Phosphorylation state-specific antibodies: applications in investigative and diagnostic pathology

Abstract

Until recently, the investigation of protein phosphorylation was limited to biochemical studies of enzyme activities in homogenized tissues. The availability of hundreds of phosphorylation state-specific antibodies (PSSAs) now makes possible the study of protein phosphorylation in situ, and is opening many exciting opportunities in investigative and diagnostic pathology. This review illustrates the power of PSSAs, especially in immunohistochemical applications to human disease and animal models. Technical considerations, including antibody specificity and lability of phosphoepitopes, are covered, along with potential pitfalls, illustrated by a case study. In the arena of oncology, PSSAs may prove especially valuable in directly demonstrating the efficacy of chemotherapies targeted at protein kinase cascades. Novel applications of PSSAs are also beginning to reveal molecular mechanisms of inflammatory, degenerative, and toxin-induced diseases.

Figure 1.

Illustration of potential pitfalls and unexpected spatiotemporal complexity of protein phosphorylation using PSSAs. A–D: Artifactual loss of phospho-ERK immunoreactivity in large tissue specimens. Glioma specimens larger than 1 cm sometimes showed strong phospho-ERK staining in the peripheral few millimeters but not the core (A, anaplastic astrocytoma; C, glioblastoma multiforme). Immunoreactivity for a phosphorylation-independent ERK antibody was always preserved throughout the tumor specimen (B and D), suggesting that phosphorylated epitopes may be lost due to ongoing dephosphorylation in the core of large specimens due to slow fixation. Bar in A, 1 cm, applies to A–D. E–F: Comparison of phospho-MEK and phospho-ERK immunohistochemistry reveals unexpected complexity of component activation within a signaling cascade. A phospho-MEK antibody (Cell Signaling Technology) shows strong and selective staining of mitotic cells in a human glioblastoma (E, arrows). In contrast, phospho-ERK immunostaining of an adjacent section revealed cytoplasmic and nuclear staining in non-mitotic cells (F). These patterns were confirmed in several non-neoplastic tissues, in both human and rodent (J.W. Mandell, unpublished data). Antibody specificity was confirmed by both Western blot and peptide competition controls (data not shown). The apparent paradox was recently revealed to be due to a mitosis-specific cleavage of MEK which renders it highly phosphorylated in mitosis, but uncoupled to ERK activation. Bar in E, 50 μm, applies to E–F.