Understanding Pseudophosphatase Function Through Biochemical Interactions.

Pseudophosphatases have been solidified as important signaling molecules that regulate signal transduction cascades. However, their mechanisms of action remain enigmatic. Reflecting this mystery, the prototypical pseudophosphatase STYX (phospho-serine-threonine/tyrosine-binding protein) was named with allusion to the river of the dead in Greek mythology to emphasize that these molecules are "dead" phosphatases. Although proteins with STYX domains do not catalyze dephosphorylation, this does not preclude their having other functions, including as integral elements of signaling networks. Thus, understanding their roles may mark them as potential novel drug targets. This chapter outlines common strategies used to characterize the functions of pseudophosphatases, using as an example MK-STYX [MAPK (mitogen-activated protein kinase) phospho-serine-threonine/tyrosine-binding], which has been linked to tumorigenesis, hepatocellular carcinoma, glioblastoma, apoptosis, and neuronal differentiation. We start with the importance of "restoring" (when possible) phosphatase activity in a pseudophosphatase, so the active mutant may be used as a comparison control throughout immunoprecipitation and mass spectrometry analyses. To this end, we provide protocols for site-directed mutagenesis, mammalian cell transfection, co-immunoprecipitation, phosphatase activity assays, and immunoblotting that we have used to investigate MK-STYX and the active mutant MK-STYX