Isolation and characterization of PP2A holoenzymes containing FLAG-tagged B subunits.
Protein serine/threonine phosphatase 2A (PP2A) is a major cellular enzyme implicated in the control of a wide variety of biological processes. The predominant form of PP2A in cells is a heterotrimeric holoenzyme (ABC) consisting of a scaffolding (A) subunit, a regulatory (B) subunit, and a catalytic (C) subunit. Although numerous signal transduction pathways are known to be regulated by PP2A, the identity of the PP2A holoenzymes controlling each pathway remains unclear. Studies aimed at elucidating substrates for individual PP2A holoenzymes have been hindered by the limited availability of purified endogenous holoenzymes and the inability to differentiate cellular roles of closely related PP2A holoenzymes. In this chapter, we describe a strategy for the functional expression of select FLAG-tagged regulatory B subunits in human embryonic kidney-293 cells and subsequent purification of PP2A holoenzymes containing the FLAG-tagged B subunit and endogenous A and C subunits (ABFLAGC). Biochemical analyses of the purified ABFLAGC holoenzymes reveal that they exhibit virtually indistinguishable specific activities and sensitivities to inhibitors as compared to the corresponding endogenous PP2A holoenzymes. The strategy described herein provides a straightforward method to purify individual PP2A holoenzymes from target mammalian cells for subsequent in vitro studies, as well as a powerful approach to identify cellular substrates and roles for each holoenzyme.
Adams
Deanna G
DG
Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN, USA.
Wadzinski
Brian E
BE
D002460:Cell Line; D015870:Gene Expression; D020035:Holoenzymes / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D010749:Phosphoprotein Phosphatases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D054648:Protein Phosphatase 2; D021122:Protein Subunits / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D020035:Holoenzymes; D021122:Protein Subunits; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2