Mutagenesis and expression of the scaffolding Aalpha and Abeta subunits of PP2A: assays for measuring defects in binding of cancer-related Aalpha and Abeta mutants to the regulatory B and catalytic C subunits.

Protein phosphatase 2A (PP2A) holoenzymes are composed of three subunits: one scaffolding A subunit, one regulatory B subunit, and one catalytic C subunit. The A subunit exists as two isoforms: Aalpha and Abeta. The C subunit also exists as two isoforms (Calpha and Cbeta) and B subunits fall into three families (B, B', and B") comprising over 15 members. The Aalpha and Abeta subunits consist of 15 nonidentical repeats, which are composed of two amphipathic alpha helices that are connected by a loop (intrarepeat loop). These loops are instrumental in binding regulatory B and catalytic C subunits. The genes encoding the Aalpha and Abeta subunits are relatively frequent targets for mutation in human cancer. The mutations often affect the intrarepeat loops and cause defects in the binding of specific B subunits or of B and C subunits. Here, we describe in vitro and in vivo binding assays for measuring these defects. Knowing which B subunits are affected in binding to the mutant A subunits sheds light on which holoenzymes might be involved in growth control and cancer.