PROTEIN PHOSPHATASE 2A SUBUNIT ASSEMBLY - THE CATALYTIC SUBUNIT CARBOXY-TERMINUS IS IMPORTANT FOR BINDING CELLULAR B-SUBUNIT BUT NOT POLYOMAVIRUS MIDDLE TUMOR-ANTIGEN

The carboxy terminus of protein phosphatase 2A (PP2A) catalytic subuni t is highly conserved. Seven out of the last nine residues, including two potential in vivo phosphorylation sites, threonine 304 and tyrosin e 307, are completely invariant in all known PP2As, Mutational analysi s of the carboxy terminus in vivo was facilitated by efficient immunop recipitation of trimeric PP2A holoenzyme via an epitope-tagged catalyt ic subunit, The results indicate that the catalytic submit carboxy ter minus is important for complex formation with the PP2A 55 kDa regulato ry B subunit, but not with polyomavirus oncogene, middle tumor antigen (MT), a viral B-type regulatory subunit, Replacing catalytic subunit threonine 304 or tyrosine 307 with a negatively charged amino acid abo lished binding of the B subunit to the dimeric enzyme core and altered substrate specificity, Certain other amino acid substitutions of diff erent size and/or charge also abolished or greatly reduced B subunit b inding, Substitution of alanine at position 304 or phenylalanine at po sition 307 did not dramatically reduce B subunit binding or phosphatas e activity in vitro, yet the latter substitutions are not found in nat urally occurring PP2As, Thus, the wild-type residues are important for a yet unknown function in vivo, Additionally, deleting the carboxy te rminal nine amino acids inhibited binding of the B subunit to the dime ric enzyme core, indicating a requirement for one or more of these ami no acids for complex formation, MT interaction with the dimeric PP2A e nzyme core was not inhibited by any of these mutations, Finally, unlik e B subunit, MT does not activate the phosphatase activity of the PP2A heterodimer towards cdc2-phosphorylated histone HI.