||Protein phosphatase 2A (PP2A) is a major intracellular serine/threonine phosphatase that has been implicated in the regulation of the cell cycle, DNA replication, cell metabolism, and signal transduction. PP2A exists as a heterotrimeric complex whose regulatory subunits determine the substrate specificity of the PP2A by directly affecting its activity and by targeting the enzyme to specific intracellular locations. This dissertation describes the cloning and characterization of human B56, a novel family of PP2A regulatory subunits. A family of five cDNAs, B56alpha-epsilon, coding for 70% identical proteins were isolated by two-hybrid interaction using the A subunit of PP2A as bait. B56 co-immunoprecipitates with the A and C subunits of PP2A along with a okadaic acid inhibitable, heparin stimulated phosphatase activity. In addition, B56 co-sediments in a glycerol gradient with the A and C subunits of PP2A as part of a 160 kDa complex. The majority of B56 proteins are phophoproteins, the exception being B56gamma1. Phosphoamino acid analysis indicates that B56 phosphorylation is on serine only. Indirect immunofluorescence microscopy demonstrated that B56 family members differentially target PP2A to different intracellular locations. B56gamma1, B56gamma3, and B56delta all target PP2A to the nucleus, and B56alpha, B56beta, and B56epsilon target PP2a to the cytoplasm. The B56 family members are expressed in a tissue specific manner, B56alpha and B56gamma mRNAs are highly expressed in muscle and heart, whereas B56beta and B56delta are highly expressed in brain. A number of experiments have indicated that PP2A-B56 may have an inhibitory effect on cell growth. First, overexpression of B56 in tissue culture cells blocked the establishment of stable cell lines. Second, transfection of B56 expression constructs inhibited expression from growth related promoters fos and AP-1 in 3T3 cells. Finally, B56beta and B56delta expression increased as neuroblastoma cells were forced to differentiate into neuron-like cells with retinoic acid. The biological significance and specific substrates that PP2A-B56 effect remain to be elucidated.