Identification, cloning and characterization of proteins associated with the Ba regulatory subunit of protein phosphatase 2A

Protein phosphatase 2A (PP2A) is a highly conserved serinehreonine phosphatase involved in the regulation of many cellular processes including signal transduction pathways and the cell cycle. PP2A in its native form consists of a heterodimer between a catalytic 36 kDa subunit (termed C) and a constant regulatory 63 kDa subunit (termed A), which is usually further complexed with one of several additional regulatory subunits (termed B, B` or B``). The interaction with the regulatory subunits modulates the substrate specificity and activity of the C subunit. For example, the regulatory B-alpha subunit greatly increases PP2A activity towards cdc2-phosphorylated histone H1, a substrate that is widely used in in vitro phosphatase assays but which most likely is not an in vivo substrate of PP2A. However, the involvement of the B subunit in a wide range of cellular processes suggests that B subunit carries out its function by directing the AC dimer to different substrates. For instance, the Drosophila mutants aar (abnormal anaphase resolution) and twins which carry mutant alleles of the Drosophila B subunit exhibited mitotic defects. Furthermore, the deletion of the Saccharomyces cerevisiae homologue of the B subunit resulted in aberrant bud morphologies, defects in cell septation/separation and a- defective spindle-assembly checkpoint. Possible substrates affected by the absence of the B subunit have not been identified as yet. In mammalian cells the loss of function of the spindle-assembly checkpoint has been found to be associated with chromosomal instability (CIN). CIN leads to abnormal chromosome numbers (aneuploidy), a characteristic- of cancer cells. The role and function of the B-subunit in the execution of the mitotic checkpoint is poorly understood primarily because the intracellular targets/substrates of a B subunit-containing PP2A complex are unknown. Therefore, the major goal of the proposed project is to identify and clone B subunit-associated proteins. Further characterization of these proteins will show whether these proteins are targets/substrates or regulators of PP2A and B subunit. The proposed project will be a step forward towards the identification of the intracellular targets of PP2A and towards the elucidation of the role and function of mammalian B subunit.

Malfunctions of protein phosphatase 2A (PP2A), a highly conserved protein-serinehreonine phosphatase (PSTPase), seem to play a role in the genesis of human diseases from cancer to Alzheimer disease. PP2A holoenzymes consist of a catalytic C subunit associated with a scaffolding A subunit and one of several regulatory B-type subunits that recruit the AC dimer to different substrates. The major goal of the project was to identify and clone novel proteins, which are targeted substrates of Ba/PR55, one of the PP2A regulatory subunits. We isolated in a two-hybrid screen with the regulatory Ba/PR55 subunit a protein, which we named hHCP-6 based on its homology to C.elegans HCP-6 and which is distantly related to the condensin subunit hCAP-D2. Condensin is a protein complex that is required for the compaction and segregation of chromosomes in mitosis. We were able to show that hHCP-6 is part of a novel condensin-related complex that associates with chromosomes in mitosis and is dephosphorylated by PP2A in a Ba/PR55 specific manner. Interestingly, genetic evidence had pointed already to possible roles of Ba/PR55 in chromosome segregation. For example, deletion of CDC55, the budding yeast homologue of Ba/PR55, causes a defective spindle assembly checkpoint, as these yeast mutants proceed with cell division in the absence of intact spindles - with lethal consequences; a phenotype also observed for yeast strains lacking RRD1 and 2, the yeast homologues of PTPA (phosphotyrosyl phosphatase activator), a protein of so far unknown intracellular function. We were able to show that the underlying defect in the RRD deletion strains is not caused by the lack of Ba/PR55-holoenzymes but rather by the inability to generate an active and specific catalytic PP2A subunit. Thus PTPA/RRD appears to fulfil an essential function in the biogenesis of PP2A. During the course of the project we generated antibodies against PP2A subunits and PP2A-associated proteins, which were necessary for the realization of the project. I concluded material license agreements with two companies for the commercial sale of these antibodies. Thus the project was scientifically as well as commercially successful. At present our work contributes "only" to basic science but at the same time it may serve as starting point for the development of therapeutic concepts for treatment of diseases caused by the malfunction of PP2A.