B cell receptor-associated protein alpha4 displays rapamycin-sensitive binding directly to the catalytic subunit of protein phosphatase 2A.
Recently, TAP42 was isolated as a high copy suppressor of sit4-, a yeast phosphatase related to protein phosphatase 2A (PP2A). TAP42 is related to the murine alpha4 protein, which was discovered independently by its association with Ig-alpha in the B cell receptor complex. Herein we show that a glutathione S-transferase (GST)-alpha4 fusion protein bound the catalytic subunit (C) of human PP2A from monomeric or multimeric preparations of PP2A in a "pull-down" assay. In an overlay assay, the GST-alpha4 protein bound to the phosphorylated and unphosphorylated forms of C that were separated in two-dimensional gels and immobilized on filters. The results show direct and exclusive binding of alpha4 to C. This is unusual because all known regulatory B subunits, or tumor virus antigens, bind stably only to the AC dimer of PP2A. The alpha4-C form of PP2A had an increased activity ratio compared with the AC form of PP2A when myelin basic protein phosphorylated by mitogen-activated protein kinase and phosphorylase a were used as substrates. Recombinant alpha4 cleaved from GST was phosphorylated by p56(lck) tyrosine kinase and protein kinase C. A FLAG-tagged alpha4 expressed in COS7 cells was recovered as a protein containing phosphoserine and coimmunoprecipitated with the C but not the A subunit of PP2A. Treatment of cells with rapamycin prevented the association of PP2A with FLAG-alpha4. The results reveal a novel heterodimer alpha4-C form of PP2A that may be involved in rapamycin-sensitive signaling pathways in mammalian cells.
Murata
K
K
Center for Cell Signaling, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA.
Wu
J
J
Brautigan
D L
DL
D000818:Animals; D019556:COS Cells; D002384:Catalysis; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D051379:Mice; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D011090:Polyenes / Q000378:metabolism*; D054648:Protein Phosphatase 2; D011947:Receptors, Antigen, B-Cell / Q000378:metabolism*; D020123:Sirolimus; D013379:Substrate Specificity
D011090:Polyenes; D011947:Receptors, Antigen, B-Cell; D005982:Glutathione Transferase; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D020123:Sirolimus