REGULATION OF PROTEIN PHOSPHATASE 2A ACTIVITY BY CASPASE-3 DURING APOPTOSIS

Although the available evidence suggests that whereas the caspase fami ly plays a major role in apoptosis, they are not the sole stimulators of death. A random yeast two-hybrid screen of a lymphocyte cDNA librar y (using caspase-3 as the bait) found an interaction between caspase-3 and the regulatory subunit A alpha of protein phosphatase 2A. This pr otein was found to be a substrate for caspase-3, but not caspase-1, an d could compete effectively against either a protein or synthetic pept ide substrate. In Jurkat cells induced to undergo apoptosis with anti- Fas antibody, protein phosphatase 2A (PP2A) activity increased 4.5-fol d after 6 h. By 12 h, the regulatory A alpha subunit could no longer b e detected in cell lysates, There was no change in the amount of the c atalytic subunit, The effects on PP2A could be prevented by the caspas e family inhibitors acetyl-Asp-Glu-Val-Asp (DEVD) aldehyde or Ac-DEVD fluoromethyl ketone. The mitogen-activated protein (MAP) kinase pathwa y is regulated by PP2A. At 12 h after the addition of anti-Fas antibod y, a decrease in the amount of the phosphorylated forms of MAP kinase was observed. Again, this loss of activated MAP kinase could be preven ted by the addition of DEVD-cho or DEVD-fmk, These data are consistent with a pathway whereby induction of apoptosis activates caspase-3, Th is enzyme then cleaves the regulatory A alpha subunit of PP2A, increas ing its activity. These data show that the activated PP2A will then ef fect a change in the phosphorylation state of the cell. These data pro vide a link between the caspases and signal transduction pathways.