DEREGULATION OF POLY(A) POLYMERASE INTERFERES WITH CELL-GROWTH

Vertebrate poly(A) polymerase (PAP) contains a catalytic domain and a C-terminal Ser-Thr-rich regulatory region. Consensus and nonconsensus cyclin-dependent kinase (cdk) sites are conserved in the Ser-Thr-rich region in vertebrate PAPs. PAP is phosphorylated by cdc2-cyclin B on t hese sites in vitro and in vivo and is inactivated by hyperphosphoryla tion in hi-phase cells, when cdc2-cyclin B is active. In the experimen ts described here, we undertook a genetic approach in chicken DT40 cel ls to study the function of PAP phosphorylation. We found that PAP is highly conserved in chicken and is essential in DT40 cells. While cell s could tolerate reduced levels of PAP, even modest overexpression of either wild-type PAP or a mutant PAP with two consensus cdk sites muta ted (cdk(-) PAP) was highly deleterious and at a minimum resulted in r educed growth rates. Importantly, cells that expressed cdk(-) PAP had a significantly lower growth rate than did cells that expressed simila r levels of wild-type PAP, which was reflected in increased accumulati on of cells in the G(0)-G(1) phase of the cell cycle. We propose that the lower growth rate is due to the failure of hyperphosphorylation an d thus M-phase inactivation of cdk(-) PAP.