Poly(A) Polymerase Phosphorylation Is Dependent on Novel Interactions with Cyclins

Abstract

We have previously shown that poly(A) polymerase (PAP) is negatively regulated by cyclin B-cdc2 kinase hyperphosphorylation in the M phase of the cell cycle. Here we show that cyclin B₁binds PAP directly, and we demonstrate further that this interaction is mediated by a stretch of amino acids in PAP with homology to the cyclin recognition motif (CRM), a sequence previously shown in several cell cycle regulators to target specifically G₁-phase-type cyclins. We find that PAP interacts with not only G₁- but also G₂-type cyclins via the CRM and is a substrate for phosphorylation by both types of cyclin-cdk pairs. PAP's CRM shows novel, concentration-dependent effects when introduced as an 8-mer peptide into binding and kinase assays. While higher concentrations of PAP's CRM block PAP-cyclin binding and phosphorylation, lower concentrations induce dramatic stimulation of both activities. Our data not only support the notion that PAP is directly regulated by cyclin-dependent kinases throughout the cell cycle but also introduce a novel type of CRM that functionally interacts with both G₁- and G₂-type cyclins in an unexpected way.

ACKNOWLEDGMENTS

We thank B. Dynlacht for the bacluloviruses expressing the GST-cyclins, W. Zhao for the vector expressing PAP I, D. F. Colgan for the baculovirus expressing PAP I, K. G. K. Murthy for the bacterial-purified PAP I, and E. Freulich for expert help with baculoviruses. We thank C. Cain, D. F. Colgan, E. Freulich, C. Gaiddon, F. Kleiman, K. G. K. Murthy, L. Ko, Y. Takagaki, and N. Baptiste for helpful discussions.

This work was supported by grants from the NIH to C.P. and J.L.M.