Cyclin D- and E-Dependent Kinases and the p57^KIP2 Inhibitor: Cooperative Interactions In Vivo

Abstract

This study examines in vivo the role and functional interrelationships of components regulating exit from the G₁ resting phase into the DNA synthetic (S) phase of the cell cycle. Our approach made use of several key experimental attributes of the developing mouse lens, namely its strong dependence on pRb in maintenance of the postmitotic state, the down-regulation of cyclins D and E and up-regulation of the p57^KIP2 inhibitor in the postmitotic lens fiber cell compartment, and the ability to target transgene expression to this compartment. These attributes provide an ideal in vivo context in which to examine the consequences of forced cyclin expression and/or of loss of p57^KIP2 inhibitor function in a cellular compartment that permits an accurate quantitation of cellular proliferation and apoptosis rates in situ. Here, we demonstrate that, despite substantial overlap in cyclin transgene expression levels, D-type and E cyclins exhibited clear functional differences in promoting entry into S phase. In general, forced expression of the D-type cyclins was more efficient than cyclin E in driving lens fiber cells into S phase. In the case of cyclins D1 and D2, ectopic proliferation required their enhanced nuclear localization through CDK4 coexpression. High nuclear levels of cyclin E and CDK2, while not sufficient to promote efficient exit from G₁, did act synergistically with ectopic cyclin D/CDK4. The functional differences between D-type and E cyclins was most evident in the p57^KIP2-deficient lens wherein cyclin D overexpression induced a rate of proliferation equivalent to that of the pRb null lens, while overexpression of cyclin E did not increase the rate of proliferation over that induced by the loss of p57^KIP2 function. These in vivo analyses provide strong biological support for the prevailing view that the antecedent actions of cyclin D/CDK4 act cooperatively with cyclin E/CDK2 and antagonistically with p57^KIP2 to regulate the G₁/S transition in a cell type highly dependent upon pRb.

ACKNOWLEDGMENTS

We thank Nicole Schreiber-Agus and Andrew Koff for critical reading of the manuscript. We also thank Sam Zigler and Joe Horwitz for antibodies against MIP26 and the crystallins; Paul Overbeek for the CPV-1 cassette; and the Analytical Imaging Facility at Albert Einstein College of Medicine.

This work was supported by the following NIH grants: RO1HD28317, RO1EY09300, RO1EY11267, and the Cancer Center Core grant T2P30CA13330; the DAMD Breast Cancer Grant (to S.J.E.); NIH training grant 2T32AG00194 (to E.G.L.); and NIH training grant T32GM07288 (to N.J.L.). R.A.D. is a recipient of the Irma T. Hirschl Award.