A structural and functional analysis of cyclin interactions with the retinoblastoma protein family member P130

Abstract

<p>pRb, p107 and p130 are structurally and functionally related polypeptides which comprise the retinoblastoma family of proteins. All three proteins are found in complexes with several cell cycle-regulating proteins, including cyclins and cyclin-dependent kinases (cdk's) which are thought to regulate the function of the pRb family members through phosphorylation. In vivo, p130 is observed in cyclin A/cdk2 and cyclin E/cdk2 complexes but not in complexes containing D-type cyclins and cdk4. This thesis examines these observations by identifying regions within the p130 sequence required for cyclin interactions. In vitro binding studies determined that D-type cyclin interactions require the majority of the "pocket domain" of p130. These interactions are disrupted upon phosphorylation of p130 by the cyclin D-associated kinase cdk4. Additional in vitro binding studies determined that a short sequence within the "spacer region" of p130 is required for interactions with cyclins A and E. This sequence contains an "RRL" motif which is present in several other cyclin A and cyclin E binding proteins. In vivo, the amino terminus of p130 is required to stabilize p130 interactions with cyclins A and E and this may be a result of inhibition of cdk2-associated kinase activity. Taken together, these results suggest that stable complexes containing p130 and cyclins A and E are not disrupted by phosphorylation of p130, perhaps because p130 inhibits the kinase activity of cdk2. In contrast, p130 interactions with D-type cyclins are disrupted in vivo because of cdk4-mediated phosphorylation of p130. This analysis concludes that D-type cyclin interactions with p130 are structurally and functionally distinct from interactions between p130 and cyclins A and E and these differences may be important in the regulation of p130 during the cell cycle.</p>