Author

Susan E. Lacy

Date of Award

6-1997

Degree Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Medical Sciences

Supervisor

Peter F. M. Whyte

Abstract

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.


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