THE HELIX-LOOP-HELIX PROTEIN ID-2 ENHANCES CELL-PROLIFERATION AND BINDS TO THE RETINOBLASTOMA PROTEIN

Cell growth and differentiation are usually antagonistic. Proteins of the basic helix-loop-helix (bHLH) family bind DNA and play important r oles in the differentiation of specific cell types. Id proteins hetero dimerize with bHLH transcription factors, blocking their activation of lineage-specific gene expression and thereby inhibiting cellular diff erentiation. To examine the effect of Id-2 on cell proliferation, we o verexpressed Id-2 in the human osteosarcoma cell line U2OS. Id-2 expre ssion in U2OS reduced the serum requirement for growth and stimulated cellular proliferation by shortening the doubling time and increasing the percentage of cells in S phase. We demonstrated that Id-2 expressi on was able to reverse the inhibition of cellular proliferation and th e block in cell cycle progression mediated by the product of the retin oblastoma tumor suppressor gene pRB. This effect was not associated wi th changes in the state of pRb phosphorylation in transfected cells. I n vitro, unphosphorylated pRb from cell lysates specifically bound Id- 2 but was not able to bind a mutated form of Id-2 lacking the HLH doma in that also did not antagonize the growth arrest by pRb. In vitro-syn thesized pRb containing mutations within the E1A/large T-binding pocke t did not bind Id-2. However, wild-type pRb was able to bind to a regi on of Id-2 corresponding to only the HLH domain. In vivo, a physical a ssociation between Id-2 and pRb was seen in cross-linked extracts from SAOS-2 cells transfected with Id-2 and pRb. Our data identify a role for Id-2 in the regulation of cellular proliferation and suggest that the interaction between Id-2 and pRb is a molecular pathway over which synchronous changes in growth and differentiation are mediated in viv o.