REGULATION OF ID3 CELL-CYCLE FUNCTION BY CDK-2-DEPENDENT PHOSPHORYLATION

The functions of basic helix-loop-helix (bHLH) transcription factors i n activating differentiation-linked gene expression and in inducing G( 1) cell cycle arrest are negatively regulated by members of the Id fam ily of HLH proteins. These bHLH antagonists are induced during a mitog enic signalling response, and they function by sequestering their bHLH targets in inactive heterodimers that are unable to bind to specific gene regulatory (E box) sequences. Recently, cyclin E-Cdk2- and cyclin A-Cdk2-dependent phosphorylation of a single conserved serine residue (Ser4) in Id2 has been shown to occur during late G(1)-to-S phase tra nsition of the cell cycle, and this neutralizes the function of Id2 in abrogating E-box-dependent bHLH home-or heterodimer complex formation in vitro (E. Hara, M. Hall, and G. Peters, EMBO J. 16:332-342, 1997). We now show that an analogous cell-cycle-regulated phosphorylation of Id3 alters the specificity of Id3 for abrogating both E-box-dependent bHLH home-or heterodimer complex formation in vitro and E-box-depende nt reporter gene function in vivo. Furthermore, compared with wild-typ e Id3, an Id3 Asp5 mutant (mimicking phosphorylation) is unable to pro mote cell cycle S phase entry in transfected fibroblasts, whereas an I d3 Ala5 mutant (ablating phosphorylation) displays an activity signifi cantly greater than that of wild-type Id3 protein. Cdk2-dependent phos phorylation therefore provides a switch during late G(1)-to-S phase th at both nullifies an early G(1) cell cycle regulatory function of Id3 and modulates its target bHLH specificity. These data also demonstrate that the ability of Id3 to promote cell cycle S phase entry is not si mply a function of its ability to modulate bHLH heterodimer-dependent gene expression and establish a biologically important mechanism throu gh which Cdk2 and Id-bHLH functions are integrated iri the coordinatio n of cell proliferation and differentiation.