Modulation of Id function during the cell cycle by Cdk-dependent phosphorylation

Abstract

The Id family (Idl-Id4) of helix-loop-helix proteins act as negative regulators of basic-helix-loop-helix (bHLH) transcription factors that drive cell lineage commitment and differentiation in diverse cell types. In general, tissue-specific bHLH proteins form heterodimers with the ubiquitously expressed bHLH proteins and activate the expression of specific genes carrying an E-box regulatory motif. Id proteins lack a basic region and act as dominant-negative antagonists of the bHLH transcription factors by forming heterodimers that are unable to bind DNA. We have previously shown that Id2 and Id3 undergo cell cycle dependent phosphorylation at a serine residue (Ser5) within a consensus target site for cyclin-dependent kinases (EMBO J. 1 6 332-342,1997). The timing of phosphorylation in the late G1 phase correlates with the activation of Cdk2 by cyclin E and/or cyclin A. We now show that Id4 can also be specifically phosphorylated by cyclin E-Cdk2 and cyclin A-Cdk2 in vitro. Analogous phosphorylation occurs in serum-stimulated F9 embryonal teratocarcinoma cells at a time in late G1 consistent with the appearance of active cyclin E-Cdk2. Phospho-mimicking (S5D) and phospho-ablating (S5A) mutants of Id4 have been constructed and are currently being evaluated in a model E-box band-shift assay and for functional properties in transfected cells. Our data suggest that modulation of Id function by Cdk-dependent phosphorylation could alter the patterns of bHLH-dependent gene expression during cell cycle progression. CLLThe transcription factors of the E2F (E2F-1 -E2F-6), cyklindependent-kinases (cdk) and cdk inhibitors play a key role in control of the cell cycle in eukaryotes. E2F proteins form heterodimers with the proteins, DP-I and DP-2. These complexes are regulated by the protein pRb, p107 and p130. E2F inhibitors are phosforylated during the cell cycle, releasing E2F/DP complexes. The phosphorylation is mediated by CyclinD/cdk4 or Cyklin D/cdk6 are mid G1 specific, Cyclin E/cdk2 late G1 specific and Cyklin A/cdk2 -S specific. The negative regulatory elements -p219(Cip), p27(Kip), p16(INK4A) block cdks activity. B-cell chronic lymphocytic leukaemia (B-CLL) is a disease with a low proliferative activity characterised by the progressive accumulation of clonal B lymphocytes in early phases (GO/Gl) of the cell cycle. Our goals were to study the mRNA expression of cell cycle proteins (E2F-1, E2F-4, DP1, DP2, cdks, p21, p27, p16), which primarily regulate the G1 phase of cell cycle, or Sphase entry, or both in B-CLL lymphocytes. We have examined mRNA expression in unstimulated and stimulated lymphocytes from 12 patients with B-CLL, used the ribonuclease protection assay. The leukaemic immunophenotype CD5/CD19 was demonstrate in more than 70% of lymphocytes in all studied cases. The constant mRNA expression E2F-4, E2F-1, DP2, cdk4 was shown in both unstimulated and stimulated lymphocytes. We did not find any mRNA expression p21, p27, p16 in patients whose stimulated PBL were in M phase of cell cycle.Objective We have previo...