CRP2 (C/EBP beta) contains a bipartite regulatory domain that controls transcriptional activation, DNA binding and cell specificity.

Two members of the C/EBP family of basic region-leucine zipper proteins enriched in the liver, C/EBP (C/EBP alpha) and CRP2 (C/EBP beta), were previously shown to transactivate the albumin promoter in a cell type-dependent manner. These proteins function efficiently in HepG2 hepatoma cells, but inefficiently in HeLa (epithelial) and L (fibroblastic) cells. Here we have investigated the mechanism for cell-specific control of CRP2 activity. We show that CRP2 contains a negative regulatory region composed of two elements, RD1 and RD2. Deletions of RD2 relieve the inhibition of CRP2 activity in L cells, but do not affect CRP2 function in HepG2 cells. These deletions also increase the DNA binding activity of CRP2 approximately 3-fold, suggesting that RD2-mediated repression of DNA binding activity is responsible for CRP2 inhibition in L cells. The adjacent RD1 element functions independently of RD2 and modulates the CRP2 activation domain, which we show to be composed of three subdomains that are conserved within the C/EBP protein family. RD1 does not affect cell type specificity, but inhibits the transactivation potential of GAL4-CRP2 hybrid proteins by 50-fold. These findings suggest that CRP2 assumes a tightly folded conformation in which the DNA binding and activation domains are masked by interactions with the regulatory domain and that to function efficiently in HepG2 cells the protein must undergo an activation step. We propose that relief of inhibition conferred by the regulatory domains also accounts for CRP2 activation in response to extracellular signals.     

Chk2 activation and phosphorylation-dependent oligomerization

The tumor suppressor gene CHK2 encodes a versatile effector serine/threonine kinase involved in responses to DNA damage. Chk2 has an amino-terminal SQ/TQ cluster domain (SCD), followed by a forkhead-associated (FHA) domain and a carboxyl-terminal kinase catalytic domain. Mutations in the SCD or FHA domain impair Chk2 checkpoint function. We show here that autophosphorylation of Chk2 produced in a cell-free system requires trans phosphorylation by a wortmannin-sensitive kinase, probably ATM or ATR. Both SQ/TQ sites and non-SQ/TQ sites within the Chk2 SCD can be phosphorylated by active Chk2. Amino acid substitutions in the SCD and the FHA domain impair auto- and trans-kinase activities of Chk2. Chk2 forms oligomers that minimally require the FHA domain of one Chk2 molecule and the SCD within another Chk2 molecule. Chk2 oligomerization in vivo increases after DNA damage, and when damage is induced by gamma irradiation, this increase requires ATM. Chk2 oligomerization is phosphorylation dependent and can occur in the absence of other eukaryotic proteins. Chk2 can cross-phosphorylate another Chk2 molecule in an oligomeric complex. Induced oligomerization of a Chk2 chimera in vivo concomitant with limited DNA damage augments Chk2 kinase activity. These results suggest that Chk2 oligomerization regulates Chk2 activation, signal amplification, and transduction in DNA damage checkpoint pathways.     

Cell cycle regulation of the c-Myc transcriptional activation domain.

The product of the c-myc gene (c-Myc) is a sequence-specific DNA-binding protein that has previously been demonstrated to be required for cell cycle progression. Here we report that the c-Myc DNA binding site confers cell cycle regulation to a reporter gene in Chinese hamster ovary cells. The observed transactivation was biphasic with a small increase in G1 and a marked increase during the S-to-G2/M transition of the cell cycle. This cell cycle regulation of transactivation potential is accounted for, in part, by regulatory phosphorylation of the c-Myc transactivation domain. Together, these data demonstrate that c-Myc may have an important role in the progression of cells through both the G1 and G2 phases of the cell cycle.