Cell Cycle-Dependent Phosphorylation of C/EBPβ Mediates Oncogenic Cooperativity between C/EBPβ and H-Ras^V12

Abstract

CCAAT/enhancer binding protein ␤ (C/EBP␤) is a widely expressed transcription factor whose activity is regulated by oncogenic Ha-Ras V12 signaling. C/EBP␤ is essential for the development of mouse skin tumors containing Ras mutations and can cooperate with Ras V12 to transform NIH 3T3 cells. Here we have investigated Ras-induced phosphorylation of C/EBP␤ in fibroblasts and report a novel proline-directed phosphoacceptor site at Ser64 within the transactivation domain. Ser64 phosphorylation was induced by activated Ras and Raf but was not blocked by chemical inhibitors of MEK1/2, phosphatidylinositol 3-kinase, JNK, or p38 mitogenactivated protein kinases. Ser64 was efficiently phosphorylated in vitro by the cyclin-dependent kinases Cdk2 and Cdc2. Thr189, previously identified as an ERK1/2 phosphorylation site that regulates C/EBP␤ activity, was also a substrate for Cdk phosphorylation. Ser64 and Thr189 phosphorylation was low in serum-starved (G 0 ) cells but was strongly increased in mid-G 1 cells and in cells arrested in S or M phase. In addition, phosphorylation on both sites was blocked by treating cells with the Cdk inhibitor roscovitine. In contrast to wild-type C/EBP␤, which enhances transformation of NIH 3T3 cells, mutants bearing alanine substitutions at Ser64 and/or Thr189 inhibited Ras V12 -induced focus formation. Our findings support a role for C/EBP␤ as a nuclear effector of Ras signaling and transformation, and they indicate that cell cycle-dependent phosphorylation of C/EBP␤ on Ser64 and Thr189 is required to promote Ras-induced transformation of NIH 3T3 cells.

Role of cdk2 in the sequential phosphorylation/activation of C/EBPβ during adipocyte differentiation

“…Because cdk2 has other roles, it is possible that its inactivation could act at another point in the cell cycle and thereby affect differentiation. It is worthy of note that C/EBP␤ can also be phosphorylated by cdk2 at other sites, i.e., S 64 and T 189 , in another context, i.e., in Ras-transformed NIH cells (19).…”

“…Several findings suggested cdk2 as the candidate priming kinase for S phase progression. First, cdk2 is known to phosphorylate rat C/EBP␤ on Thr 189 [equivalent to Thr 188 in mouse C/EBP␤ (19)]. Furthermore, the expression of both cdk2 and cyclinA is coordinately activated, concomitant with the turnover of p27/kip (a cdk2/cyclinA inhibitor) during this time window of the differentiation program (1).…”

Conditional ablation of C/EBPβ demonstrates its keratinocyte-specific requirement for cell survival and mouse skin tumorigenesis

“…For example, C/EBPb can cooperate with Ras to transform NIH 3T3 cells in a phosphorylation-dependent manner (Zhu et al, 2002;Shuman et al, 2004). The C/EBPb gene expresses three different isoforms by virtue of alternative translation initiation or regulated proteolysis (Ramji and Foka, 2002).…”

Microarray and Quantitative PCR Analysis of Gene Expression Profiles in Response to Treatment with Tomato Leaf Extract in MCF-7 Breast Cancer Cells

“…The gene was up-regulated (15.47-fold by real-time PCR) after the first 1 hour of treatment and continued to increase (51.41-fold) for another 48 hours. Recent studies had reported that this gene is the CCAAT/enhancer-binding protein-beta in human (Cao, 1991;Szpirer et al, 1992), and it is a widely expressed transcription factor whose activity is regulated by oncogenic Ha-RasV12 signaling (Shuman et al 2004). Explaining it's response to TLE treatment at the first 1 hr of treatment.…”