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Cell cycle-dependent phosphorylation of C/EBPβ mediates oncogenic cooperativity between C/EBPβ and H-RasV12

Shuman, Jon D. ; Sebastian, Thomas ; Kaldis, Philipp LU orcid ; Copeland, Terry D. ; Zhu, Songyun ; Smart, Robert C. and Johnson, Peter F. (2004) In Molecular and Cellular Biology 24(17). p.7380-7391
Abstract


CCAAT/enhancer binding protein β (C/EBPβ) is a widely expressed transcription factor whose activity is regulated by oncogenic Ha-Ras
V12
signaling. C/EBPβ 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/EBPβ 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... (More)


CCAAT/enhancer binding protein β (C/EBPβ) is a widely expressed transcription factor whose activity is regulated by oncogenic Ha-Ras
V12
signaling. C/EBPβ 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/EBPβ 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 mitogen-activated 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/EBPβ 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/EBPβ, 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/EBPβ as a nuclear effector of Ras signaling and transformation, and they indicate that cell cycle-dependent phosphorylation of C/EBPβ on Ser64 and Thr189 is required to promote Ras-induced transformation of NIH 3T3 cells.

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author
; ; ; ; ; and
publishing date
type
Contribution to journal
publication status
published
in
Molecular and Cellular Biology
volume
24
issue
17
pages
7380 - 7391
publisher
Taylor & Francis
external identifiers
  • pmid:15314150
  • scopus:4344714027
ISSN
0270-7306
DOI
10.1128/MCB.24.17.7380-7391.2004
language
English
LU publication?
no
id
15d28a1e-45e6-4f7e-88b6-406ffcea4662
date added to LUP
2019-09-18 14:28:46
date last changed
2025-10-14 13:08:57
@article{15d28a1e-45e6-4f7e-88b6-406ffcea4662,
  abstract     = {{<p><br>
                            CCAAT/enhancer binding protein β (C/EBPβ) is a widely expressed transcription factor whose activity is regulated by oncogenic Ha-Ras <br>
                            <sup>V12</sup><br>
                             signaling. C/EBPβ is essential for the development of mouse skin tumors containing Ras mutations and can cooperate with Ras<br>
                            <sup>V12</sup><br>
                             to transform NIH 3T3 cells. Here we have investigated Ras-induced phosphorylation of C/EBPβ 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 mitogen-activated 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/EBPβ activity, was also a substrate for Cdk phosphorylation. Ser64 and Thr189 phosphorylation was low in serum-starved (G<br>
                            <sub>0</sub><br>
                            ) cells but was strongly increased in mid-G <br>
                            <sub>1</sub><br>
                             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/EBPβ, which enhances transformation of NIH 3T3 cells, mutants bearing alanine substitutions at Ser64 and/or Thr189 inhibited Ras<br>
                            <sup>V12</sup><br>
                            -induced focus formation. Our findings support a role for C/EBPβ as a nuclear effector of Ras signaling and transformation, and they indicate that cell cycle-dependent phosphorylation of C/EBPβ on Ser64 and Thr189 is required to promote Ras-induced transformation of NIH 3T3 cells.<br>
                        </p>}},
  author       = {{Shuman, Jon D. and Sebastian, Thomas and Kaldis, Philipp and Copeland, Terry D. and Zhu, Songyun and Smart, Robert C. and Johnson, Peter F.}},
  issn         = {{0270-7306}},
  language     = {{eng}},
  month        = {{09}},
  number       = {{17}},
  pages        = {{7380--7391}},
  publisher    = {{Taylor & Francis}},
  series       = {{Molecular and Cellular Biology}},
  title        = {{Cell cycle-dependent phosphorylation of C/EBPβ mediates oncogenic cooperativity between C/EBPβ and H-Ras<sup>V12</sup>}},
  url          = {{http://dx.doi.org/10.1128/MCB.24.17.7380-7391.2004}},
  doi          = {{10.1128/MCB.24.17.7380-7391.2004}},
  volume       = {{24}},
  year         = {{2004}},
}