Proteomic identification of C/EBP-DBD multiprotein complex: JNK1 activates stem cell regulator C/EBPalpha by inhibiting its ubiquitination.
Functional inactivation of transcription factors in hematopoietic stem cell development is involved in the pathogenesis of acute myeloid leukemia (AML). Stem cell regulator C/enhancer binding protein (EBP)alpha is among such transcription factors known to be inactive in AML. This is either due to mutations or inhibition by protein-protein interactions. Here, we applied a mass spectrometry-based proteomic approach to systematically identify putative co-activator proteins interacting with the DNA-binding domain (DBD) of C/EBP transcription factors. In our proteomic screen, we identified c-Jun N-terminal kinase (JNK) 1 among others such as PAK6, MADP-1, calmodulin-like skin proteins and ZNF45 as proteins interacting with DBD of C/EBPs from nuclear extract of myelomonocytic U937 cells. We show that kinase JNK1 physically interacts with DBD of C/EBPalpha in vitro and in vivo. Furthermore, we show that active JNK1 inhibits ubiquitination of C/EBPalpha possibly by phosphorylating in its DBD. Consequently, JNK1 prolongs C/EBPalpha protein half-life leading to its enhanced transactivation and DNA-binding capacity. In certain AML patients, however, the JNK1 mRNA expression and its kinase activity is decreased which suggests a possible reason for C/EBPalpha inactivation in AML. Thus, we report the first proteomic screen of C/EBP-interacting proteins, which identifies JNK1 as positive regulator of C/EBPalpha.
Trivedi
A K
AK
Bone Marrow Transplantation Section, Department of Internal Medicine IV, State Center for Cell and Gene Therapy, Martin-Luther-University Halle-Wittenberg, Halle, SA, Germany.
Bararia
D
D
Christopeit
M
M
Peerzada
A A
AA
Singh
S M
SM
Kieser
A
A
Hiddemann
W
W
Behre
H M
HM
Behre
G
G
D001483:Base Sequence; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000378:metabolism*; D002460:Cell Line; D017931:DNA Primers; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D048055:Mitogen-Activated Protein Kinase 8 / Q000378:metabolism*; D010766:Phosphorylation; D020543:Proteome; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D025801:Ubiquitin / Q000037:antagonists & inhibitors* / Q000378:metabolism
D022763:CCAAT-Enhancer-Binding Protein-alpha; D017931:DNA Primers; D020543:Proteome; D025801:Ubiquitin; D048055:Mitogen-Activated Protein Kinase 8