Proteomic identification of C/EBPa multiprotein complex reveals that JNK1, an activator of C/EBPa is downregulated in patients with acute myeloid leukemia (AML)

Functional inactivation of the transcription factor CAAT Enhancer
Binding Protein Alpha (C/EBPα) either by mutation or direct
protein-protein interaction leads to acute myeloid leukemia (AML),
whereas the activation of C/EBPα restores normal myeloid cell
differentiation. We and others have shown that protein-protein
interactions of C/EBPα play a pivotal role in myeloid
differentiation and AML. In the present study we applied proteomics
based mass spectrometry to identify C/EBPα interacting proteins on
a proteome-wide scale. For this, the GST and GST-DNA binding domain
of C/EBP (GST-DBD) was incubated with nuclear extracts of U937
cells. Interacting proteins separated by two-dimensional gel
electrophoresis were identified by MALDI-TOF mass spectrometry.
Using this approach we could identify PAK6, MADP-1, ZNF45 and the
c-Jun N-terminal kinase 1 (JNK1) as C/EBPα interacting proteins.
Since JNK1 activates c-Jun, the contra-player for C/EBPα, we
hypothesized that the JNK1 and C/EBPα interaction might have some
biological relevance. We could show that JNK1 binds to the DNA
binding domain of C/EBP in GST-pull-down and to the C/EBPα in
co-immunoprecipitation assays in-vitro and in-vivo respectively.
JNK1 phosphorylates and increases the half life of the C/EBPα
protein via inhibiting its ubiquitination and thereby enhances its
transactivation and DNA binding activity. Furthermore, JNK mRNA
expression as well as its kinase activity is lower in the AML
patients in comparison to normal bone marrow mononuclear cells
which implicates a possible mechanism of C/EBPα inactivation in
certain acute myeloid leukemia subtypes. Thus our data suggest that
a JNK activity is required for C/EBPα activation in myeloid cells
and a loss of JNK regulated C/EBPα expression may contribute to
leukemogenesis.