Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a multifunctional protein known to be involved in the regulation of transcription, translation, nuclear transport, and signal transduction. To systematically obtain insight into mechanisms of hnRNP K activities, we set out to identify protein factors that interact with hnRNP K by using glutathione S-transferase-hnRNP K affinity chromatography followed by liquid chromatography/mass spectrometry/mass spectrometry analysis. Several partner proteins in the K562 cell lysates were identified through this method. One of them is a DEAD box-containing putative RNA helicase, DDX1. In vitro binding and co-immunoprecipitation studies confirmed the protein-protein interaction between hnRNP K with DDX1, and the region spanning amino acids 1-276 of hnRNP K is apparently responsible for its physical interaction with DDX1. Interestingly, their interaction was disrupted by the addition of poly(C), poly(A), and poly(U) RNA substrates. We found that DDX1 was a homopolymeric poly(A) RNA-binding protein. On the other hand, the ATPase activity of the purified recombinant DDX1 protein was stimulated by these homopolymeric RNAs and yeast total RNA but not by DNA. Moreover, the immunoprecipitated DDX1 complex but not purified DDX1 can unwind double-stranded RNA having singlestranded poly(A) overhangs.The hnRNP K 1 protein has been identified as a component of the heterogeneous nuclear ribonucleoprotein complexes. These hnRNP proteins bind pre-mRNAs directly and appear to facilitate various stages of mRNA biogenesis (1-3). hnRNP K can bind to RNA and single-stranded or double-stranded DNA. The nucleic acid binding activity of hnRNP K is mediated by three repeats of motifs termed the K homology domain, which consists of 65-70 residues (4). It contains both classical nuclear localization signal and the K nuclear shuttling domain that allow the protein to shuttle between nucleus and cytoplasm (5). hnRNP K appears to be involved in transcriptional regulation as exemplified by its binding to a C-rich sequence (the CT element) within the human c-myc promoter and subsequent activation of c-myc expression (6 -8). Additionally, its association with CCAAT/enhancer-binding protein ␤ results in the repression of CCAAT/enhancer-binding protein ␤ target genes (9). hnRNP K was also reported to bind 3Ј end of 15-lipoxygenase, and HPV 16 L2 mRNA was reported to trigger translation inhibition (10, 11). Moreover, direct protein-protein interactions between hnRNP K and some proto-oncogene products serve as clear evidence that hnRNP K acts as a docking platform to facilitate molecular interactions within signal transduction cascades (12-16).Modulation of RNA structure is an essential step in many fundamental cellular processes, including RNA synthesis, splicing, export, turnover, and translation. Recently, an increasing number of RNA helicases from different organisms, ranging from bacteria to yeast, human, and virus, has been identified (17, 18). DEAD box proteins are a family of putative RNA helicases t...

C/EBPbeta is one of the key transcription factors responsible for the induction of a wide array of genes. Like many proto-oncogenes and transcription factors, transcription of C/EBPbeta gene can be induced by multiple extracellular signals. Using nuclear extracts from lipopolysaccharide (LPS)-stimulated mouse liver, five trans-acting factor-binding motifs, URE1 (-376 to -352), URE2 (-253 to -223), URE3 (-220 to -190), URE4 (-123 to -103), and URE5 (-72 to -45) were identified by DNAse I footprinting assays. Competition and supershift analysis of the complexes formed at the URE2 and URE4 indicated that they contain CREB/ATF and AP-1 family factors. Furthermore, recombinant ATF2 and c-Jun proteins from mammalian and bacterial cells can bind to URE2 and URE4 but not URE1. Cotransfection experiments showed that ATF2 and c-Jun activate the C/EBPbeta gene expression cooperatively through URE2 and URE4, and this activation was greatly increased under the treatment of low concentration of anisomycin. During acute phase response, the phosphorylation of c-Jun and ATF2 was found to correlate with C/EBPbeta gene expression. Taken together, our results provide the evidences that both c-Jun and ATF2 are the regulators of C/EBPbeta gene.