| Two families of proteins play major roles in the regulation of the cell cycle. One family, the CDK, exerts control on downstream processes by phosphorylating selected proteins.The other family consists of specialized regulatory proteins, cyclins, that bind to cdk molecules and modulate their activity.As cells enter the cell cycle from quiescence, genes encoding D-type cyclins get activated at the beginning of the G1 phase. Cyclin D then forms an active kinase complex with the cyclin-dependent kinase CDK 4 or CDK 6, and the resulting kinase complexes are involved in phosphorylation of the proteins to regulate the downstream pathways.Cyclin D3 is found to play a crucial role not only in progression through the G1 phase as a regulatory subunit of CDK 4 and CDK 6, but also in many other aspects, such as cell growth, cell differentiation, transcriptional regulation and apoptosis. Our lab previously reported CDK11P58, a G2/M CDK, acts as a partner of cyclin D3 in G2/M phase. To further understand the cellular function in which cyclin D3 might be involved, we performed a yeast two-hybrid screening of a human fetal liver cDNA library using human cyclin D3 as bait and identified human eukaryotic initiation factor 3 p28 protein (eIF3k) as a partner of cyclin D3. The association of cyclin D3 with eIF3k was further confirmed by in vitro binding assay, in vivo coimmunoprecipitation, and confocal microscopic analysis. We found that cyclin D3 specifically interacted with eIF3k through its C-terminal domain while the other two D-type cyclins did not. When over-expressed, cyclin D3 affected eIF3k's cellular distribution leading it accumulated into the nuclear region from cytoplasm in Hela cells. Though eIF3k was observed to inhibit cellular protein synthesis at the translation level, over-expression of cyclin D3 abolished its role and up-regulated the cellular translation activity in Hela cells without affecting the mRNA levels. Taken together, these data provide a new clue to our understanding of the cellular function of cyclin D3 and eIF3k. A plethora of extracellular stimuli were transmitted into the nucleus through signal transduction pathway. The MAP kinase (mitogen activated protein kinase) pathway is at the heart of signal networks that govern cell proliferation, differentiation and apoptosis. The basic arrangement includes a three-member protein kinase cascade: MKKKs (mitogen activated protein kinase kinase kinases), MKKs (mitogen activated protein kinase kinases), and MAPKs. Ras/Raf/MEK/ERK pathway is the most elementary and well studied MAP kinase pathway that controlling the cell growth, include the regulation of cell mitosis, transformation, differentiation and apoptosis. Mammals possess three Raf proteins: A-Raf,B-Raf and c-Raf(Raf-1).The structure of the Raf kinases are highly conserved, but more and more researches indicated that their functions are not redundant, they have isozyme-specific functions.Our previous work used the yeast two-hybrid system to screen human fetal liver cDNA library for A-Raf's interaction proteins, and found one candidate protein, trihydrophobin 1(TH1), a putative new gene th1 product cloned in 2000.The function of TH1 is still unknown. TH1's interaction with A-Raf was confirmed with in vitro GST-pull down assay and their co-localization in the cells was detected using confocal fluorescence microscope. The interaction domain of TH1 was mapped to its N-domain (1-392aa) using deletion mutations. The results also showed that the TH1/A-Raf interaction was specific as no interaction between B-Raf, c-Raf and TH1 were found. It is also found that TH1 co-localizes with A-Raf not only in cytoplasm and membrane when examined by confocal microscopy,which suggested that TH1 might be a scaffold protein. These data provide a new clue to our understanding of the cellular function and mechanism of the association of TH1 with A-Raf.
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