The Screening Of Cyclin D3 Associating Partners And Study Of The Interactions Between Cyclin D3 And ERK3 Or Nm23-H2

The cell cycle is an important mechanism by which cells divide and participate in the growth and development of organism. The cell cycle is divided into four distinct phases (G1, S, G2, and M). The progression of a cell through the cell cycle is promoted by CDKs (cyclin-dependent kinases), which are positively and negatively regulated by cyclins and CKIs (CDK inhibitors), respectively. Mammalian D-type cyclins (D1, D2 and D3), which bind to and activate their associated cyclin-dependent kinases CDK4 and CDK6, are rate-limiting controllers of G1 phase progression. These three D-type cyclins share high identity and possess similar function. However, the features of individual D-type cyclins are not fully redundant. Compared with cyclin D1 and D2, cyclin D3 also exhibits its unique properties in many other physiological processes, such as development, proliferation, differentiation, apoptosis and carcinogenesis, especially for some particular types of cells or tissues. To elucidate its unique properties, we preformed a yeast two-hybrid screen of human fetal liver cDNA library using the full-length human cyclin D3 as bait. Some positive clones, including ERK3 and nm23-H2, were isolated. In this study, we identify the interaction of cyclin D3 and ERK3 or nm23-H2 and suggest the functionally potential consequences of their physical interactions.Extracellular signal-regulated kinase 3 (ERK3), an atypical mitogen-activated protein kinase (MAPK), was first characterized by virtue of its homology to MAP kinase ERK1. Little is known about the regulation and functions of ERK3. In an agreement with what was previously observed, N-terminally GFP-tagged ERK3 (EGFP-ERK3) was expressed at high levels while fusion of GFP to C-terminus was insufficient to stabilize ERK3. The interaction between cyclin D3 and ERK3 was confirmed both in vitro and in vivo. GST pull down assays showed cyclin D3 interacted directly and specifically with ERK3, showing no significant binding to the other D-type cyclins. The binding of cyclin D3 and ERK3 was further confirmed in vivo by co-immunoprecipitation assay and Confocal microscopic analysis. Furthermore, we identified C-terminal extension of ERK3 as interaction region and also showed that the entire structure of cyclin D3 was required for its interaction withERK3. These findings further expand distinct roles of cyclin D3 and in addition, suggest the potential activity of ERK3 in cell proliferation.Nm23, also known as nucleoside diphosphate kinase (NDPK), was first identified as a putative tumor metastasis suppressor and were shown to be involved in a wide variety of cellular activities including proliferation, development, and differentiation, tumor metastasis and kinase signal transduction. Nm23-H2 (NDPKB) is one of the eight members of the human nm23 family. Nm23-H2 is able to activate transcription by binding to a nuclease-hypersensitive element in the c-myc promoter and also known as PuF. In the present study, we report the identification by yeast two-hybrid screening of Nm23-H2, as a new interaction partner of cyclin D3. Similar to ERK3, nm23-H2 associates specifically with cyclin D3. This direct interaction was confirmed in vitro, using GST pull-down assays. Coimmunoprecipitation analysis from Hela cell lysates demonstrates their interaction in vivo. Irnmunofluorescence microscopy in Hela cells reveals that Nm23H2 has a cytoplasmic and nuclear localization and show extensive co-localization with cyclin D3. Furthermore, cyclin D3 inhibits the transcriptional activity of the c-myc gene by nm23-H2. Taken together, these data provide a new clue to understand the distinct roles of Cyclin D3 as a transcriptional regulator and the mechanism of nm23 regulation and function.