The Study Of The Interaction Between MEKK3 And NudC

NudC,a protein product of Nuclear distribution gene C(NudC),is highly conserved from fungus to human.NudC plays important roles in several biological processes including cell proliferation,hematogenesis and tumorigenesis.Human NudC cDNA encodes a 331-amino-acid-residue protein.The function of NudC is highly regulated by other proteins,such as Polo-like kinase 1(Plk1)that is essential for mitotic progression.Plk1 can phosphorylate NudC and has a role in mitosis.Using Co-immunoprecipitation and LC-MS/MS,we found that a number of proteins might have physical interaction with NudC,including MEKK3(MAPK/ERK kinase kinase 3,also mitogen-activated protein kinase kinase kinase 3).MEKK3 is a serine/threonine protein kinase and participates in the MAPK(mitogen-activated protein kinase)cascade.MEKK3 is involved in regulation of cell cycle and has been implicated in tumorigenesis.The aim of our research is to confirm the interaction between MEKK3 and NudC and explore the function of this interaction.We used GST-Pull down assay and Co-immunoprecipitation to verify the interaction between MEKK3 and NudC and explored in vitro kinase assay to examine if NudC can be phosphorylated by MEKK3. We firstly cloned wild type MEKK3 cDNA from HEK293 cells using RT-PCR, and then inserted the fragment into into the vectors that contain FLAG tag (pCMV-tag-2c),or GFP-fusion protein(pEGFP-C1).We also constructed the kinase-dead MEKK3(FLAG-MEKK3 KD)by PCR.The coding region of NudC cDNA was also cloned and inserted into pCMV-tag-2c vector,pEGFP-C1 vector and pGEX-5X-1 with GST-fused protein.GST-NudC vector(pGEX-5X-1-NudC)was transformed into the DH5αstrain of E.coli and GST-NudC protein was purified by GST-beads.Then,we used GST-Pull down assay to verify the interaction between MEKK3 and NudC in vitro.Co-immunoprecipitation assay demonstrated the interaction between MEKK3 and NudC in vivo.In vitro kinase assay showed that wild type MEKK3(MEKK3 WT)phosphorylated NudC,whereas the kinase-dead MEKK3 failed to phosphorylate NudC.The phosphorylated signal of GST-NudC was stripped off when immunoprecitated complex was treated by CIP(Calf Intestinal Alkaline Phosphatase),further indicating that MEKK3 may phosphorylate NudC.Moreover, we found that overexpression of MEKK3 or NudC induced similar abnormal nuclei with "semi-lunar" or "kidney" shape.As NudC participates in many process such as cell division,our data suggest that the phosphorlation of NudC by MEKK3 may has a potential role in cell cycle regulation.