Implication functional of hNek7 and its interacting proteins RGS2, alpha-tubulin, NEK9 and MNAT1 in mitosis and cytokinesis

• Grant number: 13/04386-0
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: July 01, 2013
• End date: October 31, 2013
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Jörg Kobarg
• Grantee: Edmarcia Elisa de Souza
• Supervisor: Stephen J. Doxsey
• Host Institution: Centro Nacional de Pesquisa em Energia e Materiais (CNPEM). Ministério da Ciência, Tecnologia e Inovação (Brasil). Campinas , SP, Brazil
• Institution abroad: University of Massachusetts Medical School (UMMS), United States
• Associated to the scholarship: 09/11912-5 - Expression of the human protein kinase Nek7 for structural and functional studies, BP.DR

Abstract

NIMA-related kinases (Neks) are among the largest mitotic regulators and its deregulation has been correlated strongly with the uncontrolled cell proliferation and the appearance of tumors. Human Nek7 is a regulator of cell division and plays important role in growth and survival of mammalian cell. We identified 25 interacting partners for Nek7 by yeast two-hybrid which belong to different biological processes, among them mitosis and cytokinesis which includes the proteins: MNAT1, ²-tubulin, regulator of G-protein signaling 2 and Nek9. We observed by in vitro phosphorylation that ²-tubulin, RGS2 and Nek9 are substrates of Nek7 assigning the role of Nek7 activity in maintaining of integrity of cell cycle related tasks. Furthermore, Nek7 knockdown stable selection experiments using shRNA showed the requirement of Nek7 for Nek9 and ²-tubulin accumulation in interphase. Using confocal microscopy, we observed that Nek7 colocalize with pericentrin at the spindle pole in prometaphase, with ²-tubulin at the spindle midzone in anaphase and at the cytoplasmic bridge in cytokinesis. ²-tubulin colocalize with Nek9 at the spindle pole in anaphase and cytoplasmic bridge in cytokinesis and Nek9 colocalize with pericentrin in prophase, prometaphase, anaphase and telophase, highlighting the Nek7 participation and its interacting proteins in important molecular processes throughout the different stages of cell cycle. However, given functions of MNAT1, ²-tubulin, RGS2 and Nek9 in the cell cycle, the question of how Nek7 can modulate and integrate the signaling pathways these proteins through the different stages of the cell cycle remains unanswered. This leads us to identify and characterize the molecular, cellular and functional mechanisms these proteins throughout the cell cycle, especially in mitosis and cytokinesis. (AU)