Although members of the NIMA-related kinase (NEK) family have been implicated extensively in human dis- eases including cancer and renal and cardiovascular defects, their physiological substrates are largely un- known. Moreover, little is known about the upstream and downstream components of NEK signaling pathways or about the proteins that modulate NEK activities. This has led to a fundamental gap in our knowledge concerning the molecular and cellular functions of this biomedically relevant and highly conserved family of protein kinases. The long-term goal is to characterize a recently discovered and potentially widespread function for NEKs in intracellular trafficking and extracellular matrix remodeling. The objective of this application isto characterize the trafficking functions of NEKL-2/NEK8 and NEKL-3/NEK6/7 during C. elegans development and in human cell culture systems. The central hypothesis is that NEKs regulate endocytosis and vesicle trafficking through interactions with components of the cytoskeleton including microtubule (MT)-associated proteins, molecular motors, and regulators of the acting network. Strong preliminary data support the two specific aims: 1) Elucidate the mechanisms by which NEKL-2 and NEKL-3 control trafficking; and 2) Identify molecular targets and components of the NEKL signaling pathway.
Under Aim 1, the localization of NEKLs to intracellular traf- ficking and cytoskeletal compartments will be characterized, and the specific cellular functions of NEKLs in regulating vesicular trafficking and cytoskeletal organization and dynamics will be determined. In addition, the hypothesis that ankyrin repeat (AR) binding partners of NEKLs act as signaling scaffolds will be tested. Finally, a panel of assays will be carried out to test the rles of NEK6/7, NEK8, and associated AR proteins in trafficking functions in mammalian cells.
Under Aim 2, proven RNAi-feeding screens will be carried out to identify components and candidate targets of the NEKL signaling networks. In addition, a recently developed powerful chemical genetic-proteomic strategy, which is well supported by preliminary data, will be used to directly identify in vivo substrates of the NEKLs. Follow-up studies will be carried out on select validated targets to deter- mine their functions in trafficking and cytoskeletal organization downstream of the NEKs. The approach is in- novative because other groups have not applied powerful forward genetic methods to study NEK kinases and because the function of NEKs in intracellular trafficking is entirely unknown. In addition the proposed chemical- genetic strategy to identify substrates has not been previously applied to C. elegans or to the study of NEKs in any system. The proposed research is significant because human NEKs are widely implicated in human dis- eases and have been suggested as drug targets, but their roles during interphase are not well understood nor, in most cases, are their physiological substrates known. As an additional benefit, these studies will lead to the characterization of previously unknown but conserved regulators of intracellular trafficking and extracellular matrix remodeling, both of which are highly relevant to human development, health, and disease.

Public Health Relevance

The proposed research is relevant to human health because the genes we are studying have been directly implicated in cancer, cardiovascular, and renal diseases. Findings obtained through these studies will lead to a better understanding of the roles these proteins play in human disease as well as their functions in basic biological processes that are relevant to human development. The proposed research is therefore relevant to the part of the NIH's mission that seeks to obtain fundamental biological knowledge and to reduce the burdens of human disease and disability.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM066868-10A1
Application #
9106074
Study Section
Development - 1 Study Section (DEV1)
Program Officer
Melillo, Amanda A
Project Start
2004-08-01
Project End
2020-02-29
Budget Start
2016-05-20
Budget End
2017-02-28
Support Year
10
Fiscal Year
2016
Total Cost
Indirect Cost
Name
University of Wyoming
Department
Biochemistry
Type
Earth Sciences/Resources
DUNS #
069690956
City
Laramie
State
WY
Country
United States
Zip Code
82071
Lažeti?, Vladimir; Joseph, Braveen B; Bernazzani, Sarina M et al. (2018) Actin organization and endocytic trafficking are controlled by a network linking NIMA-related kinases to the CDC-42-SID-3/ACK1 pathway. PLoS Genet 14:e1007313
Joseph, Braveen B; Blouin, Nicolas A; Fay, David S (2018) Use of a Sibling Subtraction Method for Identifying Causal Mutations in Caenorhabditis elegans by Whole-Genome Sequencing. G3 (Bethesda) 8:669-678
Lažeti?, Vladimir; Fay, David S (2017) Molting in C. elegans. Worm 6:e1330246
Dove, Katja K; Kemp, Hilary A; Di Bona, Kristin R et al. (2017) Two functionally distinct E2/E3 pairs coordinate sequential ubiquitination of a common substrate in Caenorhabditis elegans development. Proc Natl Acad Sci U S A 114:E6576-E6584
Lažeti?, Vladimir; Fay, David S (2017) Conserved Ankyrin Repeat Proteins and Their NIMA Kinase Partners Regulate Extracellular Matrix Remodeling and Intracellular Trafficking in Caenorhabditis elegans. Genetics 205:273-293
Yochem, John; Lažeti?, Vladimir; Bell, Leslie et al. (2015) C. elegans NIMA-related kinases NEKL-2 and NEKL-3 are required for the completion of molting. Dev Biol 398:255-66
Kelley, Melissa; Yochem, John; Krieg, Michael et al. (2015) FBN-1, a fibrillin-related protein, is required for resistance of the epidermis to mechanical deformation during C. elegans embryogenesis. Elife 4:
Kuzmanov, Aleksandra; Karina, Evguenia I; Kirienko, Natalia V et al. (2014) The conserved PBAF nucleosome-remodeling complex mediates the response to stress in Caenorhabditis elegans. Mol Cell Biol 34:1121-35
Kuzmanov, Aleksandra; Yochem, John; Fay, David S (2014) Analysis of PHA-1 reveals a limited role in pharyngeal development and novel functions in other tissues. Genetics 198:259-68
Polley, Stanley R G; Kuzmanov, Aleksandra; Kuang, Jujiao et al. (2014) Implicating SCF complexes in organogenesis in Caenorhabditis elegans. Genetics 196:211-23

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