Please use this identifier to cite or link to this item: http://scholarbank.nus.edu.sg/handle/10635/22880
Title: THE ROLE OF NCK IN RHO GTPASE BIOLOGY
Authors: SIT SOON TUCK
Keywords: NCK, Rho
Issue Date: 20-Aug-2010
Source: SIT SOON TUCK (2010-08-20). THE ROLE OF NCK IN RHO GTPASE BIOLOGY. ScholarBank@NUS Repository.
Abstract: The non-catalytic region of tyrosine kinase (NCK) proteins belongs to a group of src homology (SH) domain-containing adaptors that include Crk, Grap, Grb2, Grb10 and SLAP. The NCK family of proteins consists of two gene products, NCK1 and NCK2. Both NCK proteins are made up of three N-terminal SH3 domains that interact with proline-rich/polybasic motifs and a C-terminal SH2 domain that binds phosphotyrosine resides. Hence NCK serve to recruit proline-rich effectors to tyrosine-phosphorylated kinases or their substrates. While many interacting partners have been identified, the lack of a catalytic domain means that the functional impact of individual specific interactions is unclear. Genetic ablation strategies reveal that NCK play important roles in the modulation of the actin cytoskeleton, cell migration and axonal guidance in the nervous system. However, the spatiotemporal regulation of NCK activity is still poorly understood. I have identified G-protein-coupled receptor (GPCR)-kinase interacting protein-1 (GIT1) as a critical regulator of NCK1 localization and activity in the cell. Although NCK1 primarily interacts with a conserved phosphotyrosine residue (Y392) in GIT1 through its SH2 domain, I also describe a novel interaction between sequences in the GIT1 synaptic localisation domain (SLD) and the NCK1 SH3-2/SH3-3 linker region. There is strong evidence for the role of GIT1 in the adhesion recruitment of NCK1: inhibition of PAK activity enhanced adhesion targeting of both proteins; NCK1 interacts stably with endogenous PAK- PIX-GIT complexes in vivo; NCK1 only co-immunoprecipitates with paxillin in the presence of GIT1, and knockdown of GIT1 abrogates adhesion localization of NCK1. The adhesion localization of NCK1 can be blocked by a dominant-inhibitory NCK1-SH2 construct, leading to the inhibition of the migration-promoting activity of endogenous NCK. I also demonstrate that NCK proteins can potentially oligomerize through the SH3-1 domain, while the NCK1 SH3-2 and SH3-3 domains regulate the cytoplasmic retention of NCK1. I also demonstrate NCK1¿s role in regulating the GAP activity of !2- chimaerin. The NCK1-chimaerin interaction requires the NCK1 SH3-3 domain, and N-terminal chimaerin sequences preceding the SH2 domain as well as part of the SH2-C1 domain ¿linker¿ region. The NCK1 SH2 domain modulates phosphotyrosine-independent binding of both NCK1 and !2-chimaerin to EphA4 receptors, although more work is needed to work out the exact mechanism. alpha2-chimaerin was also found to interact with NCK1 that is bound to the PAK-PIX- GIT complex, with PAK enhancing the binding in a kinase-independent manner, probably through the allosteric modulation of GIT1. NCK1 binding in conjunction with phorbol ester engagement of the C1 domain, was also found to induce tyrosine-phosphorylation of the Y143 residue by Fyn, leading to the synergistic activation of alpha2-chimaerin¿s Rac-GAP activity in vivo as measured by a cell spreading assay. In conclusion, I have demonstrated the mechanism of NCK1 recruitment and function at focal adhesions, novel interactions with regions of unknown function in GIT1 and alpha2-chimaerin, and that full activation of alpha2-chimaerin GAP activity requires coincidental signals from NCK1, phorbol ester binding and Fyn phosphorylation. Further work to establish how the potential oligomerization of NCK affects the specificity and affinity of its interactions, as well as the potential interplay between the adhesion-localized GIT complex and chimaerin will help to develop a more complete understanding of NCK function in the cell.
URI: http://scholarbank.nus.edu.sg/handle/10635/22880
Appears in Collections:Ph.D Theses (Open)

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Movie 1 - GFP-Nck1-FL expressing HeLa cell.AVI1.12 MBAVI

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Movie 2 - GFP-Nck1 DELSH3-1 expressing HeLa cell.AVI1.1 MBAVI

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Movie 3 - GFP-Nck1 SH3[2M] expressing HeLa cell.AVI1.55 MBAVI

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Movie 4 - GFP-Nck1 SH3[3M] expressing HeLa cell.AVI1.41 MBAVI

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Movie 5 - GFP-Nck1-FL + mCherry-Paxillin expressing B16F1 cell (GFP channel).avi8.05 MBAVI

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Movie 6 - GFP-Nck1-FL + mCherry-Paxillin expressing B16F1 cell (mCherry channel).avi4.94 MBAVI

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Movie 7 - GFP-Nck1-FL + mCherry-Paxillin expressing B16F1 cell (Overlay).avi8.06 MBAVI

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Movie 8 - GFP-Nck1-FL expressing B16F1 Cell (GFP and DIC Overlay).mp42.06 MBMP4

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Movie 9 - GFP-Nck1 SH3(2+3m) expressing B16F1 cell (GFP and DIC Overlay).mp48.38 MBMP4

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Movie 10 - GFP-Nck1-delSH2 expressing B16F1 cell (GFP and DIC Overlay).mp44.32 MBMP4

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Movie 11 - GST-Nck1-SH2 + GFP-Paxillin expressing B16F1 cell (GFP and DIC Overlay).mp42.04 MBMP4

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Movie 12 - mCHERRY-a2-chimaerin-wt expressing B16F1 cell.AVI2.6 MBAVI

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SitST.pdf6.9 MBAdobe PDF

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