Effects of brefeldin A-inhibited guanine nucleotide-exchange (BIG) 1 and KANK1 proteins on cell polarity and directed migration during wound healing

Cardiovascular and Pulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 11/2011; 108(48):19228-33. DOI: 10.1073/pnas.1117011108
Source: PubMed


Brefeldin A-inhibited guanine nucleotide-exchange protein (BIG) 1 activates class I ADP ribosylation factors (ARFs) by accelerating the replacement of bound GDP with GTP to initiate recruitment of coat proteins for membrane vesicle formation. Among proteins that interact with BIG1, kinesin family member 21A (KIF21A), a plus-end-directed motor protein, moves cargo away from the microtubule-organizing center (MTOC) on microtubules. Because KANK1, a protein containing N-terminal KN, C-terminal ankyrin-repeat, and intervening coiled-coil domains, has multiple actions in cells and also interacts with KIF21A, we explored a possible interaction between it and BIG1. We obtained evidence for a functional and physical association between these proteins, and found that the effects of BIG1 and KANK1 depletion on cell migration in wound-healing assays were remarkably similar. Treatment of cells with BIG1- or KANK1-specific siRNA interfered significantly with directed cell migration and initial orientation of Golgi/MTOC toward the leading edge, which was not mimicked by KIF21A depletion. Although colocalization of overexpressed KANK1 and endogenous BIG1 in HeLa cells was not clear microscopically, their reciprocal immunoprecipitation (IP) is compatible with the presence of small percentages of each protein in the same complexes. Depletion or overexpression of BIG1 protein appeared not to affect KANK1 distribution. Our data identify actions of both BIG1 and KANK1 in regulating cell polarity during directed migration; these actions are consistent with the presence of both BIG1 and KANK1 in dynamic multimolecular complexes that maintain Golgi/MTOC orientation, differ from those that might contain all three proteins (BIG1, KIF21A, and KANK1), and function in directed transport along microtubules.

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Available from: Chun-Chun Li, Feb 16, 2014
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    • "The conserved protein Kank1 was identified as a human tumour suppressor [12], though exactly how it suppreses tumour growth remains unclear. So far, investigation of the mammalian Kank proteins has been carried out primarily in cell culture and they have been shown to have roles in inhibition of actin nucleation, actin organisation [13], [14], cell polarity [15] and cell growth [16]. A study in C. elegans shows that the sole Kank orthologue, VAB-19, localises to epidermal attachment structures between muscle and epidermal cells in developing nematode embryos, and later at circumferential bands that cover the length of the worm [17]. "
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    ABSTRACT: Little is known about how microtubules are regulated in different cell types during development. EB1 plays a central role in the regulation of microtubule plus ends. It directly binds to microtubule plus ends and recruits proteins which regulate microtubule dynamics and behaviour. We report the identification of Kank, the sole Drosophila orthologue of human Kank proteins, as an EB1 interactor that predominantly localises to embryonic attachment sites between muscle and tendon cells. Human Kank1 was identified as a tumour suppressor and has documented roles in actin regulation and cell polarity in cultured mammalian cells. We found that Drosophila Kank binds EB1 directly and this interaction is essential for Kank localisation to microtubule plus ends in cultured cells. Kank protein is expressed throughout fly development and increases during embryogenesis. In late embryos, it accumulates to sites of attachment between muscle and epidermal cells. A kank deletion mutant was generated. We found that the mutant is viable and fertile without noticeable defects. Further analysis showed that Kank is dispensable for muscle function in larvae. This is in sharp contrast to C. elegans in which the Kank orthologue VAB-19 is required for development by stabilising attachment structures between muscle and epidermal cells.
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    • "Closer analysis uncovered additional genes that have been shown to promote cell motility despite having not been picked up by our gene ontology analysis. These genes include PIK3R3 (McAuliffe et al., 2010), PPM1D (Wang et al., 2011), RASGRP3 (Randhawa et al., 2011; Yang, D. et al., 2010), ADAM19 (Wildeboer et al., 2006), SORBS3 (Kioka et al., 2010; Mizutani et al., 2007), ITSN1 (Ma et al., 2011), MECP2 (Degano et al., 2009; Yaqinuddin et al., 2008), VLDLR (Förster et al., 2010), HIP1 (Khatchadourian et al., 2007), PAXIP1 (Mu et al., 2008), ITGA2 (Mercurio, 2002), ARFGEF1 (Li et al., 2011; Shen et al., 2007). "
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    • "Many cancer-related genes have been found in renal cancer, including a multi-drug resistance gene (Walsh et al., 2009), anti-apoptotic genes (Bilim et al., 2009), and radiation resistant components (Kransny et al., 2010). The most characteristic genomic structure in renal cancer is the VHL-related hypoxia-inducible factor gene and its cascades shown in hereditary RCC and sporadic RCC cases (Linehan et al., 2011). The down-regulation in expression of Kank1, our main theme, was also found from the study of renal cancer and normal renal tubular cells (Sarkar et al., 2002), as we mentioned in other sections. "

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