In studies reported this year, we further defined functions of BIG1 and BIG2 in cultured HeLa cells. Among proteins that interact with BIG1, kinesin family member 21A (KIF21A), a plus-end-directed motor protein, moves cargo away from the 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, that was not mimicked by KIF21A depletion. Although colocalization of overexpressed KANK1 and endogenous BIG1 in HeLa cells was not clear microscopically, their reciprocal co-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 novel actions of both BIG1 and KANK1 in regulating cell polarity during directed migration, 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. BIG2 activates Arfs critical for continuity of intracellular vesicular trafficking by accelerating the replacement of Arf-bound GDP with GTP. Mechanisms of additional BIG2 function(s) are less clear. This year we described, the participation of BIG2 in integrin 1-cycling through actin dynamics during cell migration, identified using small interfering RNA (siRNA) and Difference Gel Electrophoresis (DIGE) analyses. After 72-h incubation with BIG2 siRNA, levels of cytosolic Arp2, Arp3, cofilin-1, phosphocofilin, vinculin, and Grb2, known to be involved in effects of integrin 1-extracellular matrix interactions on actin function and cell translocation, were increased. Treatment of HeLa cells with BIG2 siRNA resulted in perinuclear accumulation of integrin 1 and its delayed return to the cell surface. Motility of BIG2-depleted cells was simultaneously decreased, as were actin-based membrane protrusions and accumulations of Arp2, Arp3, cofilin, and phosphocofilin at the leading edges of migrating cells, in wound-healing assays. In sum, these data revealed a novel mechanism(s) through which BIG2 may coordinate actin cytoskeleton mechanics and membrane traffic in cell migration via integrin 1 action and actin functions.

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National Heart, Lung, and Blood Institute
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Chen, Pei-Wen; Jian, Xiaoying; Heissler, Sarah M et al. (2016) The Arf GTPase-activating Protein, ASAP1, Binds Nonmuscle Myosin 2A to Control Remodeling of the Actomyosin Network. J Biol Chem 291:7517-26
Li, Chun-Chun; Le, Kang; Kato, Jiro et al. (2016) Enhancement of ?-catenin activity by BIG1 plus BIG2 via Arf activation and cAMP signals. Proc Natl Acad Sci U S A 113:5946-51
Le, Kang; Li, Chun-Chun; Ye, Guan et al. (2013) Arf guanine nucleotide-exchange factors BIG1 and BIG2 regulate nonmuscle myosin IIA activity by anchoring myosin phosphatase complex. Proc Natl Acad Sci U S A 110:E3162-70
Lin, Sisi; Zhou, Chun; Neufeld, Edward et al. (2013) BIG1, a brefeldin A-inhibited guanine nucleotide-exchange protein modulates ATP-binding cassette transporter A-1 trafficking and function. Arterioscler Thromb Vasc Biol 33:e31-8
Shen, Xiaoyan; Li, Chun-Chun; Aponte, Angel M et al. (2012) Brefeldin A-inhibited ADP-ribosylation factor activator BIG2 regulates cell migration via integrin ?1 cycling and actin remodeling. Proc Natl Acad Sci U S A 109:14464-9
Le, Kang; Li, Ruifang; Xu, Suowen et al. (2012) PPAR? activation inhibits endothelin-1-induced cardiomyocyte hypertrophy by prevention of NFATc4 binding to GATA-4. Arch Biochem Biophys 518:71-8
Li, Chun-Chun; Kuo, Jean-Cheng; Waterman, Clare M et al. (2011) Effects of brefeldin A-inhibited guanine nucleotide-exchange (BIG) 1 and KANK1 proteins on cell polarity and directed migration during wound healing. Proc Natl Acad Sci U S A 108:19228-33
Meza-Carmen, Victor; Pacheco-Rodriguez, Gustavo; Kang, Gi Soo et al. (2011) Regulation of growth factor receptor degradation by ADP-ribosylation factor domain protein (ARD) 1. Proc Natl Acad Sci U S A 108:10454-9
Puxeddu, Ermanno; Uhart, Marina; Li, Chun-Chun et al. (2009) Interaction of phosphodiesterase 3A with brefeldin A-inhibited guanine nucleotide-exchange proteins BIG1 and BIG2 and effect on ARF1 activity. Proc Natl Acad Sci U S A 106:6158-63
Islam, Aminul; Jones, Heather; Hiroi, Toyoko et al. (2008) cAMP-dependent protein kinase A (PKA) signaling induces TNFR1 exosome-like vesicle release via anchoring of PKA regulatory subunit RIIbeta to BIG2. J Biol Chem 283:25364-71

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