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

Abstract

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 plusend-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 woundhealing 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.

Enhancement of β-catenin activity by BIG1 plus BIG2 via Arf activation and cAMP signals

“…In addition to roles for BIG1 in control of cell polarity during directed migration (31), and appropriate integrin β1 N-glycosylation (30) as well as for BIG2 in its intracellular trafficking (28), we show here that both BIG1 and BIG2, via multiple, different actions, contributed to PKA-catalyzed phosphorylation of β-catenin with resulting enhancement of cyclin D and c-myc transcription, whether or not initiated by Wnt signaling.…”

“…Microscopically, BIG1 was seen mainly at the trans-Golgi network (TGN), sometimes partially overlapping BIG2, which was associated also with recycling endosomes (24-26), e.g., in moving proteins and lipids among TGN, endosomes, and cell surface (24,(27)(28)(29). Although actions of BIG1 and BIG2 at the TGN were described as "redundant" (27), each protein clearly has specific roles in moving proteins and lipids that are not shared with the other (24,30,31). Overexpression in cultured cells of mutant BIG2 lacking Arf GEF activity markedly altered intracellular distributions of E-cadherin and β-catenin (32).…”

Talin-KANK1 interaction controls the recruitment of cortical microtubule stabilizing complexes to focal adhesions

“…Recently, we found that liprin-b1 interacts with KANK1 (van der Vaart et al, 2013), one of the four members of the KANK family of proteins, which were proposed to act as tumor suppressors and regulators of cell polarity and migration through Rho GTPase signaling (Gee et al, 2015;Kakinuma et al, 2008Li et al, 2011;Roy et al, 2009). KANK1 recruits the kinesin-4 KIF21A to CMSCs, which inhibits microtubule polymerization and prevents microtubule overgrowth at the cell edge van der Vaart et al, 2013).…”

Arf guanine nucleotide-exchange factors BIG1 and BIG2 regulate nonmuscle myosin IIA activity by anchoring myosin phosphatase complex

“…tion of mature integrin β1 (23), and directed migration during wound healing (24). In contrast, BIG2 was involved in recycling of internalized transferrin receptors (21) and integrin β1 (25) to the cell surface, and in regulation of tumor necrosis factor receptor release in exosome-like vesicles (26).…”

“…PP1γ (29) and phosphodiesterase 3A (30) that, respectively, dephosphorylate PKA-modified BIG1 and BIG2 and terminate cAMP signals, have been identified in complexes with BIG1 and/or BIG2. Depletion of BIG1 or BIG2 was reported to decrease cell motility as well as actin-based membrane protrusions (24,25), although the mechanisms through which these proteins altered cytoskeleton dynamics remain unclear. Here, we provide evidence that NM IIA activity in HeLa cells, which is crucial for actin stress fiber formation, is also influenced by previously unrecognized interactions with the C termini of BIG1 and BIG2, independent of Arf GEF activity, in complexes that include PP1cδ, and MYPT1.…”