Effects of brefeldin A-inhibited guanine nucleotide-
exchange (BIG) 1 and KANK1 proteins on cell polarity
and directed migration during wound healing
Chun-Chun Lia, Jean-Cheng Kuob,1, Clare M. Watermanb, Ryoiti Kiyamac, Joel Mossa, and Martha Vaughana,2
aCardiovascular and Pulmonary Branch,bCell Biology and Physiology Center, National Heart, Lung, and Blood Institute, National Institutes of Health,
Bethesda, MD 20892; andcSignaling Molecules Research Group, Biomedical Research Institute, National Institute of Advanced Industrial Science and
Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
Contributed by Martha Vaughan, October 18, 2011 (sent for review September 21, 2010)
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 microtu-
bule-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 di-
rected 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
healing (1, 2). Directed migration of cells growing on dishes,
usually in response to external chemical or mechanical cues (3),
requires complex and precisely coordinated actions, from initial
polarization and extension of protrusions in the direction of
movement to formation of adhesions at the leading edge, trans-
location of the cell body, and detachment with retraction from
an extracellular substratum at the trailing edge (1, 4). Generation
and maintenance of cell polarity, which are essential for di-
rectional migration, result from asymmetric membrane traffic
achieved by cytoskeletal reorganization to direct secretory trans-
port and delivery of additional membrane to the leading edge
(5, 6). Positioning of Golgi and microtubule-organizing center
forward movement, and supplementation of content at the cell
front are important for coordination of intracellular traffic (7, 8).
As elucidation of mechanisms of polarization and migration
continues, more molecular participants are identified (3, 9, 10).
Brefeldin A (BFA)-inhibited guanine nucleotide-exchange
protein (BIG) 1 (∼200 kDa), originally purified with BIG2 (∼190
kDa) in ∼670-kDa multiprotein complexes from bovine brain
cytosol (11), activates class I ARFs (human ARF1 and 3) by
ell motility is crucial in diverse biological events, including
embryonic development, immune surveillance, and wound
catalyzing the replacement of ARF-bound GDP with GTP to
enable critical vesicular transport (12–15). BIG1 is often found
at trans-Golgi membranes (16, 17); after BIG1 depletion, mem-
branes of the HepG2 cell Golgi trans face appeared less smooth
by electron microscopy with more vesicle-like structures (18). Boal
and Stephens also reported that Golgi structure was altered after
BIG1 depletion (19). In addition to its Golgi association, BIG1
was accumulated in nuclei of serum-deprived HepG2 cells (20) or
after their incubation with the immunosuppressive FK506 (21) or
8-Br-cAMP (22). In other conditions, BIG2 was associated with
the trans-Golgi network and recycling endosomes (17, 23).
In addition to ARF activation by specific sequence in the cen-
tral Sec7 domain (24), other parts of BIG1 molecules participate
in multimolecular complexes with nucleolin, U3 small nucleolar
RNA, and U3-binding protein fibrillarin or the RNA-binding
protein La in different nuclear complexes (25). An A kinase-an-
choring protein (AKAP) sequence in BIG1 is identical to one of
three such sequences first identified in BIG2, and antibodies
against RI or RII, regulatory subunits of cAMP-dependent pro-
tein kinase, coprecipitated endogenous BIG1 (26). ARF activa-
tion by BIG1 was decreased after its phosphorylation by PKA
in vitro (27). Reported interactions of the C-terminal region of
BIG1 with myosin IXb (28) and with kinesin KIF21A (29) suggest
that BIG1 also plays a role in cargo movement along actin fibers
Kakinuma and Kiyama reported an interaction of KIF21A and
KANK1 that influenced membrane localization of KANK1 (30).
The human KANK1 gene was first described as a growth sup-
pressor in renal carcinoma cells (31). Two KANK1 proteins,
KANK1-L (∼175 kDa) and KANK1-S (∼160 kDa), produced by
alternative splicing of exon 1 are present in many human tissues,
and levels of KANK1-L in kidney tumors were lower than those
in normal tissue (32). Predominantly cytoplasmic KANK1 (31,
33) interfered with actin polymerization by blocking interaction
of insulin receptor substrate p53 (IRSp53) and Rac1 (34). In-
hibition of RhoA activity via PI3K-Akt signaling regulated by
KANK1 binding to 14-3-3 decreased actin stress fiber formation
and cell migration (35). Participation of KANK1, with its nuclear
export (NES) and nuclear localization (NLS) signals, in nucle-
ocytoplasmic shuttling relevant to the activation of β-catenin-
dependent transcription was also reported (36). Here, we
describe the coimmunoprecipitation of endogenous BIG1 and
KANK1 from HeLa cells and subsequent exploration of actions
Author contributions: C.-C.L., J.M., and M.V. designed research; C.-C.L. and J.-C.K. per-
formed research; R.K. contributed antibody; C.-C.L., C.M.W., R.K., J.M., and M.V. analyzed
data; and C.-C.L., J.M., and M.V. wrote the paper.
The authors declare no conflict of interest.
1Present address: Institute of Biochemistry and Molecular Biology, National Yang-Ming
University, Taipei, Taiwan.
2To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| November 29, 2011
| vol. 108
| no. 48www.pnas.org/cgi/doi/10.1073/pnas.1117011108
of the two proteins revealing that depletion of either one in-
terfered seriously with regulation of HeLa cell polarity during
directed cell migration in wound-healing assays. BIG1 and
KANK1 apparently use, at least in part, the same pathway to
produce those effects, although no evidence of their direct
physical interaction in cells was obtained.
Interaction of BIG1 with KANK1. To begin to assess KANK1 in-
teraction with BIG1, we demonstrated its presence among pro-
teins immunoprecipitated from HepG2 cells with antibodies
against BIG1. The finding was confirmed in HeLa cells, which
we then used instead of HepG2 cells because of their higher
transfection efficiency and faster proliferation. BIG1 or BIG2
was partially coprecipitated from cell lysates by antibodies
against the other protein. KIF21A was also among proteins
precipitated with BIG1 antibodies (29, 37). Endogenous KANK1
specifically coprecipitated with BIG1 antibodies, but not BIG2
or control IgG (Fig. 1A). Immunoprecipitation (IP) of KANK1
yielded also BIG1 and KIF21A (Fig. 1B), and again, no BIG2
was detected; this result is consistent with the presence of BIG1
and KANK1 together in some multimolecular complexes that
differ from those containing both BIG1 and BIG2.
Because we did not have KIF21A antibodies specific enough
for IP of endogenous protein, we transiently overexpressed HA-
KIF21A, which enabled co-IP of BIG1 and KANK1 along with
HA-tagged KIF21A from those cells but not from cells expressing
empty vector (Fig. 1C). These results were in agreement with
earlier reports of BIG1-KIF21A (29) and KANK1-KIF21A asso-
ciations (30), and also with the possibility of KANK1 as a com-
ponent of some BIG1 complexes. In further experiments, we used
siRNA that decreased endogenous KANK1 to 4 ± 3% and
KIF21A to 9 ± 5% of control cells (Fig. S1C). Co-IP of endog-
enous KIF21A with BIG1 was not altered after KANK1 de-
pletion, but BIG1 IP after KIF21A depletion yielded significantly
less KANK1 than it did from control cells (Fig. 1D), consistent
with the notion that co-IP of KANK1 with BIG1 was due, in some
part, to its interaction with KIF21A (30). The small amounts of
KANK1 in BIG1 IP from cells after ∼90% depletion of KIF21A
were still ∼40% of that from control cells (Fig. 1D); however, this
result is compatible with the existence of BIG1 and KANK1 in
complexes different from those that include KIF21A.
Because the C-terminal amino acids 886–1849 of BIG1 had
interacted with KIF21A (29), we used anti-tag antibodies to in-
vestigate co-IP of KANK1-myc and HA-BIG1 or its fragments
coexpressed in HeLa cells (Fig. S2B). Despite differences in
levels of expression of the BIG1 constructs, reciprocal IP of both
N- and C-terminal fragments (but not Sec7) with KANK1-myc
was found. We concluded that any direct interaction of BIG1
and KANK1 molecules could involve only very small fractions of
both proteins and cannot involve those complexes of BIG1 that
Intracellular Localizations of BIG1 and KANK1. KANK1 is widely
viewed as a cytosolic protein, whereas BIG1 is most often asso-
ciated with Golgi structures (29, 37) identified by specific Golgi
or TGN markers (Fig. S3). However, the presence of small
amounts of each protein at other intracellular loci has been
clearly shown. Our questions about direct interaction of BIG1
and KANK1 molecules prompted a search for clues to previously
unrecognized loci of one or the other. We assessed endogenous
protein distributions by iodixanol density gradient fractionation,
confirming very little overlap between largely cytosolic KANK1
and the essentially single peak of BIG1 in fractions 7–10 (Fig.
S4). Amounts of endogenous BIG1 or KANK1 were markedly
reduced in cells treated, respectively, with BIG1- or KANK1-
specific siRNAs, although distributions of the proteins and en-
dogenous βCOP, BiP, and α-tubulin were similar to those in
control cells transfected with nonspecific siRNA.
Microscopically, endogenous BIG1 was prominently clustered
in a perinuclear region and scattered through the cytoplasm (29,
37). Insufficient specificity and sensitivity of our antibodies
or KANK1, or control IgG from extracts of HeLa cells (1 mg) were separated by SDS/PAGE before reaction of Western blots with indicated antibodies. (A and B
are enlarged in Fig. S1.) (C) Samples (50%) of proteins precipitated with anti-HA antibodies or control IgG from 200 μg of extracts prepared 24 h after
transfection of cells with HA-KIF21A (HA-21A) or empty vector (EV) were used for Western blotting with indicated antibodies. Input (20 μg) was 10% of
amount used for IP. (D) Cells transfected with 100 nM nontargeted (NT) or KANK1-specific siRNA or 100 or 150 nM KIF21A-specific siRNA or with vehicle alone
(Mock) were lysed 48 h later. Proteins precipitated with antibodies against BIG1 were analyzed by Western blotting and densitometric quantification.
Amounts of proteins from BIG1 IP in three experiments expressed relative to that of the same protein in Mock cells (=100%) are means ± SEM *P < 0.005 vs.
Mock. Arrow: protein band. Arrowhead: position of 160-kDa marker.
Immunoprecipitation of KANK1 with BIG1. (A and B) Samples of Input (2.5% of total) and 25% of proteins from IP with antibodies against BIG1, BIG2,
Li et al.PNAS
| November 29, 2011
| vol. 108
| no. 48
precluded identification of endogenous KANK1. Overexpressed
untagged KANK1, in about sevenfold the endogenous amount,
was seen at membrane ruffles and throughout the cytoplasm (Fig.
2). To further explore possible effects of BIG1 levels on distribu-
tion of KANK1, we overexpressed HA-BIG1 plus untagged
KANK1 in HeLa cells (Fig. S5). Like endogenous BIG1, HA-
BIG1 was seen in a punctate distribution with perinuclear con-
centration (Fig. S5B), and its amount did not apparently affect
KANK1 localization (Fig. S5C and Fig. 2). Overall, none of our
experiments provided unequivocal evidence of direct interactions
or reciprocal effects of total cell content of BIG1 or KANK1.
Effects of BIG1 and KANK1 on Wound Healing. Depletion of en-
dogenous BIG1 interfered with HepG2 cell attachment to col-
lagen as well as COS7 cell transwell migration (18). Kiyama and
coworkers showed that KANK1 influenced actin cytoskeleton
reorganization and lamellipodium formation via RhoA and Rac
activation (34, 35). To assess potential roles for BIG1–KANK1
interaction in cell motility, we evaluated migration in wound-
healing assays after treatment of cells with vehicle alone (Mock)
or control nontargeted or specific siRNAs (Fig. 3A). Endogenous
BIG1, BIG2, KANK1, and KIF21A levels were only 14 ± 6%,
9 ± 3%, 14 ± 5%, and 11 ± 2%, respectively, of control (Mock)
cells (mean ± SEM, n = 3) 48 h after the addition of specific
cognate siRNAs. Wound area covered by migrating monolayer
cells was quantified 6 h after wounding. BIG1, BIG2, or KANK1
siRNA treatments each delayed wound closure (Fig. 3 B and C),
but KIF21A depletion did not significantly alter any of the mi-
gration characteristics quantified in wound-healing experiments.
Individual cell paths (Fig. 3D) recorded by tracing the centers
of cell nuclei from time-lapse images and analyzing trajectories
of individual cells at the wound edge over a 6-h period are
especially useful for seeing flaws in directed migration. We found
that velocity (total length of single cell path in 6 h) was not sig-
nificantly altered by BIG1, BIG2, KANK1, or KIF21A depletion
(Fig. 3E), suggesting an absence of effects on cell motility.
Net translocation toward wound closure of cells transfected
with BIG1-, BIG2-, or KANK1-specific siRNA was significantly
less than that of control cells (Fig. 3F), but only cells depleted of
BIG1 or KANK1 made directional changes significantly more
often than did control cells. Depletion of BIG1 or KANK1
appeared to delay wound healing by interfering with directional
persistence, defined as the distance (D) between cell start and
end points divided by total migration path length (T) during 6-h
assays (Fig. 3G) of cell migration. Although some data in Fig. 3
seem to show effects of KIF21A depletion, none of these
reached statistical significance; taken together, the data indicate
transfection (24 h) with cDNA encoding untagged KANK1, cells were fixed,
reacted with rabbit anti-BIG1 and mouse anti-KANK1 antibodies, and pre-
pared for confocal immunofluorescence microscopy. In three Z-stack images
of 1-μm planes (upper, middle, and lower), overexpressed KANK1 (red) did
not apparently coincide with or alter the distribution of endogenous BIG1
(green), which is scattered in punctate collections through cytoplasm of
all cells, with greatest perinuclear concentration in the upper plane. (Scale
bar, 10 μm.)
Endogenous BIG1 and overexpressed KANK1 in HeLa cells. After
(A) Cells were lysed 48 h after transfection with nontargeted (NT), BIG1, BIG2,
KANK1, or KIF21A siRNA or vehicle alone (Mock) and amounts of proteins
quantified by densitometry of Western blots. (B) Images of cells at 0 and 6 h
after wounding as in A. (Scale bar, 70 μm.) (C) Covered area is the difference
between wound areas 0 and 6 h after wounding. Data are means ± SEM of
values from five experiments. **P < 0.005 *; P < 0.05, (two-tailed t test) for
difference from cells transfected with nontargeted siRNA. (D) Migration
paths of five representative cells at wound edges during 6-h assays (B). Ini-
tiation of cell migration = 0. (E–G) Motility characteristics in B and C were
calculated for individual cells as described in Materials and Methods. Data are
means ± SEM of values from three experiments for at least 120–150 cells in
each group. **P < 0.005; *P < 0.05, (ANOVA test) for difference from cells
transfected with vehicle alone (Mock).
Effects of BIG1, BIG2, KANK1, or KIF21A depletion on wound healing.
| www.pnas.org/cgi/doi/10.1073/pnas.1117011108Li et al.
that delayed wound healing by cells depleted of BIG1 or KANK1
resulted from defects in persistence of polarization, rather than
velocity of cell movement.
BIG1 and KANK1 Contribute to Cell Polarization During Directed
Migration. Polarized morphology of wound-edge cells is achieved
by translocation of MTOC and Golgi to precede the nucleus in
cells oriented to the direction of migration (7, 8). This enhances
microtubule growth to the lamella, enabling vesicle transport
to extend protrusions at the leading edge (2, 5) and establish
polarity. To assess these actions, the positions of Golgi (Fig. 4 A
and C) and MTOC (Fig. 4 B and C) in wound-edge cells were
recorded 6 h after wounding. Only in cells depleted of BIG1 or
KANK1 were positions of Golgi marker GM130 and MTOC
(immunoreactive γ-tubulin in the perinuclear region) significantly
different from those in controls (Mock or nontargeted siRNA).
Aberrant Golgi positions appeared correlated with extent of
BIG1 or KANK1 depletion (Fig. S6E), and effects of maximal
depletion of either one were not greater with additional depletion
of the other (Fig. S6 F and G).
Polarization of Golgi and MTOC persisted in cells depleted of
BIG2 or KIF21A, but was grossly disturbed by BIG1 or KANK1
siRNA treatment. BIG1 and KANK1 were each important for
establishing cell polarity and preserving correctly directed cell
movement during woundhealing. Thelack ofsignificant effects of
KIF21A knockdown on those characteristics means there was no
evidence that KIF21A was critical for effects of BIG1 or KANK1
on orientation of Golgi/MTOC and cell polarity (Fig. 4C).
Effects of BIG1 or KANK1 depletion on microtubule (α-tu-
bulin) and actin (phalloidin) cytoskeletons were also evaluated
(Fig. 5 A and B). Microtubules in control cells were aligned
perpendicular to the leading edge and extended well behind it,
but were short and failed to form organized networks in cells
depleted of BIG1 or KANK1. Effects of BIG2 or KIF21A siR-
NAs, although similar to one another, were clearly different from
the phenotype of cells depleted of BIG1 or KANK1 (Fig. 5A).
Stress fibers are less prominent in HeLa cells than in fibroblasts,
but differences in F-actin patterns at leading edges of control
cells (densely populated with actin-rich membrane ruffles) and
BIG1- or KANK1-depleted cells, without evidence of F-actin-
based ruffles and with nonuniform, irregular phalloidin staining,
were obvious (Fig. 5B). Microscopically, both actin and micro-
tubule skeletons differed after BIG2 or KIF21A depletion from
those of control cells (Mock or nontargeted siRNA). However,
they were also quite distinct from patterns in BIG1- and
KANK1-depleted cells. The BIG2- and KIF21A-depleted cells
still exhibited protrusions toward the wound edge and F-actin-
based ruffles within the leading edge of migrating cells.
were not significantly altered, at least some effects of BIG2 or
KIF21A depletion may be recognized as functionally relevant
when we better understand the mechanisms through which these
and numerous additional molecules act to achieve directed cell
migration via integrated operations of diverse multimolecular
machines throughout the cell.
Participation in macromolecular complexes is required for many
BIG1 and KANK1 functions. Copurification of BIG1 and BIG2
in ∼670-kDa complexes (37) and formation of homo- or heter-
odimers in multimolecular complexes (38) was suggested by
structures of specific domains of the two molecules. However,
redundancy of their actions at TGN (39) seems rather less likely
wound healing. Confluent monolayers of HeLa cells transfected 48 h before
with indicated siRNA or vehicle alone (Mock), as in Fig.3, were wounded and
fixed 6 h later for staining with DAPI and anti-GM130 (A) or anti-γ-tubulin
antibodies (B) and confocal immunofluorescence microscopy. Arrowheads
indicate GM130 (A) or γ-tubulin (B). (Scale bar, 10 μm.) (C) Percentage of
wound-edge cells with Golgi or γ-tubulin structures in forward-facing 120°
sector between nucleus and wound was recorded for at least 100 cells of
each population for Golgi localization and 30 cells for γ-tubulin in each ex-
periment. Data are means ± SEM of values from six experiments. **P < 0.02.
Depletion of BIG1 or KANK1 interfered with cell polarization during
microtubule and actin morphology 6 h after wounding. HeLa cells treated as
in Figs. 3 and 4 were fixed 6 h after wounding and reacted with anti-
α-tubulin antibodies to mark microtubules (A) or Alexa Fluor 488-conjugated
phalloidin for F-actin (B). Patterns of microtubules and F-actin were altered
in cells depleted of any of the four proteins, but effects on F-actin were most
obvious and prominent in cells treated with BIG1 or KANK1 siRNA. (Scale
bar, 10 μm.) Arrowheads indicate wound-edge membrane.
Effects of BIG1, BIG2, KANK1, or KIF21A depletion on intracellular
Li et al. PNAS
| November 29, 2011
| vol. 108
| no. 48
in light of the apparently long evolutionary histories and evident
functional differentiation of the two BIG molecules, along with
those of their specific substrates, such as human ARF1 and
ARF3, which differ by only 7 of 181 amino acids. Ishizaki et al.
(39) found that depletion of BIG1 alone had no obvious effects
on the location of Golgi or endosomal proteins that were ex-
amined, whereas depletion of BIG2 alone induced extension
from recycling endosomes of tubular structures that reacted with
antibodies against TfnR, AP-1, and Rab11. However, depletion
of both BIG1 and BIG2 led to redistribution of proteins that
were otherwise seen in TGN (e.g., TGN46, AP-1) or recycling
endosomes and blocked retrograde trafficking of furin from late
endosomes to the TGN, consistent with, as Ishizaki et al. wrote,
“redundant roles of BIG1 and BIG2 in membrane traffic be-
tween the trans-Golgi network and endosomes” (39). Several
other functions of BIG1 and BIG2 in cell organization and
membrane trafficking seem clearly not redundant; for example,
BIG1 was required to maintain usual morphology of the TGN
(18), and BIG2 was important for endosome integrity (17, 39).
Perhaps observations like those of Ishizaki et al. (39) are more
likely due to the extent of “cross-talk” among pathways in-
volving these molecules as seen after siRNA-knockdown of
pairs of different ARF molecules rather than the effects of
single siRNAs (40).
Here, we failed to demonstrate direct interaction of BIG1 with
KANK1, and concluded that BIG1 and KANK1 might exist to-
gether in multimolecular complexes different from those con-
taining both BIG1 and BIG2. Similarly, the lack of effect of BIG2
depletion on Golgi/MTOC orientation in wound healing likely
reflects differing functional interactions of BIG1 and BIG2.
Kakinuma and Kiyama reported that KIF21A interacted directly
with KANK1 (30), and the level of KIF21A protein significantly
influenced the extent ofKANK1 co-IP with BIG1 antibodies (Fig.
KIF21A, and KANK1. Depletion of KIF21A altered BIG1 and
KANK1 distributions without changing the microscopic appear-
ance of intrinsic Golgi proteins (29, 30), suggesting that BIG1
might act as a scaffold as well as a partner in KIF21A-powered
intracellular transport (29). We found no change in KANK1 dis-
tribution after BIG1 depletion, although our methodology (Fig.
S4) may have been inadequate for detection of differences in as-
sociated molecules or location of any small fraction of the total
KANK1. The effect of KIF21A depletion on migration in wound
healing did differ, at least quantitatively, from that of BIG1 or
KANK1 depletion. Molecular assemblies containing BIG1,
KIF21A, and KANK1 could exist during transport, without being
responsible for effects of BIG1 or KANK1 depletion on Golgi/
MTOC orientation. We showed that the percentage of cells with
distorted Golgi orientation during wound healing migration was
no greater after depletion of both BIG1 and KANK1 than after
either one alone, suggesting that BIG1 and KANK1 affect Golgi
polarity via the same pathways. However, further studies are re-
quired to exclude the possibility that either one might use an ad-
ditional mechanism to influence cell polarity.
We described here a previously unrecognized role for BIG1 in
the regulation of Golgi/MTOC orientation. Further studies are
needed to learn whether or how signaling upstream of BIG1 or
KANK1 controls cell polarity and to understand mechanisms
used by both proteins in maintenance or remodeling of Golgi
structure. Actin network action in Golgi structure and function
has been demonstrated (41). Cdc42 and ARF1 effects on short
actin filament dynamics in a Golgi complex pool could contribute
to the polarization of intracellular trafficking (42, 43). BIG1
interaction with myosin IXb (28) would be consistent with par-
ticipation of the motor molecule in maintenance of Golgi mor-
phology via function of the actin cytoskeleton. The report of
myosin IXb control of murine macrophage shape and motility via
its RhoGAP activity (44) appears directly relevant to the
demonstrated effects of BIG1, and perhaps KANK1, depletion
on directional persistence of HeLa cell movement.
KANK1 is known to influence the actin cytoskeleton, but the
extent to which it contributes to cytoskeleton function in main-
taining Golgi structure and protein translocation remains to be
established. Recently, Arai et al. (45) described an intrinsic
phosphoinositide signaling system in Dictyostelium that is re-
sponsible and required for random cell migration. Stochastic
variations in activities of phosphoinositide-3-kinase (PI3K) and
PTEN (phosphatase and tensin homolog) produced temporally
and spatially confined waves of individual phosphoinositide con-
centrations. Mechanisms that coordinate these phenomena with
actin-based cell movement remain to be elucidated, as do the
similarities between these systems in HeLa and Dictyostelium cells.
Regulation of actin polymerization is among KANK1 functions
seemingly involved in cell migration (33). Potential routes of
KANK1 influence on actin remodeling include PI3K/Akt signal-
ing via 14-3-3 in RhoA regulation (35) and effects of lower Rac1
activity after interaction with IRSp53 (34). Depletion of KANK1
increased RhoA activity and enhanced insulin-stimulated single
cell movement in transwell filters coated with entactin-collagen
IV-laminin (35), whereas overexpression of GFP-KANK1 inhi-
bited random migration of NIH 3T3 cells (33). It will be impor-
tant to learn why our data regarding effects of KANK1 depletion
on cell migration appear inconsistent with such reports (33–35).
In our wound-healing experiments, KANK1 depletion clearly
interfered with persistence of directional migration, which re-
quires mechanisms more complicated than those of random or
single cell migration (46, 47) for which self-organized, intrinsic
phosphoinositide signaling may suffice (45). Continuity of di-
rectional translocation and lamellipodium structure may involve
numerous regulatory influences; for example, the nature of ex-
tracellular matrix, stability of cell polarity, and multiple, diverse
intra- and intercell signals (3, 46, 47). Whether parallel differ-
ences in cell mobility and KANK1 content reflect differences in
cell type, study conditions, or the cues guiding migration remain
to be determined. Our present fragmentary understanding of
specific molecular forms of KANK1 (and other proteins) with
their posttranslation modifications is probably equally important.
Inhibition of cell migration by depletion of BIG2 could be
related to effects of BIG2 depletion on transferrin receptor
recycling (17, 23) or its interaction with Exo70, a component of
the exocyst complex required for exocytosis (48). Exo70 inter-
acted with the Arp2/3 complex, a key regulator of actin poly-
merization, and its inhibition blocked formation of actin-based
membrane protrusions plus other aspects of cell locomotion (49).
Information on exocyst participation in returning internalized
cycling proteins to the cell surface is also needed. Similarly,
understanding mechanisms through which cAMP/PKA signals
control or coordinate BIG1, BIG2, and/or KANK1 function in
actin-based cell migration will surely be important. One or more
AKAP sequences in both BIG1 and BIG2 (26) enable them to
compartmentalize PKA with other molecular complexes relevant
to cAMP metabolism and action, thereby contributing to the crit-
ical spatial and temporal specificity of cAMP signaling and/or
action (27, 50, 51). Reversible PKA-catalyzed phosphorylation of
BIG1 and BIG2 inhibits their ARF GEF activity and might be
relevant to a KANK1-14-3-3 interaction (27, 35).
Roles for both BIG1 and KANK1, which appear to act in part
via the same pathway(s) to regulate HeLa cell polarity during
oriented motion, seem clear. Direct interaction of the two mol-
ecules was not proven, although co-IP of in vitro synthesized
KANK1-myc and HA-BIG1 was consistent with both BIG1 N
and C termini binding to KANK1. Unequivocal characterization
in cells of such interactions, involving only small fractions of total
endogenous protein, will likely require the remarkable spatial and
by Waterman and Lippincott-Schwartz (52–54). Identification of
| www.pnas.org/cgi/doi/10.1073/pnas.1117011108 Li et al.
additional components of BIG1/KANK1 functional complexes Download full-text
and elucidation of detailed molecular mechanisms that accom-
plish directional migration, including potential BIG1/KANK1
interactions, require further studies.
Materials and Methods
Sourcesofantibodies andother specificreagentsnot otherwise identified are
reported in SI Materials and Methods, along with details of immunopre-
cipitation and procedures for prior, reversible cross-linking of protein com-
plexes. HeLa cells (from American Type Culture Collection) were grown, and
wound-healing assays for analyses of directed cell migration as well as
confocal immunofluorescence microscopy experiments were performed as
described in SI Materials and Methods.
ACKNOWLEDGMENTS. We are grateful to members of the Cell Biology and
Physiology Center, National Heart, Lung and Blood Institute (NHLBI),
National Institutes of Health (NIH) for invaluable help in time-lapse imaging
and to Drs. Daniela Malide and Christian Combs (Light Microscopy Core
Facility, NHLBI) for their much appreciated assistance in confocal microscopy.
This research was supported by the Intramural Research Program of the
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Li et al.PNAS
| November 29, 2011
| vol. 108
| no. 48