Keratin 8 phosphorylation regulates keratin
reorganization and migration of epithelial tumor cells
Tobias Busch1,*, Milena Armacki2,*, Tim Eiseler2, Golsa Joodi2, Claudia Temme2, Julia Jansen1, Go ¨tz von
Wichert1, M. Bishr Omary4, Joachim Spatz3and Thomas Seufferlein1,2,`
1Department of Internal Medicine I, University of Ulm, Albert-Einstein-Allee 23, 89081, Ulm, Germany
2Department of Internal Medicine I, Martin-Luther-University Halle-Wittenberg, Ernst-Grube-Str. 40, D-06120 Halle (Saale), Germany
3Max Planck Institute for Intelligent Systems, Heisenberg Str. 3, 70569 Stuttgart, Germany
4Department of Molecular and Integrative Physiology, University of Michigan Medical School, MI 48109-0622, USA
*These authors contributed equally to this work
`Author for correspondence (email@example.com)
Accepted 19 December 2011
Journal of Cell Science 125, 2148–2159
? 2012. Published by The Company of Biologists Ltd
Cell migration and invasion are largely dependent on the complex organization of the various cytoskeletal components. Whereas the role
of actin filaments and microtubules in cell motility is well established, the role of intermediate filaments in this process is incompletely
understood. Organization and structure of the keratin cytoskeleton, which consists of heteropolymers of at least one type 1 and one type
2 intermediate filament, are in part regulated by post-translational modifications. In particular, phosphorylation events influence the
properties of the keratin network. Sphingosylphosphorylcholine (SPC) is a bioactive lipid with the exceptional ability to change the
organization of the keratin cytoskeleton, leading to reorganization of keratin filaments, increased elasticity, and subsequently increased
migration of epithelial tumor cells. Here we investigate the signaling pathways that mediate SPC-induced keratin reorganization and the
role of keratin phosphorylation in this process. We establish that the MEK–ERK signaling cascade regulates both SPC-induced keratin
phosphorylation and reorganization in human pancreatic and gastric cancer cells and identify Ser431 in keratin 8 as the crucial residue
whose phosphorylation is required and sufficient to induce keratin reorganization and consequently enhanced migration of human
epithelial tumor cells.
Key words: Intermediate filaments, Mitogen-activated protein kinases, Sphingosylphosphorylcholine, Gastric cancer cells, Pancreatic cancer cells
Cell migration and invasion are markedly dependent on the
complex organization of the cytoskeleton (Ballestrem et al.,
2000). The cytoskeleton of epithelial cells is a network of three
major classes of filamentous biopolymers: microfilaments,
microtubules and intermediate filaments. Intermediate filaments
are composed of a large family of cell-specific proteins that
organize to form 10 nm filaments sharing sequence homology
and structural features. Among the cytoplasmic intermediate
filament proteins, keratins are expressed preferentially in
epithelial cells (Fuchs and Weber, 1994; Coulombe and Omary,
2002) and constitute nearly 5% of the total protein in these
cells (Omary et al., 1998). Keratin filaments are obligate
heteropolymers of at least one type I (relatively acidic keratins
K9–K28, K31–K40) and one type II keratin (relatively basic
keratins K1–K8, K71–K86) (Schweizer et al., 2006). These
filaments are usually organized into bundles, the so called
tonofibrils, which form cage-like structures around the nucleus
and extend from the perinuclear region to the cell periphery
(Hatzfeld and Franke, 1985).
K8 and K18 are the major components of intermediate
filaments of simple epithelia as found in intestine, liver and
exocrine pancreas (Fuchs and Weber, 1994; Coulombe and
Omary, 2002). The expression pattern of these proteins is
generally persistent in carcinomas arising from tissues that
normally express K8 and K18 (Oshima et al., 1996). Keratins
play a crucial role in maintaining the structural integrity and the
mechanical properties of cells and thereby protect cells from
a variety of environmental insults (Yamada et al., 2002).
Furthermore, they are major determinants for the mechanical
features of the cytoplasm and the nucleus (Maniotis et al., 1997;
Fuchs and Cleveland, 1998).
The structure and function of keratins are probably regulated
on serine residues within the so-called ‘head’ (N-terminal) and/
or ‘tail’ (C-terminal)non-a-helical
and Cleveland, 1998; Omary et al., 2006). Ser52 is the major
phosphorylation site of human K18 in vivo. This site has
been implicated in increased keratin solubility and altered
polymerization (Ku and Omary, 1994; Liao et al., 1995a), keratin
reorganization (Ku et al., 1999), apoptosis (Caulı ´n et al., 1997) and
cellular stress (Omary et al., 1998). Increased phosphorylation of
K18 has also been implicated in the reorganization of keratin
filaments in hepatocytes treated with protein phosphatase inhibitors
(Toivola et al., 1998).
Ser431 is a major in vivo phosphorylation site in human K8.
Ser431 is phosphorylated by mitogen-activated protein kinases
(MAPKs) in response to activation of the EGFR (Omary et al.,
1998). Phosphorylation at this site has also been described
during hyperosmotic stress, whereas hypo-osmotic stress leads to
Journal of Cell Science
dephosphorylation at Ser431 of K8 (Tao et al., 2006), and also
occurs in human and mouse liver upon injury resulting in
Mallory–Denk body formation (Stumptner et al., 2000) or during
mouse liver and gallbladder injury induced by a high-fat diet
(Tao et al., 2003).
Sphingosylphosphorylcholine (SPC) is a naturally occurring
bioactive lipid that acts as an intracellular and extracellular
signaling molecule in numerous biological processes including
proliferation (Seufferlein and Rozengurt, 1995), cell migration
(Boguslawski et al., 2000), wound healing (Wakita et al., 1998)
and differentiation (Kleger et al., 2007). Similar to other
bioactive lipids such as lysophosphatidic acid (LPA) or
sphingosine-1-phosphate, many of its actions are mediated by
the activation of a subfamily of low- and high-affinity G-protein-
coupled receptors (An et al., 1995; Meyer zu Heringdorf et al.,
Previously, we have shown that SPC is one of the few
naturally occurring compounds that can induce a perinuclear
reorganization of the keratin cytoskeleton in human pancreatic
cancer cells. This reorganization is accompanied by keratin
phosphorylation, including phosphorylation at K18(S52) and
K8(S431), and an increase in cellular elasticity and enhanced
migration of cancer cells through size limited pores (Beil et al.,
2003). However, the precise downstream signaling mechanisms
by which SPC induces keratin reorganization and the role of
keratin phosphorylation in this process are as yet unknown.
Here we show that the MEK–ERK signaling cascade regulates
both SPC-induced K8 phosphorylation at Ser431 and keratin
reorganization in human pancreatic and gastric cancer cells.
We identify Ser431 in K8 as the crucial residue whose
phosphorylation is required and sufficient to induce keratin
reorganization and consequently enhanced migration of human
epithelial tumor cells.
Role of the ERK cascade for SPC-induced keratin
Previously we have demonstrated that SPC reorganizes the
keratin cytoskeleton in Panc-1 and AGS human cancer cells from
a branched phenotype into a perinuclear, ring-like formation and
increases migration of epithelial tumor cells through size-limited
pores (Beil et al., 2003). These cell lines express K8 and K18 as
their major keratins, as shown using a pan anti-keratin antibody
and individual K8 and K18 antibodies (supplementary material
Fig. S1A). This effect of SPC is probably mediated by a G-
protein-coupled receptor. SPC interacts with S1P receptors 1–5,
GPR4 and OGR1 with different affinities (Meyer zu Heringdorf
et al., 2002). Both pancreatic and gastric cancer cell lines express
S1P1–S1P5, GPR4 and OGR1, as determined by RT-PCR
(supplementary material Fig. S1B).
Activation of the ERK signaling cascade has been implicated
in cell migration (Huang et al., 2004; Rajalingam et al., 2005;
Bove et al., 2008). We have previously shown that SPC
potently induces activation of ERKs in fibroblasts (Seufferlein
and Rozengurt, 1994). SPC was also able to stimulate ERK
activation in human pancreatic and gastric cancer cells, reaching
Fig. 1. SPC-induced activation of p42 and p44 (MAPK1 and
MAPK3) phosphorylation. (A) Panc-1 and AGS were incubated
with 12.5 mM SPC for the indicated time points. (B) Cells were
treated with various concentrations of SPC as indicated for 15
minutes (Panc-1) or 30 minutes (AGS). (C) Panc-1 and AGS were
preincubated with PD98059 using the concentrations indicated and
subsequently treated with 12.5 mM SPC for 15 minutes (Panc-1) or
30 minutes (AGS). (D) Panc-1 and AGS cells were preincubated
with the U0126 as indicated and subsequently treated with
12.5 mM SPC for 15 minutes (Panc-1) or 30 minutes (AGS).
Immunoblotting was performed using antibodies against p44 and
p42 (p44/42) and phosphorylated p44 and p42 (Ph-p44/42).
K8 phosphorylation in tumor cell migration2149
Journal of Cell Science
Pan-CK antibody and the phosphospecific antibody detecting K8(S431) (5B3)
were used for immunofluorescence microscopy. After the indicated stimulation,
cells were fixed with 4% formaldehyde for 10 minutes. Antibodies were added
overnight in PBS supplemented with 0.5% Triton X-100 and 0.2% Gelatin (Sigma,
St Louis, MO) at 4˚C. Cells were then incubated with Alexa Fluor 488, 568 or 647
coupled to secondary antibodies (Invitrogen). Finally, slides were embedded in
GelTol Aqueous Mounting Medium (Immunotech). Imaging was performed with
confocal laser-scanning microscope LSM510 Meta (Carl Zeiss, Jena, Germany)
equipped with a 636 1.4 NA oil objective using the indicated filters or a Keyence
BZ-8000 fluorescence microscope. Images show representative cells from at least
three independent experiments.
Cell migration through size-limited pores
Panc-1 cell migration was examined using a modified 48-well Boyden chamber
(Nucleopore, Neuro Probe, Gaithersburg, MD) and collagen-coated polycarbonate
membranes with a pore diameter of 12 mm (Nucleopore). Panc-1 cells (26105
cells ml21) in DMEM were allowed to migrate towards a gradient of the indicated
agents for a total of 4 hours in a humidified incubator (37˚C; 5% CO2). Adherent
cells on the filter membrane were fixed in 99% ethanol for 10 minutes and stained
using Giemsa dye. For a quantitative assessment of migrated cells, from three
different wells in each case, five high-power fields (15 in total) were counted. The
data shown represent the percentage of migrated cells, compared with the
Migration assays with time-lapse microscopy
Panc-1 cells were seeded on fibronectin (Roche Diagnostics, Penzberg, Germany)
covered glass-slides 3 hours before start of migration assay. Glass slides were
packed together with serum-free DMEM containing 1% penicillin-streptomycin
into a sample sandwich and sealed with wax. The sample sandwich was kept at
37˚C and time-lapse was run for 16 hours. Cells expressing K8(WT),K18(WT),
K8(SE),K18(WT) and K8(SA),K18(WT) were transfected 24 hours before the
start of migration assays. Time-lapse photos were analyzed using ImageJ (NIH).
Western blot analysis and extraction of keratins
For keratin extraction, serum-starved cultures of Panc-1 and AGS cells were
treated with factors as indicated and lyzed at 4˚C in 20 mM Tris-HCl at pH 7.4, 0.6
M potassium chloride, 1% Triton X-100 and 1 mM PMSF (Triton high-salt
buffer). Lysates were incubated for 20 minutes on ice and cleared by centrifugation
at 10,000 g for 20 minutes at 4˚C. The resulting pellet was subsequently
resuspended in the same buffer, incubated for a further 20 minutes on ice and again
subjected to centrifugation at 10,000 g for 20 minutes at 4˚C. The pellet of
insoluble proteins was resuspended in 5 volumes of 8 M urea and the same volume
of 56 SDS-PAGE sample buffer was added to the solution. Samples were then
separated by SDS-PAGE.
For western blot analysis, serum starved cultures of Panc-1 and AGS cells were
treated as indicated and lyzed at 4˚C in NP-40 lysis buffer (150 mM NaCl, 20 mM
Tris-HCl, 10% glycerol, 1% NP-40, 100 mM Na4O7P2, 100 mM NaVO4, 1M NaF,
10 mg/ml aprotinin, 100 mg/ml leupeptin, 0.7 mg/ml pepstatin). Lysates were
incubated for 10 minutes at 4˚C and subjected to centrifugation at 14,000 g for 10
minutes at 4˚C. Supernatants of the samples were resuspended in 56 SDS-PAGE
sample buffer and separated by SDS-PAGE.
Quantification of keratin morphology
Panc-1 and AGS cells were transfected as indicated and keratin morphology was
quantified by determining the percentage of cells with a predominant perinuclear
or ramified keratin organization using fluorescence microscopy. Keratin
organization was classified as predominantly perinuclear when more than 70%
of keratin, as assessed by visual judgment, was organized around the nucleus and
consequently the cytoplasm contained fewer keratin filaments. The formation of a
strict ring structure was frequently observed, but not required for the classification
as perinuclear. Keratin organization was classified as predominantly cytoplasmic
when more than 70% of keratin was organized in the cytoplasm and consequently
the cytoplasm contained more keratin filaments than the perinuclear region.
Quantification was performed in a blinded fashion in at least three independent
experiments with 50–200 cells per experiment.
Quantification of fluorescence intensity distribution
immunofluorescence or CFP-tagged keratins as described in the figure legends,
ortho-max projections of confocal image sections from top to bottom of each cell
were analyzed. Cells were imaged using a SP5 confocal microscope (Leica) with
similar settings. Three linear ROIs of equal length were placed in perinuclear and
cytoplasmic regions of each cell (LAS AF Lite Software, Leica). The normalized
intensity profile for all ROI was integrated by calculating area under the curve and
the mean intensity ratio of perinuclear to cytoplasmic ROIs for each cell was used
an indicator for cytokeratin distribution under different conditions. Height of
keratin distribution visualizedbyindirect
cells (Z-Volume) was calculated from confocal image stacks. Calculations and
statistical analysis was performed using GraphPad Prism version 5.00.
Reverse transcriptase and PCR
mRNA was prepared from either Panc-1 or AGS cells and semi-quantitative RT-
PCR analysis was conducted with specific primers for SPC receptors as described
previously (Kleger et al., 2007).
K8 and K18 mRNA levels were measured using the LightCycler System (Roche
Diagnostics, Mannheim, Germany) or the ICyclerIQ system from Bio-Rad.
Primers used in real-time PCR were as follows: human keratin 18: QuantiTect
Primer Assay Hs_KRT18_2_SG. Human keratin 8: 59-GCCGTGGTTGTGAA-
GAA-39 and 59-CTGTTCCCAGTGCTACCCT-39. Human HMBS: 59-CCCTG-
GAGAAGAATGAAGTGGA-39 and 59-TGGGTGAAAGACAACAGCATC-39.
For K8 knockdown, the siRNA probe: GGGUGACCCAGAAGUCCUA labeled
with 39-fluorescein or 39-Alexa-Fluor-488 was used. As a control, an AllStar
siRNA labeled with Alexa Fluor 546 (Qiagen, Hilden, Germany) was used. To
deplete K18, we used a mixture of two different siRNA constructs: Stealth RNAi
KRT18-HSS142770 (cat. no. 5194538, Invitrogen) and Hs-KRT18-3 CK (59-
ccgccggatagtggatggcaa-39, 59-gccggauaguggauggcaatt-39) (Qiagen). AllStar siRNA
labeled with Alexa Fluor 546 (Qiagen) was used as a control. For knockdown of p44
and p42 MAPKs, ERK1/2 siRNA from Cell Signaling was used. Panc1 cells were
transfected using Hyperfect (Qiagen) according to manufacturer’s instructions.
To obtain amino acid exchange of K8(Ser431) and K18(Ser52), site-directed
mutagenesis with pK8–eCFP and pK18–eYFP (Rudolf Leube, Universita ¨tsklinikum
Aachen, Germany) (Wo ¨ll et al., 2005) as matrices were performed using
QuikChange XL-sdm-Kit (Stratagene, La Jolla, CA) according to the instruction
manual. To replace K8(S431) and K8(S52) with Alanine (A) or Glutamic acid (E),
primers as follows were used: K8(S431A): TATGGGGGCCTCCAGCCCCC-
GGCCTCA; K8(S431E): CTATGGGGGCCTCACAGAACCCGGGCTCAGCT-
ACAG; K18(S52A): ATCTCCGTGTCCCGGTCGACCGCCTTCAGGGGC; K-
Serum-starved Panc-1 cells were subjected to a random migration on fibronectin
and imaged by the time-lapse video microscopy. Glass bottom culture dishes
(MakTek Corporation) were coated with 50 mg/ml of fibronectin (Roche). Cells
were allowed to spread for 3 hours on the fibronectin-coated dishes in DMEM with
1% FCS. Imaging was performed using a BZ-8000 Keyence microscope. During
imaging cells were kept at 37˚C in an atmosphere containing 5% CO2. A motion
picture (AVI format) was created from time-lapse images using the BZ-Analyzer
software (Keyence Corporation). Cell movement was analyzed using tracking
routines implemented in ImageJ software. Three independent experiments were
done for each condition.
Isolation and analysis of keratin fractions
Keratins were isolated from three cellular fractions. The cytosolic fraction was
obtained after disrupting cells by centrifugation at 100,000 g for 90 minutes in
Buffer A [PBS with 10 mM EDTA and protease and phosphatase inhibitor cocktail
(Roche)]. The pellet was then solubilized using 1% NP-40 in buffer A (30 minutes
at 4˚C) followed by centrifugation (16,000 g, 15 minutes, 4˚C) and collecting the
NP-40 fraction. The remaining cytoskeletal fraction was solubilized in 50 mM
Tris-HCl, pH 7.4, 2 mM EDTA with 9.5 M urea.
We thank Rudolf Leube (Aachen) for providing the keratin vectors
pK8–eCFP and pK18–eYFP, and Claudia Ruhland and Ulrike Mayr-
Beyrle for expert technical assistance.
This work was supported by the Deutsche Krebshilfe [grant number
107344 to T.S.]; and National Institutes of Health [grant number
DK47918 to M.B.O.]. Deposited in PMC for release after 12 months.
Supplementary material available online at
Journal of Cell Science 125 (9)2158
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