From the Veterans Affairs Palo Alto Health Care System and Stanford University Digestive Disease Center, Palo Alto, California 94304 and § CLONTECH Laboratories, Palo Alto, California 94303
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ABSTRACT |
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Keratin polypeptide 19 (K19) is a type I
intermediate filament protein that is expressed in stratified and
simple-type epithelia. Little is known regarding K19 regulation or
function, and the only other type I keratin that has been studied in
terms of regulation is keratin 18 (K18). We characterized K19
phosphorylation as a handle to study its function. In vivo,
serine is the major phosphorylated residue, and phosphopeptide mapping
of 32PO4-labeled K19 generates one major
phosphopeptide. Edman degradation suggested that the radiolabeled
phosphopeptide represents K19 Ser-10 and/or Ser-35 phosphorylation.
Mutation of Ser-10 or Ser-35 followed by transfection confirmed that
Ser-35 is the major K19 phosphorylation site. Transfection of Ser-35
The cytoskeleton of most mammalian cells consists of three
distinct major protein families: microtubules, microfilaments, and
intermediate filaments (1). Among these three cytoskeletal protein
groups, intermediate filaments
(IF)1 have several unique
features (2-6). For example, IF proteins make up a complex and diverse
family; they are expressed in a tissue-preferential manner; their
functions are poorly understood; they have a characteristic structure
that consists of a central coiled-coil Although the likely multidimensional functional roles of keratins are
still under active study, two important functions are well established,
and keratin phosphorylation appears to play a role in both functions.
The first function is maintaining cell mechanical integrity as has been
clearly demonstrated from the skin and ocular keratin-associated
diseases (reviewed in Refs. 4-6) and from transgenic models that
manifest a liver phenotype (reviewed in Ref. 14). In the case of the
liver, there is a strong correlation between exposure to a variety of
cell stresses, with resultant hepatocyte toxicity, and a significant
increase in keratin phosphorylation (reviewed in Refs. 15 and 16). To
that end, expression of K18 that is mutated at its major
phosphorylation site (serine 52) in transgenic mice is associated with
a significant increase in susceptibility to hepatotoxic injury as
compared with transgenic mice that overexpress wild type K18 (17). A
second functional role for keratins is the interaction with other
cellular proteins such as the 14-3-3 protein family, which presumably
regulates the known interaction of 14-3-3 proteins with a number of
their binding partners (18). The keratin/14-3-3 association is cell cycle-regulated in that it occurs with K18 (but not K19 or K8) only
during the S and G2/M phases of the cell cycle in a
reversible fashion (19) upon phosphorylation of K18 serine 33 (18).
Therefore, the characterization of keratin phosphorylation and its
significance is emerging as a productive approach for understanding not
only keratin regulation but also its function.
In this report we focus on characterizing K19 phosphorylation and begin
to address its significance. The only keratins that have been studied
in detail with respect to their phosphorylation are K1 (20), K8, and
K18 (reviewed in Refs. 15 and 16). However, most if not all of the 20 keratins are likely to be phosphorylated based on several criteria
including: (i) the presence of multiple keratin-charged isoforms upon
analysis by two-dimensional gels, (ii) the presence of numerous
serines/threonines (Ser/Thr), many of which are in proximity to
arginine/lysine/proline residues that indicate potential kinase
targets, and (iii) biochemical evidence for the phosphorylation of
several of the epidermal keratins (21, 22). Here we identify Ser-35 as
the major K19 phosphorylation site during interphase and show that
mutation of this site interferes with K8/19 filament assembly in
transfected NIH-3T3 cells. K19 phosphorylation is dynamic and is likely
to involve multiple sites based on the increase in the number of K19
phosphopeptides and 32PO4 incorporation upon
treatment of cells with the Ser/Thr phosphatase inhibitor okadaic acid
(OA). Although K19 and K18 type I keratins have some similarities in
their phosphorylation properties, we show that they also have unique
features that are likely to have functional correlates.
Cell Culture, Reagents, and Metabolic Labeling--
HT29
(human), BHK (hamster), and NIH-3T3 (mouse) cells were obtained from
the American Type Culture Collection (Manassas, VA). K19 cDNAs were
kindly provided by Drs. Pierre Coulombe (The Johns Hopkins University)
and Richard Eckert (Case Western Reserve University). Antibodies used
in this work were monoclonal antibody (mAb) L2A1, which recognizes
human K18 (23); rabbit antibody 8592, which recognizes human K8 and K18
(24); mAbs 4.62 and VIM-13.2, which recognize human K19 and vimentin,
respectively, from several species (Sigma). Other reagents used were OA
(Alexis Corp., San Diego, CA), immobilized protein A-Sepharose and the BCA protein determination kit (Pierce), trypsin and chymotrypsin (Worthington), enhanced chemiluminescence (ECL) reagent (Amersham Pharmacia Biotech), 32PO4-phosphoric acid (NEN
Life Science Products).
HT29 cells were cultured using RPMI 1640 medium; BHK cells and NIH-3T3
cells were cultured in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and
100 µg/ml streptomycin at 37 °C. BHK cell culture medium also
included 100 mM methotrexate. For okadaic acid treatment, cells were cultured with 1 µg/ml OA for 2 h (37 °C). Cell
cycle analysis was carried out after fixing cells with 70% ethanol and then staining with propidium iodide as described (23). HT29 or
transfected BHK cells were labeled with 32PO4
(250 µCi/ml, 5 h, 37 °C) in phosphate-free RPMI 1640 medium containing 8% dialyzed fetal calf serum and 2% (v/v) normal
phosphate-containing medium. For labeling in the presence of OA, the
phosphatase inhibitor was added during the last 2 h of labeling.
Immunoprecipitation--
Cells, with or without different
treatments, were collected and washed with phosphate-buffered saline
(PBS), pH 7.4. Cells were solubilized with 1% Nonidet P-40 in PBS (pH
7.4) containing 5 mM EDTA, 0.5 mM
phenylmethanesulfonyl fluoride, 10 µM pepstatin A, 10 µM leupeptin, 25 µg/ml aprotinin, and 0.5 µg/ml
okadaic acid (buffer A) (45 min, 4 °C), followed by pelleting
(16,000 × g, 5 min, 4 °C). For some experiments,
the residual pellet was subsequently solubilized with 1% Empigen BB
(Emp) in buffer A (1 h, 4 °C) (25). The supernatants were used for
immunoprecipitation with mAb L2A1 (covalently conjugated to protein
A-Sepharose) or with mAb 4.62 (plus protein A or G-Sepharose for 6 h at 4 °C) followed by washing five times with 0.5% Nonidet P-40 in
PBS.
Biochemical Analysis Methods--
Immunoprecipitates were
analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) using 10% polyacrylamide gels under nonreducing conditions
(26). Proteins were stained with Coomassie Blue R-250. Two-dimensional
gel electrophoresis was carried out as described (27) with isoelectric
focusing in the first dimension (horizontal) and SDS-PAGE in the second dimension (vertical). The stoichiometry of K19 phosphorylation was
estimated by densitometric scanning of Coomassie-stained isoelectric spots 1, 2, and 3 and then calculating the optical density ratio of
phosphorylated spot 3 to the total phosphorylated and unphosphorylated K19 spots (1 + 2 + 3). For peptide mapping, K19 bands were isolated from Coomassie-stained gels followed by electroelution, concentration, acetone precipitation, and digestion with trypsin or chymotrypsin as
described (28, 29). Peptide mapping was done using cellulose thin layer
chromatography plates, with separation in the horizontal dimension by
electrophoresis and in the vertical dimension by chromatography (28).
For manual Edman degradation, the major cellulose-containing
32PO4-labeled spot was removed from cellulose
plates followed by water extraction and lyophilization. The dried
peptide was subjected to manual Edman degradation for 5-15 cycles as
described (29). The released materials after each cycle and the disks
containing residual undigested peptide were counted.
Construction of the K19 Serine Mutant and Cell
Transfections--
K19 Ser-35 Immunofluorescence Staining--
Transfected NIH-3T3 cells were
cultured on coverslips for 3 days and then fixed in methanol (3 min,
Characterization of K19 Phosphorylation--
We studied and
characterized K19 phosphorylation to begin understanding its regulation
and function. Metabolic labeling of colonic HT29 cells with
32PO4 followed by immunoprecipitation of K8/19
showed incorporation of phosphate into both K8 and K19 (Fig.
1A). Phosphoamino acid analysis of 32PO4-labeled K19 indicated that
all of the incorporated radioactivity resided on serine (Fig.
1C), which is similar to what has been noted for the
epidermal keratins (20, 21, 30) and for K8 and K18 (23, 31).
Previous phosphorylation studies of K8 and K18 showed that K8/18
phosphorylation increases during mitosis and that there is a
phosphorylation gradient in that the membrane/cytosolic keratin fraction has a higher specific activity of phosphorylation as compared
with the remaining cytoskeletal compartment (15, 19). The
membrane/cytosolic-enriched fraction is isolated using nonionic detergents (e.g. Nonidet P-40), and the remaining
cytoskeletal fraction is efficiently extracted, although maintaining
antigenicity for immunoprecipitation, using the zwitterionic detergent
Empigen (25). We tested if K19 has similar phosphorylation properties to K8 and K18. As shown in Fig. 1A, K19 manifests a
phosphorylation gradient similar to K8 and K18 in that its
phosphorylation increases in mitotically enriched cells (Fig.
1A, compare lane 2' with 1' and
lane 3' with 4'). In addition, K19 (and K8)
phosphorylation is increased in the Nonidet P-40 fraction as compared
with the Emp fraction (Fig. 1A, compare lane 1'
with 3' and lanes 2' with 4').
Furthermore, two-dimensional gel analysis (Fig. 1B) shows an
acidic shift of K8 (isoform 2) and K19 (isoform 3) in K8/19 that is
isolated from the Nonidet P-40 fraction as compared with the Emp
fraction. Similar acidic shifts in K19 and K8 were also noted in
mitotically enriched cells after two-dimensional gel analysis (not
shown). The stoichiometry of K19 phosphorylation in the Nonidet P-40
and Emp fractions under basal conditions is ~24 and 18%,
respectively (see "Experimental Procedures"). Of note, K19 isoform
2 does not appear to be phosphorylated as determined by the lack of
incorporation of 32PO4 upon metabolic labeling,
which is incorporated into isoform 3 only (not shown). A similar
nonphosphorylated "isoform 2" was also observed for K18 (17), but
the identity of the acidic modification(s) that accounts for this
isoform is presently unknown.
Identification of Serine 35 as the Major K19 Phosphorylation
Site--
We used biochemical and molecular means to identify the K19
phosphorylation site(s). Metabolic labeling of K19 with
32PO4 followed by protease digestion with
trypsin or chymotrypsin and peptide mapping generated one major
radiolabeled phosphopeptide (Fig.
2A). Manual Edman degradation
of the purified phosphopeptide showed a single dominant radioactive
serine that corresponds to the third amino acid position of the tryptic
phosphopeptide (Fig. 2B) and the fourth amino acid position
of the chymotryptic phosphopeptide (Fig. 2C). Inspection of
the known K19 amino acid sequence (10, 11) and incorporation of the
Edman degradation results and the predicted digestion sites of trypsin
and chymotrypsin indicated that the major K19 phosphopeptide can only
represent serine 10 (6YRQSS) and/or serine 35 (31FRAPS) phosphorylation. Mutation of K19 at
Ser-10 or Ser-35 followed by transfection into BHK cells, radiolabeling
with 32PO4, and tryptic peptide mapping showed
that Ser-35 is the major phosphorylation site of K19 (Fig.
3D, note absence of the
Ser-35-containing spot).
Role of K19 Serine 35 in Keratin Filament Organization--
Given
that keratin phosphorylation can play a role in keratin filament
organization, we asked if K19 Ser-35 has an effect on keratin filament
assembly. As shown in Fig. 4,
transfection of Ser-35 Relative Dynamic Nature of K8, K18, and K19
Phosphorylation--
Our earlier observations of K8/18 phosphorylation
showed that K8 phosphorylation is ~3-4-fold higher than K18 (19).
The relatively similar basal phosphorylation of K8 and K19 (Fig.
1A, lanes 1' and 3') prompted us to
formally examine the relative stoichiometry and dynamic nature of K8,
K18, and K19 phosphorylation. We also compared the effect of the
phosphatase I and IIA inhibitor OA (32) on K8, K18, and K19
phosphorylation. As shown in Fig. 5A (with quantitation in Table
II), basal K19 and K8 phosphorylation are
nearly equal, although basal K18 phosphorylation is significantly less
than that of K8. Protein phosphatase inhibition by OA increases the
phosphorylation of all keratins, but the increase in K19
phosphorylation is significantly less than that of K8 or K18 (Table II
and Fig. 5A). The "relatively low" K19
hyperphosphorylation in the presence of OA is also evident by the small
change in migration in SDS-PAGE gels as compared with K8 and K18 (Fig.
5A, hyperphosphorylated K8, K18, and K19 bands are
highlighted by an asterisk). The results of these
comparisons between K19 and K18 phosphorylation suggest that K19
in vivo phosphorylation is regulated very differently than
K18 and that K19 in vivo dephosphorylation involves
different phosphatase(s) than those involved in K8 or K18
dephosphorylation.
Although K19 has one major phosphorylation site basally
(i.e. Ser-35), it is likely that several other
phosphorylation sites occur in vivo under different
conditions. For example, human K19 has 38 serines (9-11), 20 of which
are located in the head and tail domains, which are the most likely
domains to undergo post-translational modifications based on what is
known regarding IF protein phosphorylation (15, 16, 33, 34). Support
for the presence of other K19 phosphorylation sites is evident after
tryptic peptide mapping of K19 that is isolated from OA-treated
32PO4-labeled HT29 cells (Fig. 5B,
compare panels a and b). Under these conditions,
four other phosphopeptides become major radiolabeled peptides in
addition to the Ser-35-containing phosphopeptide.
General Properties and Significance of K19
Phosphorylation--
Our results showed that K19 phosphorylation,
under basal interphase conditions, can be accounted for by the
predominant phosphorylation at a single, serine 35. This was confirmed
by phosphoamino acid analysis (Fig. 1C), tryptic and
chymotryptic peptide mapping (Fig. 2A), and manual Edman
degradation (Fig. 2, B and C), which narrowed down the number of potential phosphorylation sites from the total of 38 K19 serines to 2 serines (Ser-10 or Ser-35). Confirmation of Ser-35 as
the major K19 phosphorylation site was carried out by mutational
analysis coupled with tryptic peptide mapping (Fig. 3). More
importantly, mutation of K19 Ser-35 resulted in a filament assembly
defect that consisted of perinuclear filament collapse and a propensity
to form short filaments in transfected cells (Fig. 4 and Table I). The
assembly defect that we observed differs from other K8 (Ser-73,
Ser-431, and Ser-23) and K18 (Ser-33 and Ser-52) phosphorylation sites
that appear to have normal filament assembly upon cell transfection of
their corresponding phosphorylation mutants (18, 19,
35).2 Notably, this assembly
defect was unique to Ser-35 of K19 because mutation of Ser-10 in K19
did not have any noticeable impact on filament assembly (Fig. 4 and
Table I). Defects in filament organization may be envisioned to occur
at the level of assembly as noted for K19 Ser-35 or at the level of
stimulus-induced filament reorganization as noted for K18 Ser-52 (35)
and for nuclear lamin A Ser-22 (36).
Several potential explanations may be envisioned that could account for
the observed assembly defect. The most attractive potential is the
higher stoichiometry of K19 Ser-35 phosphorylation, in terms of single
site phosphorylation, as compared with the only three other keratins
whose phosphorylation has been studied in some detail. These keratins
are: K8, which has similar overall phosphorylation stoichiometry to K19
but is divided nearly equally among 3-4 phosphorylation sites (29);
K1, which also has multiple phosphorylation sites (8 serines and 1 threonine, Ref. 20); and K18, which has one major phosphorylation site
but 3-4-fold lower phosphorylation stoichiometry. In this context, K19
Ser-35 phosphorylation could play a significant role in the in
vivo assembly of K19-containing filaments possibly by impacting on
the structure and/or the protein-protein interaction of K19. In
addition, K19 appears to be basally significantly less stable than
other type I keratins in terms of its ability to form stable complexes
with type II keratins including K8 (37). This raises the possibility that K19 may be more susceptible to modulation by modifications such as
phosphorylation changes. Other less likely possibilities that could
account for the observed phenotype include an effect of the Ser-35
A role for phosphorylation in intermediate filament assembly has
been demonstrated in vitro using purified keratins and a number of other IF proteins (reviewed in Refs. 15 and 16). This
regulation can be modulated by affecting the solubility of keratins or
by regulating keratin-keratin interaction or keratin interaction with
other associated proteins. For example, in vitro phosphorylation of rat K8/18 by several kinases results in keratin filament disruption and increased solubility (38), and microinjection of cAMP into BHK cells results in IF reorganization (39). Furthermore, there are many examples of increased K8/18 solubility in association with increased keratin phosphorylation (reviewed in Ref. 16). Aside
from solubility, keratin phosphorylation can play an essential role in
modulating the interaction with an associated protein. For example, K18
Ser-33 phosphorylation during mitosis regulates the binding of 14-3-3 proteins to K8/18 with consequent modulation of keratin filament
organization (18, 19). At this stage, the mechanism of filament
assembly modulation by K19 Ser-35 phosphorylation is not known, and we
have not observed any easily detectable proteins that associate with
K19 in a Ser-35 phosphorylation-dependent fashion. Notably,
there is a solubility gradient of K8/19 that correlates with increased
K8/19 phosphorylation. For example, the relative K19 phosphorylation in
the more soluble Nonidet P-40 fraction is higher than that in the less
soluble Emp ("cytoskeletal") fraction (Fig. 1, A and
B).
Comparison of the Known Phosphorylation Properties among Type I
Keratins--
The sequence context of K19 Ser-35
(32RAPSIH) is conserved in multiple species
including human (10, 40, 41), bovine (9), mouse (42), and potoroo (43)
but is unique when compared with other keratins. Based on the sequence
context and known kinase consensus sequences (44), several kinases may
be potential in vivo phosphorylating enzymes including
cAMP-dependent protein kinase, protein kinase C, and
calmodulin kinase II.
Although K19 phosphorylation may have features that are
reminiscent of K18 phosphorylation, there are several distinguishing features that are highlighted by this study. The similarities among K18
and K19 phosphorylation are: (i) a single major interphase serine
phosphorylation site (Ser-52 for K18, Ref. 35; and Ser-35 for K19, Fig.
3), (ii) a phosphorylation gradient with a relatively higher K18 and
K19 phosphorylation in the cytosolic/membrane compartments (i.e. Nonidet P-40 solubilized) versus the
remaining cytoskeletal compartment (i.e. Emp solubilized)
(see Ref. 19 for K18 and Fig. 1 for K19), and (iii) the emergence of
multiple phosphorylation sites upon phosphatase inhibition (see Ref. 45
for K18, and Fig. 5 for K19). The unique features of K19
phosphorylation, as compared with K18, are its higher basal
phosphorylation state and the limited response to protein phosphatase
types I and IIA inhibition. The relatively limited response of K19
phosphorylation to OA suggests that at least a significant portion of
K19 phosphorylation is likely to be regulated by a unique set of
kinases and phosphatases as compared with K8 and K18. This distinct
regulation supports the hypothesis that K19 serves specific function(s)
in epithelial cells, which is also supported by the unusual K19
structural feature of having a very short 13-amino acid tail domain as
compared with other keratins (e.g. the next smallest
keratin, K18, has a 39-amino acid tail, Refs. 46 and 47).
Ala K19 showed a filament assembly defect as compared with normal
or with Ser-10
Ala K19. Comparison of K18 and K19 phosphorylation
features in interphase cells showed that both are phosphorylated
primarily at a single site, preferentially in the soluble
versus the insoluble keratin fractions. K19 has higher
basal phosphorylation, whereas K18 phosphorylation is far more
sensitive to phosphatase type I and IIA inhibition. Our results
demonstrate that Ser-35 is the major K19 interphase phosphorylation
site and that it plays a role in keratin filament assembly. K19 and K18
phosphorylations share some features but also have distinct properties
that suggest different regulation of type I keratins within the same cells.
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-helical domain (termed
"rod") that is flanked by globular N-terminal (termed "head")
and C-terminal (termed "tail") domains; and several mutations have
been identified in IF proteins that result in a variety of skin, oral,
and ocular diseases. Another feature of IF proteins is their expression
in the nucleus (i.e. the lamins) and in the cytoplasm
(2-4). Among the cytoplasmic IF proteins, keratins make up the largest
family and are expressed specifically in epithelial cells in a
cell-specific manner (7, 8). Keratins (K) include more than 20 unique
gene products (termed K1-K20) that are divided into type I (K9-K20)
and type II (K1-K8) (7, 8). Most epithelial cells express at least one
type I and one type II keratin as their predominant IF protein complement in an epithelial cell-specific manner (7). For example, basal keratinocytes express K5/14 with little if any K1/10, whereas suprabasal keratinocytes lose most if not all of their K5/14 and express K1/10. In addition, "simple-type" epithelia as found in the
liver, exocrine pancreas, and intestine express K8/18 with various
levels of K19 and K20. Among the keratins, K19 is unique in that it has
a very short 13-amino acid tail domain, it is expressed in stratified
and simple-type epithelia and in hair follicles, and it may serve as a
skin stem cell marker (9-13).
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Ala and Ser-10
Ala mutants were
generated using a TransformerTM kit (CLONTECH, Palo
Alto, CA). The cDNA for human K19 was used as a template. Mutation
was confirmed by sequencing followed by subcloning into the pMRB101
mammalian expression vector with an hCMV promoter-directed expression.
BHK or NIH-3T3 cells were cotransfected with wild type human K8 and one
of three K19 constructs (wild type, Ser-35
Ala, or Ser-10
Ala).
Given the phenotype that we observed with the Ser-35 K19 mutant, we
also sequenced the entire K19 Ser-35
Ala construct to confirm the absence of any other inadvertent mutations.
20 °C). Fixed cells were blocked with PBS that contained 2%
bovine serum albumin and a 1% cytoplasmic extract of nontransfected
NIH-3T3 cells (15 min). Blocked cells were coincubated with mAb 4.62 (1:50 dilution) and rabbit antibody 8592 (1:1000 dilution) in PBS
containing 2% bovine serum albumin for 30 min. After washing, cells
were blocked again with PBS containing 2% bovine serum albumin and 2%
goat serum for 15 min followed by incubating with Texas Red and
fluorescein isothiocyanate-conjugated goat anti-mouse and goat
anti-rabbit antibodies, respectively (30 min), and then washed five
times with PBS. Cells were visualized using a Nikon TE300 microscope coupled to a Bio-Rad MRC1024 confocal microscope. Quantitation of the
keratin filament assembly phenotype was done by assigning the
visualized cells (done by an independent investigator without prior
knowledge of the transfection construct type to prevent bias) as having
normal filaments, collapsed filaments (perinuclear), or short
filaments. All fields on the slide were counted, and cells that did not
double stain with the anti-K19 (mAb 4.62) and antibody 8592 were excluded.
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Fig. 1.
Characterization of human K19
phosphorylation. A, HT29 cells that are near confluent
(enriched in the G0/G1 stage of the cell cycle)
or growing in log phase (enriched in the S/G2/M phases of
the cell cycle) were labeled with 32PO4
followed by sequential solubilization with Nonidet P-40
(NP40) and Emp as described under "Experimental
Procedures." The Nonidet P-40 and Emp cell lysates were then used to
immunoprecipitate K19 with mAb 4.62 followed by separation by SDS-PAGE,
Coomassie Blue staining, and autoradiography. Lanes 1-4
show the Coomassie stain of the corresponding lanes
1'-4' of the radiograph. Note the increase in K8/19
phosphorylation in log phase versus confluent cells and the
relatively higher specific activity of K19 phosphorylation in the
Nonidet P-40 versus the Emp fraction. Cell cycle analysis of
duplicate dishes of the near-confluent and log phase cells showed 37 and 47% of S/G2/M cells, respectively (not shown).
B, precipitates that are similar to those shown in
A were obtained from
non-32PO4-labeled HT29 cells followed by
isoelectric focusing (IEF) in the horizontal dimension,
SDS-PAGE in the vertical dimension, and then Coomassie staining of the
gel. Note that the relative Coomassie intensity ratio of K19 isoform 3 (the only phosphorylated isoform under basal conditions) to the sum of
K19 isoforms is higher in K19 that is isolated from the Nonidet P-40
fraction as compared with K19 that is isolated from the Emp fraction.
C, HT29 cells were labeled with
32PO4 (250 µCi/ml, 5 h) followed by
solubilization with Emp, precipitation of K8/19, separation of K19 by
SDS-PAGE, and phosphoamino acid analysis as described under
"Experimental Procedures." pS, phosphoserine;
pT, phosphothreonine; pY, phosphotyrosine.
Dotted circles highlight the absence of detectable
phosphothreonine and phosphotyrosine.
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Fig. 2.
Phosphopeptide mapping of human K19 and
manual Edman degradation of tryptic and chymotryptic peptides.
A, HT29 cells were metabolically labeled with
32PO4-phosphate (5 h, 250 µCi/ml) followed by
immunoprecipitation of K8/19, separation of K8 from K19 using
preparative SDS-PAGE gels, electroelution of the K19 bands, and acetone
precipitation. Purified K19 was digested with trypsin or chymotrypsin
followed by separation of the peptides using electrophoresis in the
horizontal direction and then chromatography in the vertical direction.
x indicates the origin of samples spotted. B,
manual Edman degradation was carried out on the tryptic peptide T (from
A) as described under "Experimental Procedures." The
histogram shows the counts released after each cycle. C,
manual Edman degradation of peptide C (from A).
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Fig. 3.
The major human K19 phosphorylation site is
Ser-35. A, BHK cells were cotransfected with wild type K8
and one of three K19 constructs that corresponded to wild type K19
(WT), Ser-10 Ala K19 (S10A), or Ser-35
Ala K19 (S35A). After 3 days, cells were solubilized
followed by immunoprecipitation of K8/19 using mAb 4.62. B-D, BHK cells were cotransfected with K8 and one of the
three K19 constructs shown in A. After 3 days, cells were
metabolically labeled with 32PO4 (5 h, 250 µCi/ml) followed by isolation of K19 and tryptic peptide mapping as
in Fig. 2. Note that transfected K19 in BHK cells undergoes
phosphorylation at several sites. The arrows in B
and C correspond to the major tryptic peptide that is also
phosphorylated in HT29 human cells (confirmed by mixed peptide mapping,
not shown). The dotted arrow in D highlights the
absence of a labeled phosphopeptide that corresponds to Ser-35, thereby
indicating that Ser-35 is the major K19 phosphorylation site.
Ala K19 into NIH-3T3 cells had a significant
effect on filament assembly as compared with WT or with Ser-10
Ala K19. As such, the Ser-35 K19 mutant resulted in a preponderance of
cells that manifested perinuclear collapse (e.g. Fig.
4C) or short cytoplasmic filaments (e.g. Fig.
4D). For example, WT and Ser-10
Ala K19 transfectants
had 70 and 69%, respectively, of their cells with a normal-appearing
filament organization, whereas the Ser-35
Ala K19 transfectants had
only 26% of their cells with normal-appearing filaments (Table
I). Similar results were also obtained in
transfected BHK cells (determined qualitatively, not shown). We chose
NIH-3T3 cells for detailed quantitation of the immunofluorescence
experiments instead of BHK cells (which were used primarily for protein
isolation and peptide mapping experiments) because the latter have a
significantly higher transfection efficiency (at the protein level) but
far fewer cells that have normal-appearing filaments for all three
constructs (particularly Ser-35
Ala K19), thereby making assessment
of an assembly phenotype in BHK cells inadequate. The keratin filament
disruption that is noted in transfected NIH-3T3 cells does not affect
the endogenous IF network (vimentin), as determined by double
immunofluorescence staining using anti-vimentin and anti-keratin
antibodies (not shown).
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Fig. 4.
Effect of the Ser-35 Ala K19 mutation on filament organization. NIH-3T3 cells
were cotransfected with WT K8 and one of the following three K19
constructs: A, WT K19; B, S-10
A K19;
C and D, S-35
A K19. Three days after
transfection, cells were double-stained with mAb 4.62 (anti-K19) and
with antibody 8592, which recognizes K8 and K18 as described under
"Experimental Procedures." The figure shows only the anti-K19
antibody staining, which manifested complete overlap with antibody 8592 staining (not shown). A and B demonstrate
normal-appearing filamentous arrays, whereas C and
D show examples of perinuclear collapse (C) and
short cytoplasmic filaments (D) (highlighted by
arrows). Quantitation of the keratin network morphology in
the transfected cells is shown in Table I.
Filament assembly phenotype of wild type and mutant K19 contructs in
transfected cells
Ala, and Ser-10
Ala. After 3 days, cells
were double stained as described in Fig. 4 and then scored as having
normal, collapsed, or short filaments. The percent ± S.E. is
shown for each filament type and construct and represents an average of
three experiments. The transfection efficiency was similar for each
experiment and ranged from ~5 to 10%. The number of cells counted
per experiment (and per construct) was 60-120.
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Fig. 5.
Effect of okadaic acid on K8/18/19
phosphorylation and tryptic peptide mapping of K19 that is isolated
from okadaic acid-treated HT29 cells. A, HT29 cells were
labeled with 32PO4 for 5 h in duplicate
dishes. OA (1 µg/ml) was added during the last 2 h of labeling
to one of the duplicate dishes. Labeled cells (±OA) were solubilized
with Emp followed by precipitation of K8/19 using mAb 4.62 (4.62 immunoprecipitate (i.p.)), or of K8/18 using mAb L2A1 (L2A1
immunoprecipitate). Immunoprecipitates were analyzed by SDS-PAGE,
Coomassie Blue staining, and autoradiography. Note that the
heteropolymeric nature of the keratins results in minimal but
detectable levels of K19 that coprecipitate with K8/18 (although mAb
L2A1 recognizes K18), and similar small amounts of K18 coprecipitate
with K8/19 despite the K19 specificity of the 4.62 mAb. The specific
activity of 32PO4 incorporation into K8, K18,
and K19 is shown in Table II. B, K19 was isolated from HT29
cells that were labeled with 32PO4 in the
presence or absence of OA (as in A) followed by tryptic
peptide mapping as described in Fig. 2. Peptide map b shows
a mix of K19 that was isolated from cells in the absence of OA (map is
identical to that shown in Fig. 2) + K19 that was isolated from cells
treated with okadaic acid.
Efficiency of K8, K18, and K19 32PO4 labeling in
cultured cells in the presence and absence of okadaic acid
OA), were noted in two other separate experiments (not shown).
The relative amount of the major type I and type II keratin per
immunoprecipitate was nearly equal, as determined by densitometric
scanning (not shown), so that the indicated counts represent the
specific activity.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Ala mutation, independent of phosphorylation, or differences in the
expression level of the various transfected K19 constructs. To that
end, K19 Ser-10
Ala did not have any significant effect on keratin
assembly (Fig. 4 and Table I), and the transfection efficiency of the
different keratin constructs was similar as determined by
immunofluorescence staining and immunoblotting of transfected cells
(not shown).
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. Pierre Coulombe and Richard Eckert for the cDNA constructs, Dr. Diana Toivola for assistance with confocal microscopy, Kris Morrow for preparing the figures, and Romola L. Breckenridge for assistance with word processing.
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FOOTNOTES |
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* This work was supported by Veterans Affairs Career Development Award, National Institutes of Health Grant DK47918, Training Grant DK07056, and Digestive Disease Center Grant DK38707.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom reprint requests should be addressed: Palo Alto Veterans
Affairs Medical Center, 154J, 3801 Miranda Ave., Palo Alto, CA 94304.
¶ To whom correspondence should be addressed: Palo Alto Veterans Affairs Medical Center, 154J, 3801 Miranda Ave., Palo Alto, CA 94304. Fax: 650-852-3259.
2 N-O. Ku and M. B. Omary, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: IF, intermediate filament(s); Emp, Empigen BB; K, keratin; mAb, monoclonal antibody; OA, okadaic acid; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; WT, wild type.
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