Institute of Molecular Biology, Department of Cell Biology, Austrian Academy of Sciences, Billrothstrasse 11, A-5020 Salzburg, Austria
* Author for corrspondence (e-mail: mgimona{at}server1.imolbio.oeaw.ac.at )
Accepted 5 March 2001
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Summary |
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Key words: Calponin, Repeats, Regulation, Localization, Actin binding
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Introduction |
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CaP interacts in vitro with a large variety of cytoskeletal components,
including myosin (Szymanski and Tao,
1993; Szymanski and Tao,
1997
), caldesmon
(Vancompernolle et al., 1990
;
Graceffa et al., 1996
), desmin
(Mabuchi et al., 1997
), and
tubulin (Fujii et al., 1997
),
and also with phospholipids (Bogatcheva and
Gusev, 1995
; Fujii et al.,
1995
), extracellular regulated Ser/Thr kinases (ERK) involved in
MAP kinase signaling pathways (Menice et
al., 1997
; Leinweber et al.,
1999a
) and protein kinase C
(Leinweber et al., 2000
). One
common biological property of all three CaP isoforms is binding to actin
filaments; however, the subtle differences in biological functions, which can
be predicted from the tissue-specific expression patterns of the individual
isoforms, have not been determined.
The interaction of CaP with actin is mediated by two independent
actin-binding sites: a strong (`high affinity') binding site (ABS1)
encompassing residues 142-163 and a second, `low affinity' binding site (ABS2)
consisting of three copies of the 29 residue CLIK
(calponin-like repeat) motif and extending to residue
266 in h1 CaP (Mino et al.,
1998; Gimona and Mital,
1998
). Sequences beyond Cys273 are different in, and unique for,
all three mammalian CaP variants and for the Xenopus XCaP H3
molecule. In h2 and ac. CaP these C-terminal tail sequences contain a
number of acidic residues that account for the observed differences in the
total isoelectric points of the different CaP isoforms. The role of the
N-terminal type 3 calponin homology (CH) domain in CaP is not well understood
(Gimona et al., 2002
) and a
number of different functions have been assigned to this region including the
binding to phospholipids and ERK1. In general, however, the CH domain is
dispensable for actin binding in vitro and in vivo and probably plays a role
in targeting CaP to the cell cortex or in recruiting additional CaP-binding
partners.
Three dimensional image reconstructions of CaP in complex with F-actin have
revealed the position of the N-terminal CH domain in a position similar to
that occupied by other CH proteins such as -actinin or fimbrin
(Hodgkinson et al., 1997
;
Tang et al., 2001
;
Hanein et al., 1998
). However,
the remaining mass of the CaP molecule, still comprising two thirds of the
molecule, remained undetected and this region has been suggested to be
unstructured or disordered. Similar conclusions were drawn from structural
predictions of the CaP C-terminus, which appears to be mostly random coil.
Thus, the precise location of CaP, and particularly its C-terminal region,
along the filament remain to be elucidated. In our earlier studies we have
shown that deletion of the C-terminal tail sequences strongly enhances
cytoskeletal association, especially for the h2 variant of CaP
(Danninger and Gimona, 2000
).
The amino acid sequence of h2 CaP differs from that of h1 or
ac. CaP in two critical positions (K151 and K156) within the region of the
ABS1. A K154E mutation introduced into chicken
CaP (equivalent to the
K156 position in the mouse h1 CaP sequence) has been shown to
significantly reduce the actin-binding capacity of recombinant chicken gizzard
CaP in vitro. Together, these data point towards a non-functional ABS1 in
h2 CaP and thus actin binding of h2 CaP can be achieved only
via a functional ABS2. From this we concluded that the tail regions regulate
the ABS2 of CaP. This hypothesis was underscored by findings of Bartegi and
colleagues who demonstrated a global conformational change in the C-terminal
domain of CaP upon actin binding (Bartegi
et al., 1999
). Their data already emphasized the potential
participation of the C-terminal region (including the tandem CLIK repeats) not
only in binding to actin directly, but also in the regulation of the
interaction of CaP with actin filaments and possibly other, as yet unknown
components of the cytoskeleton.
We have now tested our hypothesis that the tail sequences regulate the actin-binding site(s) in CaP by deletion and site-directed mutagenesis studies. We demonstrate that the tails regulate the ABS2 region in all three genetic CaP isoforms and that this regulatory function requires the negatively charged residues present in the tail sequences. These results have significant implications for the understanding of CaP function and regulation in vivo and provide important information for the interpretation of structural data.
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Materials and Methods |
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ABS1 REV: 5'-AAC GTC TCT GGC TCA AAG TTC CGC TCT TGT; h2 ch-knockout: H2Charge FWD: 5'-CCC AAG TAC TGC CCA CAG GGA TCT GCA GCT AAC GGG GCT CCT GCG GGT AAC G; H2Charge REV: 5'-GAG AGA ATT CTC AGT AAC CGG CTT GCT GCT GGC AGT AGG CTA GGT ACT GTG GGG CTT GGC C. Tail switch mutants were constructed by multiple digestion and ligation steps using the full length CaP cDNAs as templates. All constructs were sequenced using a LI-Cor model 4000 automated sequencer (MWG Biotech, Germany).
Cell culture, transfection and immunofluorescence microscopy
A7r5 rat smooth muscle cells were grown in low glucose (1000 mg/l) DMEM
without phenol red supplemented with 10% FBS (PAA, Austria),
penicillin/streptomycin (Gibco, Austria) at 37°C and 5% CO2.
For transient expression, cells were grown in 60 mm plastic culture dishes and
transfected using Superfect (Qiagen, Hilden) at 70% confluence, essentially as
described elsewhere (Kranewitter et al.,
2001). Expression and stability of the constructs was assessed by
Western blotting using a monoclonal antibody against GFP (Clontech, Germany).
Cells were replated onto 15 mm coverslips 16 hours post-transfection and
prepared for immunofluorescence microscopy after an additional 48 hours on
glass coverslips. Cells were washed three times in PBS (138 mM NaCl, 26 mM
KCl, 84 mM Na2HPO4, 14 mM KH2PO4,
pH 7.4), extracted in 3.7% paraformaldehyde (PFA)/0.3% Triton X-100 in PBS for
5 minutes and fixed in 3.7% PFA (Merck, Germany) in PBS for 30 minutes. Alexa
568 phalloidin was from Molecular Probes (Leiden, NL). Fluorescent images were
recorded on a Zeiss Axioscope equipped with an Axiocam driven by the
manufacturer's software package (all Zeiss, Vienna) using a 63x oil
immersion lens.
Electrophoresis and western blotting
Analytical SDS gel electrophoresis on 8-22% gradient polyacrylamide
mini-slab gels and western blotting onto nitrocellulose (Amersham, Austria)
was performed as described elsewhere
(Gimona et al., 1990).
Transferred proteins were visualized using horseradish-peroxidase-coupled
secondary antibodies and the ECL chemiluminescence detection system (Amersham,
Austria).
Cell extraction and fractionation
Transiently transfected A7r5 cells grown in 60 mm petri dishes were washed
twice with ice-cold PBS and extracted with 200 µl extraction buffer (10 mM
Hepes pH 7.5, 1.5 mM MgCl2, 1 mM DTE, 0.1 mM PMSF, 0.67 µg/ml
pepstatin, 1.67 µg/ml leupeptin, 0.5% Triton X-100, containing either 10,
120 or 300 mM KCl) for 30 minutes. Cells were scraped off the dish with a cell
scraper and the extract was centrifuged at 100,000 g for 30 minutes
at 4°C in a Sorvall RC M150 GX centrifuge (Inula, Austria). The
supernatant was centrifuged again at 100,000 g for 20 minutes. To the
supernatant of this latter centrifugation one-quarter volume of 5x SDS
sample buffer was added. Pellets were resuspended in an equal amount of SDS
sample buffer. The distribution of GFP CaP proteins in the pellet and
supernatant was estimated by densitometric scanning of western blots using a
monoclonal antibody against GFP and Adobe PhotoShop 5.02 and HEROLAB Easy
WinTM software (Herolab, Wiesloch, Germany).
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Results |
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Expression and subcellular localization of h1 and
h2 calponin mutants
To study the influence of the C-terminal sequences on actin binding of the
three CaP isoforms, we constructed a series of EGFP-tagged mutants
(Fig. 1C). We have shown
previously that EGFP can serve as a suitable tag for both biochemical and
fluorescence studies (Danninger and Gimona,
2000; Kranewitter et al.,
2001
). A7r5 cells were transfected with the indicated constructs
and cells were either plated on coverslips for observation by
immunofluorescence microscopy, or extracted under different ionic conditions.
The cytoskeletal and soluble fractions were separated by centrifugation for
western blot analysis. As demonstrated before, both h1 CaP and the
h1
t mutant localize to the central stress fibers in A7r5
cells. No GFP signal is detected at the ends of stress fibers or at the cell
periphery (Fig. 2). While there
is no obvious difference in stress fiber association between the h1
and h1
t mutant, the extraction profile indicates that the
mutant lacking the C-terminal tail sequences is more strongly retained in the
insoluble cytoskeleton fraction (Fig.
2; Fig. 3A),
pointing towards an increased actin association.
|
|
By contrast, removal of the tail sequence in h2 CaP causes a
significant shift in the localization pattern of the protein from the cell
periphery to the central actin stress fibers
(Fig. 4), resembling the
h1 and h1t variants. At the same time, extractability
of the h2
t CaP construct is decreased significantly
(Fig. 3B), again arguing for an
increase in cytoskeletal association due to the removal of an autoinhibitory
sequence.
|
Since deletion of the tail apparently increased the association of CaP with the actin cytoskeleton, we asked whether the inhibitory action depends merely on the presence of a C-terminal extension, or whether the individual tail sequences have isoform-specific function(s). To address this question, we exchanged the tails of h1 and h2 CaP with that of one of the three CaP variants and assayed for localization and cytoskeleton association as above. In the case of h1 CaP, fusion to either h2 CaP or ac. CaP tails reduces the amount of h1 CaP retained in the cytoskeletal fraction, with the more negatively charged h2 CaP sequence having a more pronounced effect (Fig. 3A). The localization pattern of the GFP constructs in transfected A7r5 cells is also altered: the previously observed, strictly central stress fiber localization is lost and a fraction of the h1 CaP mutant protein is localized in the more peripheral regions of the cells (Fig. 5).
|
Again, the opposite effect is seen with the tail-switched constructs of h2 CaP. Neither the h1 CaP nor the ac. CaP tail can cause a decrease in the actin association in h2 CaP equivalent to the native h2 CaP sequence (Fig. 3B), and the cellular localization is likewise altered in comparison to full length h2 CaP (Fig. 6). Notably, the ac. CaP tail, containing a significantly greater number of negatively charged residues and exhibiting an isoelectric point closer to that of h2 CaP (Table 1), is more efficient in `downregulating' CaP than the neutral h1 CaP tail at high ionic strength (300 mM KCl) extraction conditions (Fig. 3B), but not at the lower ionic conditions of 10 and 120 mM KCl, respectively. Despite a longer tail sequence, containing a larger number of negatively charged amino acids, the net isoelectric point of the ac. CaP tail is slightly more basic than that of h2 CaP tail. Thus, the regulatory potential of the tails followed the calculated net charge of the tail sequences, the most acidic h2 CaP tail (pI 3.09) being more efficient than the less negatively charged ac. CaP tail (pI 3.53), whereas the h1 CaP tail (pI 5.88) had little effect in downregulating actin association.
|
|
Since the three CaP variants differ essentially only in their sequences C-terminal of Cys273, the results of the `tail-switch' experiments suggest that there may be specific information within the individual CaP tails, sufficient for differential regulation or isoform-specific function(s). We tested this hypothesis by introducing six point mutations in the h2 CaP tail, changing the aspartate and glutamate residues to asparagine and glutamine, respectively (mutant h2 ch-knockout). The isoelectric point of this mutant sequence is basic at 7.99 (Table 1). This `neutralized' tail now causes h2 CaP to associate preferentially with the central actin stress fibers in transfected A7r5 cells (Fig. 4) and significantly increases the actin association in the extraction assay (Fig. 3B), Notably, the effect is almost identical to that observed with the h2-h1t mutant, supporting our initial theory that the negatively charged residues indeed play a role in the autoinhibition of the actin-binding site(s).
While the above experiments document an autoinhibitory role for the tail in
h2 CaP, the situation is less clear for h1 CaP. This is
primarily due to the presence of two autonomous functional actin-binding
sites, ABS1 and ABS2, in this isoform. Gong et al. have shown that a mutation
in the critical lysine residue at position 154 in the chicken sequence (156 in
mouse) causes a significant reduction in actin binding
(Gong et al., 1993). This
residue and the more N-terminal arginine 151 are both absent in the ABS1
sequences of mouse h2 CaP (Fig.
1B), suggesting that this site is non-functional in this variant.
Therefore, we introduced the identical mutations into the ABS1 sequence of
h1 CaP in order to eliminate the function of this site. In accordance
with the previous report (Gong et al.,
1993
), this mutant displays a significantly reduced cytoskeletal
association in extraction experiments, and the localization on actin stress
fibers is less defined in transfected A7r5 cells
(Fig. 2;
Fig. 3A). However, actin
binding is not abolished completely, indicating that the ABS2 is intact and
also functional in h1 CaP. With this latter mutation (h1
ABS1-knockout) in hand, we were then able to further test the influence of the
specific tail sequence of h1 CaP on this isoform's actin-binding and
localization behavior. In agreement with the results obtained with the
h2 CaP mutants, we expected that deletion of the tail would release
the presumptive block on the ABS2. Indeed, additional deletion of the tail
sequence from the h1 ABS1-knockout mutant completely restored both
the extraction profile and subcellular localization to almost wild-type
levels, despite the mutations in the ABS1.
We also attempted to verify the increased actin association of the t
mutants by direct biochemical analysis employing actin co-sedimentation
assays. For this purpose, we re-cloned the three constructs into a vector
suitable for expression in bacteria. However, the tailless constructs for
h2 and ac. CaP were unstable in solution, thus preventing detailed
analysis (not shown). However, note that h1
t CaP bound more
strongly to purified smooth muscle CaP than its wild-type counterpart and
showed increased actin-bundling activity (G.B., W.J.K. and M.G., unpublished),
supporting our data obtained in the localization and extraction
experiments.
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Discussion |
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Mutational analysis in h1 CaP
Most of the subtle changes concerning the actin-binding/cytoskeletal
association in this major CaP isoform are obscured by the function of the
strong binding ABS1. Deletion of the tail sequences had little detectable
effect on actin binding, owing to the high intrinsic actin affinity of the
intact molecule. However, altering the critical residues K151 and K156 in the
ABS1 from the h1 to the h2 type severely affected both the
subcellular localization and extraction behavior for this mutant, but did not
abolish actin binding. Notably, the extraction profile for the h1
ABS1-knockout mutant resembled that of the `charge-neutralized' h2
ch-knockout construct. Thus, as for h2 CaP, the ABS2 appears to
function as an autonomous binding site in this variant. In accordance with our
original hypothesis, additional deletion of the regulatory tail largely
restored binding back to levels similar to that found in the h2
ch-knockout and h2t mutant. Thus, these mutations allow us to
separate the relative contributions of the two ABSs in h1 CaP.
Kolakowski et al. proposed the existence of two independent actin-binding
sites on the calponin molecule and hypothesized further that one site may be
involved in the binding/bundling activity of the CaP molecule, whereas the
second may be involved in regulating the actomyosin ATPase activity
(Kolakowski et al., 1995
). As
discussed below, these functional requirements may be performed by the ABS2
and ABS1, respectively, as outlined in this study. However, the resolution of
our immunofluorescence studies is not sufficient to answer these questions
unequivocally and the localization experiments must thus be seen together with
the data obtained from the extraction studies. Although the ABS2 can clearly
function as an actin-binding site in h2 CaP, it remains to be shown
whether the ABS1 forms an independent site or contributes to an overall
extended actin-binding interface in the h1 CaP isoform.
Mutational analysis in h2 CaP
For h2 CaP the consequences of deletion of the tail were much more
evident due to the nonfunctional ABS1. Removal of the C-terminal 35 amino acid
residues unmasked the ABS2, significantly increasing the actin association of
this mutant. In addition, neutralization of the negative charges in the tail
had a similar, yet weaker effect suggesting that the negative charges in the
CaP tails are essential for the regulation of the ABS2. Additional tail switch
analysis further support our hypothesis that the tails regulate only the ABS2,
and not the ABS1, the latter being functional only in h1 and ac. CaP.
Whereas both h1 CaP and ac. CaP tails were capable of downregulating
actin association for the h2 CaP variant, the ac. CaP tail had almost
no effect on the extraction of h1 CaP. Surprisingly, however, the
tail sequences of h2 CaP caused a reduction in actin association of
h1 CaP, best seen at the high ionic extraction conditions. These
latter results are in good agreement with those obtained from the h1
CaP mutants discussed above: altering the ABS1 sequence to an h2 CaP
type results in a mutant resembling h2 CaP fused to the h1
CaP tail. Indeed, the extraction profiles and subcellular localization of the
h2-h1t and h1 ABS-knockout mutant are almost
indistinguishable. These data clearly point towards an autoregulatory function
of the isoform specific CaP tails.
Regulation of protein function by structurally flexible tail regions is a
common theme in cytoskeletal proteins. The focal adhesion protein vinculin
exposes a cryptic actin-binding site in its C-terminus upon binding to actin
and phosphatidylinositol (4,5)-bisphosphate, and/or phosphorylation by protein
kinase C (Weeks et al., 1996;
Steimle et al., 1999
). This
F-actin-binding site is regulated by an intramolecular interaction between the
acidic, globular head region and the basic vinculin tail. ERM proteins (ezrin,
radixin, moesin and merlin) undergo a similar structural transition upon
phosphorylation of a threonine residue in the C-terminal tail region by
Rho-kinase, which releases a block on the F-actin-binding site residing in the
C-terminal part (Tsukita et al.,
1997
; Matsui et al.,
1998
; Pearson et al.,
2000
). It remains to be shown whether the interactions of the CaP
tails also involve the globular, all helical N-terminal CH domain of the
molecule. A molecular domain arrangement similar to that of CaP (an N-terminal
CH domain and a negatively charged C-terminal tail domain) is present in the
microtubule plus-end binding protein EB-1, suggesting that head-to-tail
interactions play a role in the regulation of CH domain family proteins.
Together with previous biochemical fluoresecence data
(Bartegi et al., 1999), our
results allow new interpretations of some of the important and controversial
findings about calponin. One possibility is that the tail sequences interact
with the positively charged residues of the CLIK repeats and cause a
conformational change in this region of the molecule. Consequently, the ABS2
becomes regulated and inacessible/nonfunctional. Upon deletion of the tail in
our mutant constructs (or the regulated removal of the tail by conformational
alterations), a further conformational change in the CLIK repeats causes the
formation of a functional second actin-binding site. These interpretations
support the conjecture of Bartegi and co-workers, who suggested that the
C-terminal tail region and the actin-binding site(s) in CaP communicate with
each other and that deletion of the C-terminal sequences can transmit
information to the actin-binding region
(Bartegi et al., 1999
). Our
data indicate that this `information' prevents autoinhibition of the ABS2 by
the tail sequences. The ABS2 probably occupies a novel site along the actin
filament, away from the CH domain docking site (J. L. Hodgkinson and M.G.,
unpublished), as the interactions of both CaP and the C. elegans
protein UNC-87, which contains seven copies of the CLIK module, are not
perturbed by saturating amounts of
-actinin, filamin or tropomyosin
(Kranewitter et al., 2001
).
This site may be similar to that recently identified for myosin light chain
kinase (Hatch et al., 2001
).
Alternatively, the tail may interact (1) intramolecularly with the N-terminal
CH domain of CaP (see above) or (2) via intermolecular association with
specific binding proteins such as serine/threonine kinases. CaP is a substrate
for both Rho-kinase (Kaneko et al.,
2000
), and PKC
and
in vitro
(Leinweber et al., 1999a
;
Leinweber et al., 2000
).
Helical reconstructions of cryo-EM images from CaP-coated F-actin filaments
have revealed the position of the CH domain of CaP. This location is in good
agreement with those reported for two other CH domain proteins, namely fimbrin
and -actinin (Volkmann et al.,
2001
; McGough et al.,
1994
). Thus, the position of the CaP CH domain on the actin
filament may indeed be similar. Crosslinking studies have further revealed
that the ABS1 of h1 CaP, adjacent to the CH domain, interacts with
the C-terminus of actin around Cys374
(Mezgueldi et al., 1992
).
Consequently, the remaining mass of the CaP molecule was not detected in the
helical image reconstructions and therefore was hypothesized to point away
from the filament. The identification of the ABS2 in CaP did not agree with
this interpretation. From our studies, we conclude that the presence of the
tail in both native and recombinant CaP captures the molecule either in the
closed (tail interacting with the CLIK repeats) or open (CLIK repeats forming
the ABS2) conformation. However, the presence of the ABS1 alone is sufficient
to attach CaP with considerable affinity to the filament. The co-existence of
an open and closed conformation of the CaP molecules in solution will
inevitably interfere with cryo-EM image analysis, depicting the flexible
C-terminal portion of CaP as `unstructured'. Further, several groups,
including ours, have tried for many years to grow crystals of either native or
recombinant CaP for X-ray diffraction studies, but with little success. The
presence of a highly flexible tail and the conformational alterations of the
unstructured CLIK repeats in the presence of the tail sequences may account
for the failure to determine the 3D structure of CaP. Thus we expect that our
tailless (
t) mutants should help to overcome these problems for both
image reconstruction and crystallization studies.
Although several studies have provided evidence for the specific sorting of
ß-cytoplasmic actin and of ß-actin mRNA to the cell periphery and
the ruffling membrane regions of motile cells
(Herman, 1993;
Bassell et al., 1998
),
stationary cells such as REF-52 fibroblasts or A7r5 smooth muscle cells appear
to incorporate both ß- and
-cytoplasmic, as well as smooth muscle
-actin into their stress fibers, without detectable regional
preference. Hence, the observed specific interaction of CaP with the central
stress fibers may reflect a structural difference of the actin filament itself
(Egelman and Orlova, 2001
)
rather than actin isotype sorting. Katoh and colleagues
(Katoh et al., 2001a
;
Katoh et al., 2001b
) have
argued that in human fibroblasts myosin light chain kinase (MLCK) and Rho
kinase impact differentially on the structural organization of the actin
cytoskeleton. Whereas MLCK may regulate peripheral stress fibers, Rho-kinase
is involved in the modulation of the central stress fibers. The two types of
stress fibers exhibit differential contractile properties, but potential
differences in their molecular composition remain to be determined. In
addition, Lehman (Lehman,
1991
) demonstrated that caldesmon and calponin are present on
distinct types of thin filaments in avian smooth muscle in vivo, suggesting a
functional segregation of thin filament subpopulations in smooth muscle. Taken
together, these results provoke the speculation that CaP may play a role as a
signal transduction molecule for only a certain subset of actin filaments
under the control of the Rho/Rhokinase pathway and primarily involved in
cytoskeleton stabilization.
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Conclusions |
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Acknowledgments |
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