From the Tumor Biology Program, Department of Biochemistry and Molecular Biology, Mayo Graduate School and Mayo Clinic Cancer Center, Mayo Clinic/Foundation, Rochester, Minnesota 55905
Received for publication, November 6, 2000, and in revised form, January 16, 2001
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ABSTRACT |
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Human calmodulin-like protein (CLP) is an
epithelial-specific Ca2+-binding protein whose
expression is strongly down-regulated in cancers. Like calmodulin, CLP
is thought to regulate cellular processes via
Ca2+-dependent interactions with specific
target proteins. Using gel overlays, we identified a ~210-kDa protein
binding specifically and in a Ca2+-dependent
manner to CLP, but not to calmodulin. Yeast two-hybrid screening
yielded a CLP-interacting clone encoding the three light chain binding
IQ motifs of human "unconventional" myosin X. Pull-down experiments
showed CLP binding to the IQ domain to be direct and Ca2+-dependent. CLP interacted strongly with IQ
motif 3 (Kd ~0.5 nM) as determined by
surface plasmon resonance. Epitope-tagged myosin X was localized
preferentially at the cell periphery in MCF-7 cells, and CLP
colocalized with myosin X in these cells. Myosin X was able to
coprecipitate CLP and, to a lesser extent, calmodulin from transfected
COS-1 cells, indicating that CLP is a specific light chain of myosin X
in vivo. Because unconventional myosins participate in
cellular processes ranging from membrane trafficking to signaling and
cell motility, myosin X is an attractive CLP target. Altered myosin X
regulation in (tumor) cells lacking CLP may have as yet unknown
consequences for cell growth and differentiation.
Human calmodulin-like protein
(CLP)1 is encoded by an
intronless gene localized on chromosome 10p13-ter (1), a chromosomal region known to be lost in a number of breast and other cancer types
(2-6). Initial studies failed to show expression of the CLP gene in a
number of tissues and cell types (7); however, in an independent study
aimed at identifying mRNAs coding for transformation-sensitive
proteins, CLP was recloned as a protein named NB1 (for
Normal-Breast-1) from a normal
primary breast epithelial cell line that had been subtracted with RNA
purified from the same cell line after chemical transformation (8). CLP
was subsequently shown to be expressed in a highly tissue-specific
manner in basal and suprabasal epithelial cells of the normal breast,
cervix, prostate, and skin, and to be down-regulated in naturally
occurring breast, cervix, and prostate tumors as well as in
artificially immortalized and transformed breast epithelial cells in
culture (9). Recently, CLP was found to be down-regulated in 80% of primary breast tumors of both noninvasive as well as invasive and
metastatic stages (10). Given the apparent correlation between its
down-regulation and the tumorigenic state, the available data support
the notion that CLP expression may be incompatible with the transformed
state of a cell. The mechanism responsible for this incompatibility is
presently unknown, as is the physiological function of CLP, though they
are likely related.
CLP's deduced amino acid sequence of 148 residues is identical in
length and shows 85% identity to calmodulin (CaM) (7, 11).
Accordingly, CLP shows a number of characteristics similar to those of
CaM; however, it also displays unique features that suggest its
functional divergence (11-13). For example, CLP displays a
Ca2+-dependent electrophoretic mobility shift
that is typical of CaM, and it binds four Ca2+ as does CaM,
albeit with an ~8-fold lower affinity (11, 12). Purified CLP is able
to fully activate several known CaM-regulated enzymes, including CaM
kinase II and cyclic nucleotide phosphodiesterase (although the
Km for CLP activation of the phosphodiesterase is
higher than that for CaM). By contrast, CLP is ineffective at
stimulating other CaM-dependent enzymes such as
calcineurin, NO synthase, and smooth muscle myosin light chain kinase
(11, 13). At present there are no known CaM targets that show higher affinity for CLP than for CaM. On the other hand, the unique expression pattern of CLP and its possible involvement in specific cellular processes (e.g. cell differentiation) raise the possibility
that it may have its own distinct targets. Knowing the identity of the
proteins with which CLP normally interacts will greatly aid in
understanding the normal physiological function(s) of CLP as well as
the consequences of its down-regulation in transformed cells. Here, we
therefore set out to identify such proteins using gel overlays and
yeast two-hybrid interaction screening, and report the cloning and
characterization of a human unconventional myosin (myosin X) as a
specific target of CLP.
Expression and Purification of Recombinant Human CLP--
Human
CLP was expressed in Escherichia coli JM109(DE3) from
plasmid pGem-CLP and purified as described (14). Site-directed mutagenesis (15) was performed to generate an expression vector for
CLPF99Y in which phenylalanine 99 was replaced by tyrosine. A 475-bp EcoRI/BamHI cassette from pGem-CLP was
first subcloned into M13mp18. Recombinant phage were propagated in
E. coli RZ1032, and uracil-containing single-stranded DNA
was isolated for in vitro hybridization to the mutant
oligonucleotide 5'-d(GCG CTG ACG TAG CCG TTG CC)-3'. The hybrid was
elongated by T7 DNA polymerase (Sequenase 2.0, United States
Biochemical Corp.) ligated by T4 ligase and transformed into E. coli JM101. The mutated cassette was then excised from
double-stranded replicative form DNA of an appropriate M13 clone and
religated into EcoRI/BamHI-digested pGem-CLP,
yielding expression vector pGem-CLPF99Y.
CLPF99Y was expressed as described (14).
CLP Overlays--
CLPF99Y was radiolabeled using
"Enzymobeads" according to the manufacturer's instructions
(Bio-Rad, Brussels, Belgium) in 50 mM MOPS (pH 7.5), 100 mM NaCl, 2 mM CaCl2, 0.1% glucose
(w/v), and 1 mCi of Na125I/100 µg of recombinant protein.
The specific activity was ~1.5 × 106 cpm/µg
protein. For biotin labeling, purified recombinant CLP was dissolved at
12 mg/ml in 20 mM HEPES (pH 7.0), 5 mM
Plasmid Construction--
pAS2-CLP and pAS2-CaM were made by
subcloning a NcoI/XbaI fragment carrying the
entire coding sequence from pKK-CLP and pKK-CaM, respectively (11), into pAS2-1 (CLONTECH,
Palo Alto, CA). pGADGH-IQ123K corresponds to the original plasmid
isolated from the HeLa Matchmaker cDNA library
(CLONTECH). It contains nt 2333-2674 of the human myosin X sequence (accession no. AF234532) fused with a linker of
sequence 5'-d(GAA TTC GGC ACG AG)-3' at the 5' end and 5'-d(GAG CTC
(T)15)-3' at the 3' end to the pGADGH vector at cloning
sites EcoRI and XhoI, respectively. The resulting
vector expresses amino acids 705-817 of myosin X as a C-terminal
fusion protein with the yeast Gal4 activation domain. In addition, the
myosin X sequence is followed by lysines resulting from the oligo(dT)
primer used in library construction as well as a few extra C-terminal
residues encoded by the vector. pGADGH-IQ0 was constructed by deletion of an ApaI fragment from this construct and expressed amino
acids 705-751 of myosin X in frame with the Gal4 activation domain. pGADGH-IQ1, -IQ12, and -IQ123 were constructed by amplifying
appropriate fragments from pGADGH-IQ123K using a pGADGH-specific
upstream primer and downstream primers terminating at nt 2511 (IQ1), nt 2579 (IQ12), or nt 2674 (IQ123). The resulting PCR products were cut
with BamHI (cuts within the upstream pGADGH sequence), and then cloned into pBluescript, which had been cut with EcoRV,
T-tailed, and BamHI cut. The
BamHI/XhoI inserts from this vector were then transferred to pGADGH. The resulting constructs expressed amino acids
705-763 (IQ1), 705-785 (IQ12), or 705-817 (IQ123) of myosin X in
fusion with the Gal4 activation domain. pEGFP-HA-His and pEBFP-HA-His
were constructed by inserting a double-stranded oligonucleotide with
the (top strand) sequence 5'-d(pGAT CCT ACC CCT ATG ATG TGC CTG ACT ATG
CCC ACC ACC ACC ACC ACC ACC TGG TGC CCA GGG GCA GCA)-3' into the
BspEI site of plasmids pEGFP and pEBFP
(CLONTECH), thereby reconstituting the
BspEI site at the 3' end only, and introducing the peptide
sequence SYPYDVPDYAHHHHHHLVPRGSR, which contains an HA tag followed by
a His tag, to the C terminus of the GFP/BFP tag. The full-length myosin
X coding sequence from nucleotide position 233 to 6405 (accession no.
AF234532) was then cloned as a KpnI/XbaI fragment
from pBluescript-MyoX into pEBFP-HA-His, creating plasmid
pEBFP-HA-His-MyoX, which expresses the human myosin X with an
N-terminal BFP, HA, and His tag. pEYFP-CLP and pEYFP-CaM were
constructed by PCR amplification using pKK-CLP and pKK-CaM (11) as
templates together with primers pKK233-2K (5'-d(GTG AGC GGA TAA CAG
GTA CCC ACA GGA AAC AG)-3') and pKK233-2r (5'-d(CCG CCA AAA CAG CCA
AGC TTG CAT GCC TG)-3'). The KpnI/XmaI fragment
from each PCR product was then ligated into pEYFP
(CLONTECH). In-frame GST fusion constructs GST-IQ1,
GST-IQ12, and GST-IQ123 were made by amplifying sequences corresponding
to nt 2344-2511 (IQ1), 2344-2579 (IQ12), or 2344-2674 (IQ123) of
myosin X from plasmid pGADGH-IQ123K, TA-cloning the resulting PCR
fragments into pCR2.1 (Invitrogen), and subcloning the EcoRI
insert fragments from this vector into pGEX-2TK (Amersham Pharmacia
Biotech). All vector inserts were completely sequenced to confirm their integrity.
Yeast Two-hybrid Interaction Analysis--
Yeast two-hybrid
screening was performed according to the Matchmaker Two-Hybrid System 2 instructions (CLONTECH). Yeast were transformed
using the lithium acetate method (17) with sheared herring sperm DNA as
carrier. Selection of initial positives was done after 14 days of
growth on SD/ Cloning of Full-length Human Myosin X--
5'-Rapid
amplification of cDNA ends was performed on HeLa cell
Marathon-Ready cDNA as recommended by the supplier
(CLONTECH), using nested primers 5'-d(GTC TTC CCC
AGC TGC CAC TCG CTG TTG G)-3' and 5'-d(AGG CAT CAT AGA GCT GCA GCA GGC
TCG)-3' based on the sequence from the yeast two-hybrid clones. This
resulted in a ~2-kb PCR product, which was sequenced directly using a
primer walking strategy and extended through the initiator codon and ~30 bp of the 5'-untranslated region of the myosin X cDNA. The entire 6-kb human myosin X open reading frame (ORF) was then
PCR-amplified using information from this sequence and from human ESTs
homologous to bovine myosin X sequence. Because the 6-kb PCR product
proved resistant to standard cloning techniques, we cloned the myosin X
ORF cDNA in pieces, which were then reassembled. The 3'-most 2 kb
of the ORF were amplified from Marathon-Ready cDNA using Taq DNA polymerase and cloned as an
EcoRI/XbaI fragment into pBluescript. The
5'-terminal 4 kb of the ORF was amplified from Marathon-Ready cDNA
using Pwo DNA polymerase (Roche Molecular Biochemicals) and cloned as a KpnI/EcoRI fragment into pBluescript.
This fragment contained two mutations, one of which resulted in a
frameshift and was corrected by site-directed mutagenesis using the
GeneEditor kit (Promega, Madison, WI). The 3' fragment was then
subcloned into the EcoRI and XbaI sites of this
vector. The resulting full-length cDNA in pBluescript
(pBluescript-MyoX) contains one discordance from the original sequence
(accession no. AF234532), an A GST Pull-down Assays--
GST and GST fusion
proteins were expressed in E. coli BL21(DE3) from pGEX-2TK
derived vectors upon
isopropyl-1-thio- Surface Plasmon Resonance Analysis--
Surface plasmon
resonance analysis was performed on a BIAcore 1000 instrument using
Sensor Chip SA (streptavidin) chips (Biacore Inc, Piscataway, NJ).
Affinity was measured for the N-terminally biotinylated IQ3 peptide
(biotin-RFLHLKKAAIVFQKQLRGQIARRVYRQ-NH2) synthesized in the
Mayo peptide core facility. Approximately 1000 RU of peptide were bound
to the chip, and varying CLP and CaM concentrations were passed over
the chip. The concentration of purified CLP (14) was measured by UV
absorbance at 276 nm using a molar extinction coefficient of 1500 M Co-immunoprecipitation--
COS-1 cells were obtained from Dr.
John T. Penniston (Mayo Clinic, Rochester, MN) and were grown in
Dulbecco's modified Eagle's medium containing 10% fetal bovine serum
(FBS) and supplemented with 1% antibiotic-antimycotic (all cell
culture reagents from Life Technologies, Inc.). Cells were transfected
at ~80% confluence using Qiagen (Valencia, CA) purified plasmid DNA
and LipofectAMINE 2000 (Life Technologies, Inc.) according to the
manufacturer's directions. Plasmids pEYFP, pEYFP-CLP, or pEYFP-CaM
were cotransfected with either pEBFP-HA-His or pEBFP-HA-His-MyoX, using
a DNA ratio of 1:8 (w/w). Cells were grown for 2 days
post-transfection, washed three times in DPBS (Life Technologies, Inc.)
and scraped into lysis buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 10 mM imidazole, 2 mM
CaCl2, 1% Nonidet P-40, 0.5% sodium deoxycholate (NaDOC), 0.1 mM Na3VO4, 100 µg/ml Pefabloc
(Roche), 40 µg/ml leupeptin, 40 µg/ml aprotinin). Either
Ni2+-NTA-agarose (Qiagen) or anti-HA-agarose beads (Roche)
were added to the lysate, and the lysate was rocked end-over-end for
2 h at 4 °C. The beads were then washed once with lysis buffer
and four times with CaTBST, and finally resuspended in Laemmli buffer containing 5 mM EDTA. Precipitated proteins were detected
by Western blot using anti-GFP antibodies (Santa Cruz Biotechnology
Inc., Santa Cruz, CA), which recognize both YFP and BFP.
Immunofluorescence--
MCF-7 cells were grown on glass
coverslips and transfected with purified plasmid DNA as described
above. Transfected cells were grown for 2 days, then rinsed in DPBS
(Life Technologies, Inc.) and fixed for 20 min in 0.1 M
PIPES (pH 6.95), 1 mM EGTA, 3 mM
MgSO4, 3% formaldehyde. Coverslips were then rinsed three times (3 min each time) in DPBS, permeabilized for 2 min in 0.2% Triton X-100 (Curtis-Matheson Scientific), and blocked for 1 h in
blocking buffer (5% v/v normal goat serum and 2% w/v BSA in DPBS).
Coverslips were then incubated for 1 h in primary antibody (polyclonal rabbit anti-HA IgG, Upstate Biotechnology, Lake Placid, NY)
diluted 1:50 (10 µg/ml) in blocking buffer, rinsed three times in
DPBS, and incubated for 1 h in secondary antibody (5 µg/ml Alexa-350 goat anti-rabbit IgG; Molecular Probes, Eugene, OR) and 25 ng/ml rhodamine-phalloidin (Sigma) in blocking buffer. Following three
rinses in DPBS and a final rinse in dH2O, coverslips were
mounted with ProLong (Molecular Probes) on glass slides.
Identification of a ~210-kDa CLP-binding Protein by Gel
Overlay--
To identify specific CLP targets in total tissue
extracts, we used a gel overlay method proven to be successful in the
identification of CaM target proteins in various tissues and
subcellular fractions (20-22). In previous studies with CaM, the best
results were obtained when 125I-labeled protein was used as
probe. Unfortunately, in CLP a Phe residue replaces the
Tyr99 iodinated in CaM, leading to a dramatic reduction in
the success of CLP iodination. We therefore engineered (by
site-directed mutagenesis) a CLPF99Y expression construct.
Purified CLPF99Y was then labeled with 125I to
a high specific activity and used as probe in overlay experiments, assuming that the single conservative amino acid replacement would not
lead to a major change in target binding specificity. This approach
identified a relatively small number (two to four major bands) of
putative CLP-binding proteins in human breast, cervix, and lung (Fig.
1). The most prominent was a protein with
an estimated molecular mass of 210 kDa. This protein (p210) was also
detected by biotinylated CLP (data not shown), confirming that
detection does not result from the F99Y substitution. p210 reacted only weakly or not at all with CaM in the overlays (Fig. 1).
CLP-binding proteins, including a band for p210, were also detected in
the 100,000 × g microsomal membrane fraction from
cultured HeLa cells (Fig. 2A),
as well as from several other normal and transformed epithelial cell
lines (data not shown). Importantly, the 210-kDa protein interacted
with CLP in a specific, Ca2+-dependent manner;
labeled CLP could be competed off the blot with excess unlabeled CLP,
but not with unlabeled CaM (Fig. 2), and binding was detected in the
presence of Ca2+, but not in the presence of EDTA (data not
shown).
Identification of a Specific CLP-interacting Protein by Yeast
Two-hybrid Screening--
We turned to the yeast two-hybrid approach
(23) to identify CLP interacting proteins and to potentially clone the
p210 protein. We used the MatchmakerTM yeast two-hybrid
system (CLONTECH) to screen an oligo(dT)-primed, directionally cloned HeLa cell cDNA library with a CLP "bait" fused in frame to the Gal4 DNA binding domain in the plasmid pAS2-1 (CLONTECH). Screening 1.7 × 107
initial transformants yielded 1070 colonies, of which 266 were
Using sequence information from the original yeast two hybrid clone, we
performed a 5'-rapid amplification of cDNA ends using HeLa cell
Marathon-Ready cDNA (CLONTECH). The resulting
PCR product contained the entire N-terminal coding sequence of human
myosin X including the initiator codon and ~30 bp of 5'-untranslated region. Using this sequence and sequences present in the human expressed sequence tag data base homologous to the bovine myosin X
sequence, we successfully PCR-amplified the entire 6-kb human myosin X
ORF (accession no. AF234532). Comparison of this cDNA with
subsequently posted human genomic sequence revealed no discordances and
allowed for the mapping of the intron-exon structure (Fig. 3). The human myosin X heavy chain
contains 2058 amino acids with a calculated molecular mass of 238 kDa.
Amino acid sequence comparison of the human with the murine (accession
no. CAB56466) and bovine (accession no. T18519) myosin X heavy chain
amino acid sequence revealed 96% and 93% identity, respectively.
Correspondence of p210 to Myosin X and Confirmation of Interaction
with CLP--
Identification of myosin X as a potential CLP interactor
suggested that myosin X and p210 may be the same protein. Several lines
of evidence support this notion. The calculated molecular mass of
myosin X (238 kDa) matches well with that of p210, considering that the
Mr of p210 was estimated based on its migration
in an SDS-polyacrylamide gel with only few marker proteins in the high molecular weight range. In addition, we tested extracts from yeast expressing the original Gal4AD-myosin X "prey" fusion protein for
reactivity to biotinylated CLP. These extracts show a CLP reactive band
of 36 kDa, which is the expected size for the Gal4AD-myosin X IQ-domain
fusion protein. This band shows a similar affinity for CLP and CaM as
does p210 as determined by competitive overlay (Fig. 2B).
Taken together these data strongly suggest that myosin X is at least
one component and most likely the only component of p210.
We next characterized CLP's interaction with myosin X in more detail.
Coexpression of BFP-HA-His-tagged myosin X and YFP-tagged CLP in MCF-7
cells showed intense localization at the cell periphery for both
proteins with some cytoplasmic localization also evident (Fig.
4B). By contrast, YFP-CaM
demonstrated general cytoplasmic and nuclear localization and much
poorer colocalization with myosin X (Fig. 4C). This mostly
cytoplasmic and nuclear localization was also evident in cells that
were singly transfected with YFP-CLP (Fig. 4A), suggesting
that CLP localization to the plasma membrane was enhanced and perhaps
"driven" by coexpression of myosin X.
CLP and, to a lesser extent, CaM coprecipitated with myosin X (Fig.
5). BFP-HA-His-tagged myosin X or control
BFP-HA-His were coexpressed with YFP-CLP, YFP-CaM, or YFP in
transiently transfected COS-1 cells. BFP-HA-His-myosin X or control
BFP-HA-His were then precipitated with Ni2+-NTA-agarose or
with immobilized anti-HA antibody. Equivalent amounts of precipitated
BFP-HA-His-myosin X and BFP-HA-His were loaded onto SDS-PAGE gels, and
these precipitates were analyzed by Western blot using an anti-GFP
antibody that recognizes both BFP and YFP. CLP and, to a lesser extent,
CaM coprecipitated with myosin X (Fig. 5B, lanes
2 and 3) while no YFP was precipitated (Fig.
5B, lane 1) even though equivalent
amounts of all three proteins were expressed in total cell lysates
(Fig. 5A). On the other hand, when BFP-HA-His was
precipitated, essentially no YFP-CLP or YFP-CaM were coprecipitated
(Fig. 5B, lanes 4 and 5).
An interesting observation was the detection of a second, more slowly
migrating YFP-CLP band, which coprecipitated with myosin X (Fig.
5B, lane 3).
CLP Binds with High Affinity to IQ3 of Myosin X--
We next
wished to determine which region of myosin X binds specifically to CLP.
The yeast two-hybrid screen strongly suggested that the IQ motifs are
involved in CLP binding. This is because the original yeast two-hybrid
clone (pGADGH-IQ123K) only encodes a small portion of the head domain,
all three IQ motifs, and a few amino acid residues of the coiled coil,
followed by an artificial polylysine stretch introduced by the
oligo(dT) primer used in library construction. Yeast two-hybrid vectors
were constructed containing about half of the first IQ motif
(pGADGH-IQ0, amino acids 705-751 of hMyoX), the first IQ motif
(pGADGH-IQ1, amino acids 705-763 of hMyoX), the first two IQ motifs
(pGADGH-IQ12, amino acids 705-786 of hMyoX), and all three IQ motifs
plus eight amino acids of the coiled coil domain (pGADGH-IQ123). Y190
or CG1945 cells were cotransformed with these vectors and either pAS2-CLP or pAS2-CaM baits and assayed for growth on
SD/
These results suggested that the third IQ motif was critical for CLP
binding. We used GST fusion proteins in a pull-down experiment to
demonstrate direct interaction between CLP and the myosin X IQ motifs
(Fig. 7). GST-IQ1, GST-IQ12, GST-IQ123,
or GST alone as control was incubated with bacterial lysates containing
various concentrations of CaM or CLP. At high CLP and CaM
concentrations (5 µM) strong CLP binding to GST-IQ123 was
observed and weaker binding of CaM was also observed. Both of these
binding events were Ca2+-dependent as complex
formation was inhibited in the presence of EDTA (Fig. 7B).
At high CLP concentrations (500 nM), CLP bound to all three
fusion constructs, but as CLP concentration was reduced, binding was
lost, first from GST-IQ1 and then from GST-IQ12. At the lowest CLP
concentration tested (5 nM), only weak binding of CLP to
GST-IQ12 was observed, whereas strong binding to GST-IQ123 was still
observed (Fig. 7A). In no case did we observe CLP or CaM
binding to GST alone (Fig. 7A). These results indicate that CLP, and to a lesser extent CaM, can bind directly to the IQ motifs of
myosin X, that this binding is Ca2+-sensitive, and that the
third IQ motif is required for the highest affinity CLP binding.
Finally, we used surface plasmon resonance to measure the affinity of
CLP for a peptide corresponding to the third IQ motif of myosin X (Fig.
8). Using the known extinction
coefficients for CLP (11) and CaM (19), dilutions of these proteins
were made and their binding to the biotinylated IQ3 peptide immobilized on a streptavidin surface chip was measured in a BIAcore 1000 instrument (Fig. 8A). After blank subtraction, these values
were used to calculate a binding affinity of CLP for the IQ3 peptide using a Scatchard plot (Fig. 8B). The resulting value of 0.5 nM for the Kd gives an indication of the
affinity that CLP likely has for the intact protein. In addition, we
measured the affinity of CaM for the IQ3 peptide and found that CaM
binds to the peptide-coated chip surface with an affinity of ~1
µM.
To test the hypothesis that specific targets for CLP exist, we
performed overlay experiments on different human tissue extracts including breast, lung, and cervix. These overlays demonstrated the
existence of a prominent band at ~210 kDa (p210) that bound to
labeled CLP, but not to labeled CaM. Competition experiments using an
excess of unlabeled CLP or unlabeled CaM further demonstrated the
specificity of the CLP-p210 interaction. Yeast two-hybrid screening
identified human myosin X as a CLP interactor and suggested its
identity to p210. In hindsight, this finding is perhaps not surprising
given the known association of CaM with a number of unconventional
myosins (28). Early overlay experiments had demonstrated weak
interaction between CLP and the chicken skeletal muscle myosin used as
a marker for gel electrophoresis. In addition, some binding of CLP to
purified chicken brush border myosin I was also observed, though this
binding could be effectively competed away by
CaM.2
Myosin X was originally identified by PCR screening of cDNAs using
degenerate primers specific for the myosin head domain (29, 30). The
resulting cDNA fragments included one for a novel myosin named
myosin X. This sequence was subsequently localized to chromosome 15 in
mice and to chromosome 5 in humans (31). Very recently, the cloning and
initial characterization of full-length myosin X from mice, cows, and
humans has been reported (26, 27). Although little is known about
myosin X function, some possibilities have been inferred from its
domain structure (see Refs. 26 and 27; Fig. 3) and its relationship to
other myosins. Like other myosins, myosin X is a molecular motor able
to translocate actin filaments in an in vitro motility assay
(32) and it cosediments with F-actin in an ATP-dependent
manner (27). Like myosins VII and XV (28), myosin X also contains a
FERM domain (band
4.1/ezrin/radixin/moesin, also known as talin homology domain), which appears to be involved in
interaction with transmembrane proteins linked to the cytoskeleton. In
mammals, myosins VII and XV are implicated in deafness syndromes in
which deafness is caused by a disturbance of the stereociliar cytoskeleton (33, 34). An involvement of myosin X in actin cytoskeletal
organization is therefore plausible and has gained recent support
through the demonstration of colocalization of endogenous myosin X with
actin bundles in lamellopodia and membrane ruffles in Madin-Darby
bovine kidney cells as well as in several other cell lines (27). In
addition, the data reported by Berg et al. (27) suggest that
myosin X may relocate with dynamic actin redistribution. The
localization we observed of recombinant myosin X (and of CLP) in
transiently cotransfected MCF7 cells (Fig. 4) matches very well with
that reported for the endogenous myosin X. In addition to the FERM
domain, myosin X contains three pleckstrin homology domains (27).
Pleckstrin homology domains are known to interact with
phosphatidylinositol phosphates, and these lipids are concentrated at
the ruffled edge of chemotactic cells, among other places (35). The
notable biochemical property of p210 (alias myosin X) of poor
solubility in aqueous buffers without detergents fits well with a
possible membrane lipid interaction of this protein.
The interaction of myosin X with CLP was confirmed by a number of
methods. In cells cotransfected with tagged myosin X and CLP, CLP
precipitated along with myosin X when the latter was pulled down with
either anti-HA beads or Ni2+-NTA beads. This indicates that
the two full-length proteins can bind to each other in vivo.
We consistently observed a second CLP band coprecipitating with myosin
X (Fig. 5). Although Ca2+-CLP runs differently than apo-CLP
in SDS-PAGE gels (11), the additional band is unlikely to represent a
Ca2+-bound form of CLP because the sample loading buffer
contained 5 mM EDTA, which is sufficient to cause CLP and
CaM to run in their Ca2+-free form (11). Mobility shifts
such as the one we observed are common among phosphorylated proteins,
and it is therefore possible that a fraction of the myosin X-bound CLP
is phosphorylated. Indeed, CLP shares with CaM several serine and
threonine residues (Thr79, Ser101,
Thr117) as well as a tyrosine residue (Tyr138)
known to be targets for phosphorylation in CaM (36-38). A substantial fraction of CaM tightly associated with the plasma membrane and underlying cytoskeleton has been shown to be phosphorylated by membrane-associated protein kinases (39). Assuming that CLP can be
similarly phosphorylated, and given the
cytoskeletal/membrane-associated location of myosin X, it may well be
that a significant fraction of the myosin X-bound CLP in
vivo is phosphorylated. Both yeast two-hybrid and GST pull-down
experiments implicate IQ3 as a major CLP binding site. Although the
two-hybrid screen indicated that IQ2 can also bind some CLP, the
pull-down assay allowed us to estimate more closely the affinity of CLP
for IQ1 and IQ2. In particular, we observed loss of CLP binding to
GST-IQ12 as we reduced the CLP concentration from 50 to 5 nM, suggesting an affinity of CLP for IQ2 on the order of
tens of nanomolar. Using surface plasmon resonance we found CLP's
affinity for a synthetic IQ3 peptide to be 0.5 ± 0.2 nM, which is in the range of high affinity CaM targets (40)
and unequivocally confirms that myosin X is a bona
fide CLP interacting protein. The affinity of CLP for myosin X as estimated by competitive filter overlay is somewhat lower than
this (low nanomolar). The overlay technique, however, typically underestimates true affinities (20). In addition, affinities measured
between peptides and CaM typically overestimate CaM affinities for the
parent protein. For example, CaM binds to the intact plasma membrane
calcium ATPase with 10-fold lower affinity than it binds to the CaM
binding peptide from that protein (41, 42). Thus, the actual affinity
of CLP for intact myosin X will be less (i.e. the
Kd will be higher) than 0.5 nM, but
still in the low nanomolar range.
It should be noted that full-length myosin X (and its IQ123 domain) can
also bind CaM (as demonstrated by coprecipitation and yeast two-hybrid
mating assays), albeit with reduced affinity when compared with CLP.
The interaction of CLP with the myosin X IQ domains is
Ca2+-dependent. This contrasts with the binding
of CaM to some other myosins, which is often
Ca2+-independent and frequently inhibited by
Ca2+ binding (25). Thus, the conditions used to analyze CLP
binding may not be optimal for CaM interaction with myosin X. Because there are three IQ motifs, each potentially able to bind a CaM-like light chain, the "occupancy" state and actual light chain
composition of myosin X may be complex and may change depending on the
free Ca2+ and relative concentration of potential light
chains (e.g. CaM versus CLP). The distribution of
myosin X has recently been investigated in murine, bovine, and human
tissues (26, 27), indicating that myosin X is fairly ubiquitous but
generally expressed at low levels. Because CLP expression is confined
to epithelial cells, it obviously cannot be the sole light chain of
myosin X. In cells that express significant amounts of CLP
(e.g. mammary epithelial cells, keratinocytes), CLP may well
serve as one of the light chains of myosin X, whereas CaM (and/or
additional, yet to be discovered light chains) will likely fulfill this
purpose in other cell types. Indeed, several other CaM-related proteins
have recently been discovered that serve as light chains of
unconventional myosins. For example, the CaM-related EF-hand protein
Mlc1p has been shown to function as a light chain for Myo2p in
Saccharomyces cerevisiae (43) and MICLC (another CaM-related
"noncalmodulin" protein) has been identified as a light chain for
Acanthamoeba myosin IC (44). In addition, chicken myosin V
binds to at least two other noncalmodulin proteins that likely
correspond to light chains (45). On the other hand, the abundance of
CLP in several epithelial cells (e.g. skin or breast) seems
to exceed the level required if CLP were to serve only as a light chain
for the much less abundant myosin X. It is therefore possible that
additional specific target proteins besides myosin X exist for CLP in
these specialized tissues.
The identification of myosin X as a specific CLP target represents an
important step toward the elucidation of CLP's biological function and
potential involvement in tumor suppression. As mentioned above, the
domain structure of myosin X suggests a role in actin organization at
ruffling membrane edges. As these are frequently associated with
chemotactic responses, it may well be that myosin X is involved in the
organization of cellular membranes in response to specific cellular
signals. In this context it is of interest that keratinocytes exhibit a
rapid chemotactic response, which is decreased upon differentiation, a
time when CLP is up-regulated in these cells. This, along with the
observation that CLP is down-regulated in tumors, suggests that CLP may
function to inhibit the membrane cytoskeletal reorganization required
for some types of cell motility, perhaps via its interaction with
myosin X. The identification of myosin X as a CLP target represents a
crucial step toward elucidating the reason for CLP's consistent
down-regulation in human cancers and may point the way toward novel
therapeutic strategies.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol, 5 mM CaCl2, and biotin
X-NHS (Calbiochem, San Diego, CA) was dissolved at 20 mg/ml in
Me2SO. Biotin solution was added to the CLP solution to
20% (v/v), and the mixture incubated for 1 h at room temperature. The reaction was then quenched by adding ammonium acetate to 1 M, and labeled CLP was separated from free label by gel
filtration using Sephadex G-25 (Sigma). Breast and cervix tissues from
biopsy specimens, and lung from autopsy were obtained from Drs. C. Moll and R. Caduff (Department of Pathology, University Hospital, Zurich, Switzerland). 1 g of tissue was minced with a razor blade and homogenized with a Polytron at 4 °C in 4 ml of 10 mM
Tris-HCl (pH 7.0), 150 mM KCl, 10 mM
1,4-dithio-DL-threitol, 2 mM EDTA, and 0.5 mM phenylmethylsulfonyl fluoride. The homogenate was passed through four layers of cheesecloth and centrifuged for 1 h at 24,000 × g and 4 °C. The pellet was resuspended in
the same buffer and stored at
20 °C until use. For overlays,
protein samples were run in SDS-PAGE gels and blotted to PVDF membranes
(Millipore, Bedford, MA) using standard Western blotting procedures
(16). Blotted membranes were incubated for 5 min in CaTBST (2 mM CaCl2, 50 mM Tris-HCl (pH 7.4),
150 mM NaCl, 0.05% Tween 20) + 1.5%
H2O2, and blocked overnight in 5% nonfat dry
milk in CaTBST. Membranes were probed in 105 cpm/ml (5-10
nM) of iodinated CLPF99Y or in 75 nM biotinylated CLP in CaTBST for 90 min, and then washed
four times (5 min each time) in CaTBST, and exposed to Fuji x-ray film
at
70 °C if 125I-labeled CLPF99Y was used.
If biotin-labeled CLP was used, the blot was probed with
biotin-avidin-horseradish peroxidase (1:3000, Amersham Pharmacia
Biotech), washed as before, and developed using RenaissanceTM chemiluminescent reagent (PerkinElmer Life Sciences).
Trp/
Leu/
His agar plates. 5 mM or 40 mM 3-aminotriazole (Sigma) was added to all
His media used for yeast strains CG1945 or Y190, respectively. Positive clones
were propagated on SD/
Trp/
Leu media. Yeast media components were
from CLONTECH and Difco.
-Galactosidase assays
were performed by freeze-thaw of yeast colonies on filter lifts of
3-day-old streak plates. Bait plasmid was dropped out by cycloheximide
counterselection, using concentrations of 1 and 10 µg/ml for strains
CG1945 and Y190, respectively. Yeast matings were performed in liquid
culture using microtiter plate volumes and yeast strains Y187 (mating type
) and either Y190 or CG1945 (a-type). Plasmid inserts from positive yeast clones were directly amplified after picking 3-day-old yeast colonies into TE containing 0.25 unit/µl lyticase (Sigma). Following 2 h at 37 °C, the yeast were subjected to three
rounds of freeze-thaw and heated to 95 °C for 5 min. They were then
chilled on ice, pelleted, and the supernatant used as template for PCR with pGADGH-specific primers. Library plasmids were prepared from counterselected yeast containing only "prey" plasmids by an
alkaline lysis method (18) and transformed directly into E. coli strain DH5
.
G transition at base 2261, which
results in an arginine for glutamine substitution at amino acid 679. During all PCR reactions, DNA polymerase was not added until the tubes
reached 94 °C (hot start PCR).
-D-galactopyranoside induction as
described (16). Cells were pelleted, resuspended in Tris-buffered
saline (50 mM Tris-HCl (pH 7.4), 150 mM NaCl) plus protease inhibitors (0.2 mM phenylmethylsulfonyl
fluoride, 1 µg/ml pepstatin A, 2 µg/ml leupeptin, 0.2 µg/ml
aprotinin, 10 mM EDTA) and 30 mM
-mercaptoethanol, and lysed by the addition of sarkosyl
(Curtis-Matheson Scientific, Houston, TX) to a final concentration of
1.5%. After 15 min on ice, the lysate was cleared by centrifugation at
15,000 × g and supplemented by the addition of Triton
X-100 to 3%. This lysate was then bound to glutathione-Sepharose (Sigma) and washed with TBST and Tris-buffered saline. The quantity of
bound fusion protein was estimated by Coomassie Blue staining of
SDS-PAGE gels of known amounts of fusion protein-containing glutathione-Sepharose beads, and glutathione-Sepharose was added to
equalize the amount of fusion protein/ml of beads among various fusions. Varying amounts of CLP or CaM were mixed with glutathione Sepharose beads containing equal quantities (~5 µg) of GST-IQ123, GST-IQ12, GST-IQ1, or GST alone in the presence of 5 mM
CaCl2 (Ca) or 5 mM EDTA in TBST and allowed to
bind overnight at 4 °C. The beads were washed four times with CaTBST
or with 5 mM EDTA in TBST and solubilized in Laemmli buffer
(16). These samples were then analyzed by Western blot for CLP using
affinity-purified rabbit anti-human CLP antibody TG-7, or for CLP/CaM
using unpurified TG-7 antiserum, which recognizes both CLP and CaM
equally well (10).
1 (11). Bovine brain CaM was
from Calbiochem (San Diego, CA) and quantitated by UV absorbance at 276 nm using a molar extinction coefficient of 3300 M
1 (19). CLP and CaM were diluted
from 0.1 mM stocks immediately prior to sensorgram runs.
All sensorgram runs were performed in a running buffer of 50 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM
-mercaptoethanol, 0.02% w/v NaN3,
0.005% Surfactant P-20 (Biacore Inc.), and 1 mM CaCl2. Regeneration was performed using running buffer with
2 mM EDTA in place of CaCl2 and was complete as
determined by comparing blank buffer injections to 1 µM
injections of CLP or CaM. Sensorgrams were blank subtracted and
baseline-adjusted, then RU at equilibrium were measured. For each of
three experiments, a Scatchard plot was done and the
x-intercept of a least squares fitted line was calculated.
Each point in that experiment was then normalized to the
x-intercept (i.e. divided by the value of the
x-intercept and multiplied by 100), all data points were
pooled, and a Scatchard plot representing all three experiments was
plotted. Least squares fitting was then used to calculate a best fit
line, and the affinity of CLP for the IQ3 peptide was estimated from
the slope of that line. Kinetic rate constants were not estimated
because analysis of flow rate dependence of the apparent rate constants
demonstrated that CLP binding was mass transfer limited up to a flow
rate of 100 µl/min.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (60K):
[in a new window]
Fig. 1.
Overlay of electrophoretically separated
tissue extracts with 125I-labeled CLPF99Y or
CaM. 50 µg of protein from the indicated tissues were
electrophoresed per lane on a 7% SDS-polyacrylamide gel, blotted onto
PVDF membrane and processed for an overlay with iodinated
CLPF99Y or CaM. Lanes 1,
3, and 5 were probed with CLPF99Y;
lanes 2, 4, and 6 with CaM.
Molecular mass standards are indicated in kDa on the
right.
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[in a new window]
Fig. 2.
Competition analysis of the binding of
biotinylated CLP to its putative target proteins. Overlay samples
containing HeLa microsomes (5 µg/lane; A) or total yeast
extract from Y190/pGADGH-IQ123K (20 µg/lane; B) were run
in standard SDS-PAGE gels and blotted to PVDF membranes. Membranes were
probed in 75 nM labeled CLP in CaTBST for 90 min in the
presence of 0, 750 nM (10-fold excess), or 7500 nM (100-fold excess) unlabeled CLP or 7500 nM
CaM and washed in CaTBST. The blot was then probed with
biotin-avidin-horseradish peroxidase, washed, and developed using
Renaissance chemiluminescent reagent as described under "Experimental
Procedures." Molecular mass standards are indicated on the
left in kDa.
-galactosidase positive in a colony lift assay. Following
cycloheximide counterselection to cure strains of the bait plasmid, two
colonies for each
-galactosidase-positive transformant were mated
against Y187 yeast cells containing bait plasmids coding for CLP or a laminin fusion protein (negative control bait). Library plasmids remaining positive for interaction with CLP and testing negative for
interaction with laminin were rescued, and their inserts were amplified
by PCR and sequenced. 18 of the library plasmids positive for CLP
interaction contained a ~350-bp insert whose sequence showed high
similarity (86.3% identity) to a cDNA sequence for bovine myosin X
(GenBankTM accession no. U55042). This insert obviously represented
the cDNA of the corresponding human myosin X, which had not yet
been cloned. Each of the insert sequences was primed from an internal
A-rich stretch of the myosin X RNA and encoded a region mainly made up
of the three IQ domains of the myosin X heavy chain. IQ domains are
named for the isoleucine-glutamine (single-letter code IQ) pair, which
is part of the signature sequence characterizing these domains (24).
Importantly, IQ domains are known binding sites for calmodulin (and for
other Ca2+ binding light chains) in several
"unconventional" myosins (24, 25).
View larger version (12K):
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Fig. 3.
Schematic structure of human myosin X. Top, scheme of the deduced mRNA structure with
the start and stop codon indicated and exons shown as
alternating white boxes. The scale is
given in kilobase pairs. Bottom, scheme of the protein with
the various structural motifs indicated. The coding sequence present in
the original CLP interacting clone from the yeast two-hybrid screen is
indicated as a black bar. The scale is given in
amino acids.
View larger version (49K):
[in a new window]
Fig. 4.
Colocalization of human CLP and myosin X in
transfected MCF-7 cells. Cells were transiently transfected with
pEYFP-CLP (A and B) or pEYFP-CaM (C)
and with pEBFP-HA-His-MyoX (B and C). After 2 days, cells were fixed, permeabilized, and stained with anti-HA
antibodies followed by Alexa 350 secondary antibodies to detect myosin
X, as well as with rhodamine-phalloidin to stain actin. A,
cells transfected with YFP-CLP alone (green fluorescence);
B, YFP-CLP (green fluorescence) and HA-His-MyoX
(blue fluorescence) cotransfected cells; C,
YFP-CaM (green fluorescence) and HA-His-MyoX
(blue fluorescence) cotransfected cells. Stained cells were
visualized by confocal epifluorescence microscopy. Note that areas of
overlap between the red, green, and blue fluorescence appear
white (merge).
View larger version (57K):
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Fig. 5.
Coprecipitation of CLP with myosin X. COS-1 cells were transfected with pEBFP-HA-His-MyoX (BFP-His
MX), pEBFP-HA-His (BFP-His), pEYFP, pEYFP-CaM, and
pEYFP-CLP as indicated on top, grown, and then lysed as
described under "Experimental Procedures." His-tagged proteins were
precipitated with Ni2+-NTA-agarose, and the proteins were
analyzed by SDS-PAGE followed by Western blotting with anti-GFP
antibodies (which also recognize YFP and BFP). A, total cell
lysates. B, top panel, precipitates
run on a 5% gel to visualize the BFP-tagged myosin X;
bottom panel, precipitates run on a 10% gel to
visualize the YFP-tagged CaM and CLP and the control BFP-His. The
position of the proteins is marked on the left, and
molecular mass standards are indicated in kDa on the
right.
Trp/
Leu/
His medium or for
-galactosidase expression in a
filter lift assay (Fig. 6). In these
experiments a strong association was only observed between CLP and
IQ123 or IQ123K. In the less stringent Y190 yeast strain, we observed
weak interaction between CaM and IQ12, IQ123, or IQ123K as well as
between CLP and IQ12. Similar results were obtained when single
pGADGH-IQ "prey" transformants were mated with Y187 cells
containing either pAS2-CLP or pAS2-CaM.
View larger version (40K):
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Fig. 6.
Identification of CLP binding sites on myosin
X by yeast two-hybrid analysis. Top, Y190 and
CG1945 cells were cotransformed with the indicated plasmids, and growth
on SD/ Trp/
Leu/
His/+3-aminotriazole medium was scored after 4 days
as no colonies (
), very small/few colonies (+/
), some colonies (+),
and many colonies (+++). Bottom, Y190 and CG1945 cells were
cotransformed with the same plasmids as above, grown for 2 days on
SD/
Trp/
Leu medium, and then assayed for
-galactosidase on filter
lifts.
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Fig. 7.
Demonstration of direct CLP-myosin X
interaction by GST pull-downs. A, 5 µg of purified
GST alone or of different GST-IQ fusion proteins (indicated on
top of the gel) were mixed with 1 µg of CLP in a final
volume of 12 ml of CaTBST for a final protein concentration of 5 nM CLP. After rocking overnight, the beads were collected
and washed four times with CaTBST, and one-fifth of the total was
analyzed by Western blot using anti-CLP antibodies. B, 5 µg of GST-IQ123 were mixed with 50 µg of CLP or CaM in a final
volume of 600 µl of CaTBST (Ca) or 5 mM EDTA
in TBST (E) for a final CLP or CaM concentration of 5 µM. After washing, one-fifth of the total was analyzed by
Western blot using unpurified anti-CLP antiserum TG7, which recognizes
CLP and CaM equally well.
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Fig. 8.
Surface plasmon resonance analysis of the
binding of CLP and CaM to the IQ3 peptide of myosin X. Biacore
analysis was performed on a BIAcore 1000 instrument using streptavidin
chips coated with biotinylated IQ3 peptide. A, typical
sensorgrams using 1000 and 100 nM CLP and CaM.
B, Scatchard plot of CLP-IQ3 binding using the equilibrium
phase RU for amount bound. For each of three experiments
(diamond, triangle and square
symbols), a Scatchard plot was done and the
x-intercept of a least squares fitted line was calculated.
Each point in that experiment was then normalized to that
x-intercept, all data points were pooled, and a Scatchard
plot containing all three experiments was plotted as shown. Least
squares fitting was used to calculate the best fit lines shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Johannes A. Rhyner for the preparation of CLPF99Y and for performing the 125I labeled CLP overlays. We thank Dr. John T. Penniston (Mayo Clinic, Rochester, MN) for the COS-1 cells and Dr. John Lust (Mayo Clinic, Rochester, MN) for allowing us to use the BIAcore 1000 instrument. Thanks are also due to Steven J. DeMarco for help with the yeast two-hybrid analysis and for many fruitful discussions.
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Addendum |
---|
While this manuscript was in preparation, the full-length sequence and domain structure of the mouse and human myosin X were reported by Yonezawa et al. (26) and Berg et al. (27), respectively. Our data on the human HeLa cell myosin X are in complete agreement with these reports.
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FOOTNOTES |
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* This work was supported in part by a grant from the Fraternal Order of Eagles' Cancer Research Fund (Eagles #176) and by the Mayo Clinic Cancer Center.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF234532.
Supported by United States Army Medical Research and Materiel
Command Predoctoral Training Grant DAMD 17-94-J-4116 and NCI, National
Institutes of Health Predoctoral Training Grant T32CA75926. Present
address: Div. of Surgical Research, Children's Hospital, Harvard
Medical School, Boston, MA 02115.
§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Mayo Clinic/Foundation, 200 First St. S.W., Rochester, MN 55905. Tel.: 507-284-9372; Fax: 507-284-2384; E-mail: strehler.emanuel@mayo.edu.
Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M010056200
2 J. A. Rhyner and E. E. Strehler, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: CLP, calmodulin-like protein; CaM, calmodulin; HA, hemagglutinin; RU, resonance units; NTA, nitrilotriacetic acid; bp, base pair(s); kb, kilobase pair(s); MOPS, 4-morpholinepropanesulfonic acid; YFP, yellow fluorescence protein; PCR, polymerase chain reaction; GST, glutathione S-transferase; DPBS, Dulbecco's phosphate-buffered saline; BFP, blue fluorescence protein; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; nt, nucleotide(s); TBST, Tris-buffered saline with Tween 20; CaTBST, Tris-buffered saline with Tween 20 plus CaCl2; ORF, open reading frame.
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