Journal of Cell Science 105, 613-628 (1993)
Printed in Great Britain © The Company of Biologists Limited 1993
613
Differential localization patterns of myristoylated and nonmyristoylated
c-Src proteins in interphase and mitotic c-Src overexpresser cells
Thérèse David-Pfeuty1,*, Shubha Bagrodia2 and David Shalloway2
1Institut Curie-Biologie, Centre Universitaire, Bâtiment 110, 91405 Orsay Cédex, France
2Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY 14853,
USA
*Author for correspondence
SUMMARY
Myristoylation of pp60src is required for its membrane
attachment and transforming activity. The mouse monoclonal antibody, mAb327, which recognizes both
normal, myristoylated pp60 c-src and a nonmyristoylated
mutant, pp60c-src/myr−, has been used to compare the
effects of preventing myristoylation on the localization
of c-Src in NIH 3T3-derived overexpresser cells using
immunofluorescence microscopy. During interphase,
pp60c-src partitions between the plasma membrane and
the centrosome, while pp60c-src/myr− is predominantly
cytoplasmic but also partly nuclear. The cytoplasmic,
but not the nuclear, staining can be readily washed out
by brief pretritonization of the cells before fixation, indicating that the cytoplasmic pool of pp60c-src/myr−, in contrast with the nuclear one, does not associate tightly with
structures that are insoluble in the presence of nonionic
detergents. We have previously shown that during G2
phase, pp60c-src leaves the plasma membrane and is
redistributed diffusely throughout the cytoplasm and to
two clusters of patches surrounding the two separating centriole pairs. In contrast, we now find that
pp60c-src/myr− translocates to the nucleus in late G2 or
early prophase prior to there being any clear evidence of
nuclear membrane breakdown or nuclear lamina disassembly. Similar nuclear translocation of pp60c-src/myr−,
but not of pp60c-src, is also observed when cells are
arrested in G0 or at the G1/S transition. Furthermore,
during mitosis, pp60c-src is found primarily in diffuse
and patchy structures dispersed throughout the cytoplasm while pp60c-src/myr− more specifically associates
with the main components of the spindle apparatus
(poles and fibers) and inside the interchromosomal
space.
These results suggest that a possible role for myristoylation might be to prevent unregulated nuclear transport of proteins whose nonmyristoylated counterparts
are readily moved into the nucleus. They also raise the
possibility that a subfraction of wild-type pp60c-src may
behave, at specific times, like its nonmyristoylated counterpart, and may translocate to the nucleus and exert
specific functions in that location.
INTRODUCTION
1990). In particular, significant fractions of myristoylated
pp60c-src and pp60v-src have been found to associate with
perinuclear membranes (Resh and Erikson, 1985) and
myristoylated, yet cytosolic, variants of pp60v-src have been
described (Garber et al., 1985). On the other hand, wildtype myristoylated pp60c-src overexpressed in NIH 3T3 cells
has been shown to partition between the plasma membrane
and the centrosomal area in interphase cells and colocalize
with endocytosed concanavalin A (ConA) at all stages of
the ConA-induced endocytotic process (David-Pfeuty and
Nouvian-Dooghe, 1990). More recently, pp60c-src was also
shown to be enriched in a population of late endosomes in
Rat-1 c-Src overexpresser cells (Kaplan et al., 1992) and in
PC12 synaptic vesicles (Lindstedt et al., 1992).
Many v-src mutations that prevent myristoylation and
plasma membrane association also abrograte v-src transforming activity (for review, Jove and Hanafusa, 1987); this
The transforming protein of Rous sarcoma virus, pp60v-src,
and its cellular homolog, pp60c-src, are cotranslationally
myristoylated at the amino-terminal glycine 2 residue
during protein synthesis (Buss et al., 1984; Buss and Sefton,
1985; Wilcox et al., 1987; Deichaite et al., 1988). Most
pp60v-src molecules are so modified although it has been
estimated that as much as 16% of pp60v-src may not be
myristoylated (Buss and Sefton, 1985). For both proteins,
myristoylation is required for association with the plasma
membrane (Buss et al., 1986; Schuh and Brugge, 1988;
Reynolds et al., 1989) but not all myristoylated proteins are
associated with the plasma membrane (for review, Sefton
and Buss, 1987; McIlhinney, 1990). Independent domains
of Src may cooperate with myristoylation to specify association with distinctive cellular membranes (Kaplan et al.,
Key words: c-Src, c-Src overexpresser cells, myristoylation, cell
cycle
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T. David-Pfeuty, S. Bagrodia and D. Shalloway
supports the hypothesis that important cellular targets of
Src are located at the plasma membrane. However, nonmyristoylated, transformation-defective, cytosolic v-Src
proteins still have mitogenic properties, promoting growth
of cells to high densities and growth of some normally nondividing cells of neuronal origin (Kamps et al., 1986;
Calothy et al., 1987). Myristoylation and plasma membrane
association are not absolutely required for src transforming
activity: two nonmyristoylated Src proteins encoded by
recovered avian sarcoma viruses rASV157 and rASV1702
fractionate as soluble cytosolic enzymes yet are transformation-competent (Krueger et al., 1982, 1984). In this case,
the transforming activity of these unusual mutants depends
on the presence of env signal sequences at the amino termini of the retrovirus-encoded fusion proteins (Garber and
Hanafusa, 1987). Moreover, a slow, time-dependent cellular transformation of cells infected with temperature-sensitive mutants of RSV has been reported to occur when the
infected cells are continuously grown at the nonpermissive
temperature, under conditions in which the ts-pp60v-src is
impaired in its capacity to associate with the plasma membrane and, consequently, accumulates in the cytoplasm, particularly in the centrosomal area (David-Pfeuty and Nouvian-Dooghe, 1992). The activities of cytoplasmic (either
myristoylated or nonmyristoylated) mutants could reflect
non-specific low-level Src-mediated phosphorylation of
plasma membrane-associated targets. Alternatively, it may
indicate that Src also acts at cellular sites distinct from the
plasma membrane.
The observation that the activity of c-Src is transiently
stimulated during mitosis (Chackalaparampil and Shalloway, 1988), partly as an indirect result of phosphorylation by p34cdc2 or the related kinase (Morgan et al., 1989;
Shenoy et al., 1989, 1992; Bagrodia et al., 1991; Kaech et
al., 1991), suggests that c-Src may have special functions
in this phase of the cell cycle (for review, see Taylor and
Shalloway, 1993). This observation may be related to the
fact that wt pp60c-src in NIH 3T3 overexpresser cells delocalizes from the plasma membrane in late G2 to condense
around the two separating centriole pairs and then to disperse in a diffuse and patchy way in the cytoplasm throughout mitosis (David-Pfeuty and Nouvian-Dooghe, 1990).
These considerations prompted us to perform a detailed
comparison, using immunofluorescence techniques, of the
subcellular localization of wild-type (wt) and myristoylation-defective (myr−) c-Src proteins throughout the cell
cycle both to determine which aspects of the cell cycledependent relocalization of c-Src depend on myristoylation
and to get an insight into the behavior of the potential, small
subfraction of nonmyristoylated Src molecules present in
normal cells. This study revealed unexpected localization
properties of myr− c-Src that differed from those of wt csrc: (1) although primarily cytoplasmic, myr− c-Src exhibits
detectable nuclear localization during the G1 and S phases
of the cycle; (2) it translocates to the nucleus during late
G2 or early prophase; and (3) it concentrates in the spindle
apparatus and interchromosomal space during mitosis.
These findings raise the possibility that nonmyristoylated
forms of other normally myristoylated proteins might also
be targeted to the nucleus.
MATERIALS AND METHODS
Cells and plasmids
Plasmid-transfected NIH 3T3 cells that overexpress wild-type
chicken c-Src (NIH(pMc-src/focus)B cells) have been previously
described (Johnson et al., 1985). To generate cells expressing the
myr− mutant, NIH 3T3 cells were cotransfected with pcLN, a
chimeric plasmid containing Moloney murine leukemia virus long
terminal repeats and the Src coding sequence from plasmid
pRSVc-SrcLN (Schuh and Brugge, 1988), and G418-resistance
plasmid pSV2neo (Southern and Berg, 1982) and subjected to
G418 selection and cloning as described (Kmiecik and Shalloway,
1987). A mass-culture [NIH(pcLN/ pSV2neo/MC)C] derived from
about 50-100 G418-resistant colonies was used.
Cell culture and synchronization
Cells were cultured in 35 mm dishes on 22 mm2 coverslips in
Dulbecco’s modified Eagle’s medium containing 5% newborn calf
serum and antibiotics in 10% CO2, at 37°C, for at least two days
before immunofluorescence observation. The average cell density
was 2 ×103 to 10 4 cells cm −2.
Cells were synchronized at the G1/S boundary by a thymidineaphidicolin double-block; cells were grown in normal medium for
two days, then sequentially incubated with 2.5 mM thymidine (16
h), normal medium (8 h) and 5 µg/ml aphidicolin (16 h). Immunofluorescence with anti-tubulin antibody and DAPI (4′,6′diamidino-2-phenylindole) showed that G2 cells started to appear
6 h after release from the double-block and were most prevalent
7 h after release. Mitotic cells were most prevalent 7-8 h after
release; a second peak of mitotic cells appeared 27-29 h after
release.
Immunofluorescence and antibodies
Monoclonal antibody mAb327 (Lipsich et al., 1983) was from
Clinisciences (Paris, France). Visualization of intracellular
cytoskeletal structures was achieved using rat monoclonal antitubulin (Biosys, France) for microtubules and nitrobenzoxadiazole
(NBD)-phallacidin (Molecular Probes, Inc., Beaverton, OR) for
microfilaments.
Anti-p60 polyclonal rabbit antiserum (Resh and Erikson, 1985),
monoclonal antibody GD11 (Parsons et al., 1984), human autoantibody to lamin B (Guilly et al., 1987) and anti-clathrin polyclonal
rabbit antiserum were generous gifts from Marilyn Resh, Sarah
Parsons, Françoise Danon and Paul-Henri Mangeat, respectively.
Secondary antibodies were goat IgG Fab fragments from antibodies directed against either mouse or rat IgG. (The anti-rat goat
IgG was preabsorbed against a mouse IgG column to eliminate
cross-reactivity; a reciprocal preabsorption was used for the antimouse goat IgG.) These fragments were conjugated with either
fluorescein isothiocyanate (FITC; Interchim, France) or tetramethyl rhodamine isothiocyanate (TRITC; Interchim, France).
These secondary antibodies gave essentially undetectable background fluorescence.
Single- or double-fluorescence cell labeling was performed as
described (David-Pfeuty and Singer, 1980) following fixation with
3% formaldehyde and subsequent permeabilization by treatment
with 0.1% Triton X-100 at room temperature. When specified,
cells were pre-Tritonized by treatment with PHEM buffer (0.1%
Triton X-100, 45 mM PIPES, 45 mM HEPES, 10 mM EGTA, 5
mM MgCl2, 1 mM PMSF, pH 6.9) for 30-45 s before fixation
(Bailly et al., 1989). Fluorescence microscopy was performed with
a Leitz microscope equipped with fluorescein, rhodamine and
DAPI filters using a ×40 oil objective. Photographs were taken
with Kodak TMax 400 film.
Localization of wt and myr− c-Src
RESULTS
Differential localization of myristoylated and
nonmyristoylated c-Src proteins in asynchronous
cells
NIH 3T3-derived cells, which express wild-type and nonmyristoylated chicken c-Src from transfected chimeric plasmids containing Moloney murine leukemia virus long terminal repeats, and wt and mutant src genes, were used for
all studies. They expressed Src at levels 10- to 20-fold
above the level of endogenous c-Src (data not shown), so
almost all immunofluorescently visualized protein was plasmid-expressed. The mutant gene used for expression of
myr− Src contained codons for four additional residues at
the amino terminus of the protein. It encodes the aminoterminal sequence Met-Ala-Ala-Ala-Met-Gly-... (where the
underline denotes the first two amino acids encoded by the
wt src gene). This mutant has been studied previously in
chicken embryo fibroblasts by Schuh and Brugge (1988),
who found, as expected, that it was not localized to the
plasma membrane but had normal specific protein-tyrosine
kinase activity. This was confirmed in NIH 3T3 cells (data
not shown). Anti-Src monoclonal antibodies mAb327 (Lipsich et al., 1983) and, occasionally, anti-p60 polyclonal
rabbit antiserum (Resh and Erikson, 1985) or mAb GD11
(Parsons et al., 1984), which react with a broad range of vSrc and chicken, mouse and human c-Src variants, were
used in these studies.
In asynchronous wt c-Src overexpresser cells, mAb327
revealed the three characteristic, previously documented
(David-Pfeuty and Nouvian-Dooghe, 1990) distributions:
(i) uniformly dispersed at the inner face of the plasma membrane (open arrows, Fig. 1C); (ii) inside a dense spot situated at the focal point of the interphasic microtubules in the
centriolar area (thin arrows, Fig. 1C and D); (iii) inside a
cluster of patches surrounding the nucleus and embedded
within a microtubule meshwork (arrowheads, Fig. 1C and
D). In G2 cells, the mAb327 staining typically remained
concentrated in part near the two separating centriole pairs
(thin arrows, Fig. 1H and I).
A very different pp60c-src localization pattern was
observed in asynchronous myr− c-Src overexpresser cells.
Here, mAb327 gave a rather intense and uniformly distributed cytoplasmic staining (arrowheads, Fig. 1A) identical
to that described by Reynolds et al. (1989). However, we
also detected a nuclear staining (arrows, Fig. 1A), which
was low (compared to the cytoplasmic staining) in the
majority of the cells, but conspicuous in a minor cell population (~2%). Triple labeling of the cells with anti-Src,
anti-tubulin antibodies and DAPI (large arrows, Fig. 1E-G)
clearly indicates that prominent nuclear staining occurs in
late G 2 cells (these can be unambiguously distinguished by
the presence of the two centriole pairs brightly labeled with
the anti-tubulin antibodies (thin arrows, Fig. 1F) and the
appearance of the typically punctate chromosomal DAPI
staining (large arrow, Fig. 1G) that signals the beginning
of chromosome condensation). In addition, anti-Src staining concentrated in the spindle apparatus of mitotic cells
(arrowheads, Fig. 1E-G).
These first observations indicated that pp60c-src/myr− is
615
located predominantly in the cytoplasm of interphase cells
but that it translocates to the nucleus, presumably in G2
phase.
Localization of pp60c-src/myr in synchronized cells
Translocation of myr−c-Src during first S and G2
following release from G1/S block
myr− and wt c-Src overexpresser cells were synchronized
at the G1/S boundary by thymidine-aphidicolin doubleblock (see Materials and Methods) and their distributions
were observed at various times (tR) after release from the
double-block. Unexpectedly, at tR = 0 (Fig. 2A and B) a
very intense nuclear staining (generally partly excluded
from the nucleolar region; small arrows), largely supplanted
the cytoplasmic staining in the majority of the myr− c-Src
cells. The percentage of cells exhibiting this feature varied
between 70 and 100% from one experiment to the other
and decreased as cells were released from the block at later
−
times. This abundant nuclear accumulation of pp60c-src/myr
did not reappear at the following G1/S phase transition,
which occurred about 20 h later. We infer that pp60c-src/myr−
collects intensely in the nucleus during the abnormally long
arrest in G 1 phase caused by thymidine-aphidicolin doubleblock but not in normal G1 phases. A similar, abnormal,
myr− c-Src nuclear accumulation also occurred during arrest
in G0 induced by 48 h serum starvation; no nuclear accumulation of wt c-Src was observed after release from a
double-block (Fig. 2E and F) or during a G0 arrest.
−
The strong nuclear concentration of pp60c-src/myr did not
prevent passage into G2, which started approximately 7 h
after release from the double-block. A natural control
indicating that the observed nuclear accumulation of
pp60c-src/myr− is not an artifact was provided by the observation that anti-Src staining was totally excluded from the
nucleus, but not from the cytoplasm, of confluent cells
(which do not progress into G 2 after release from the block;
Fig. 2C and D).
G1 and S phases following the first round of mitosis
after release from a G1/S block
The first round of mitosis occurred 7 to 9 h after release
from the thymidine-aphidicolin double-block. During the
following G1 and S phases, that is between 9 and 27 h after
release, mAb327 staining in wt and myr− c-Src overexpresser cells did not differ significantly from that exhibited
by unsynchronized cell populations (cf. Figs 1A to 3A, and
Figs 1C to 3E).
Brief permeabilization of the cells with Triton X-100 for
30-45 s (pre-Tritonization) before fixation (see Materials
and Methods) washed out almost all of the cytoplasmic
mAb327 staining but preserved the nuclear one in subconfluent cultures (arrows, Fig. 3C). Such treatment did not
severely alter the interphasic microtubule network (Fig.
3D), or the organization of actin into microfilament bundles (not shown). This experiment showed that the cyto−
plasmic pool of pp60c-src/myr does not associate tightly with
structures that are insoluble in the presence of nonionic
detergents. In contrast, it showed that the nuclear myr− cSrc does interact with nuclear structures that are insoluble
in Triton X-100.
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T. David-Pfeuty, S. Bagrodia and D. Shalloway
Fig. 1. Double immunolabeling of asynchronous wt (C, D, H-J) and myr− (A, B, E-G) c-Src overexpresser cells with anti-Src (A, C, E, H)
and anti-tubulin (B, D, F, I) antibodies. Each row of panels shows the same cells visualized by immunofluorescence with different filters.
Cells in (G) and (J) display chromosomal DAPI fluorescence following triple staining. Black arrows and arrowheads in A identify nuclear
and diffuse cytoplasmic concentrations of myr− c-Src, respectively. Small arrows, arrowheads and open arrows in (C) and (D) designate
pericentriolar, perinuclear and plasma membrane-associated pp60c-src, respectively. A selected region containing late G2 cell (large
arrows) from each of these cultures is displayed in E, F, G (myr−) and H, I, J (wt). The small black arrows in F, H, I identify the two
separating centriole pairs in the G2 cells; the arrowheads in (E, F, G) identify a metaphase cell.
For comparison, the effect of brief pre-Tritonization
before fixation on wt c-Src overexpresser cells is shown in
Fig. 3E-H. When the cells were fixed without pre-Tritonization, c-Src partitioned between the centriolar area
(thin arrows, Fig. 3E), the perinuclear region (arrowheads,
Fig. 3E) and, uniformly at the inner face of the plasma
membrane (open arrow, Fig. 3E). Strikingly different c-Src
features appeared when the cells were permeabilized with
Localization of wt and myr− c-Src
617
Fig. 2. Immunolabeling of wt (E) and myr− (A, C) c-Src overexpresser cells with anti-Src antibodies following release from a thymidineaphidicolin double-block. Time following double-block release (tR) is 0. Prominent nuclear staining with anti-Src antibodies of a majority
of myr− c-Src overexpresser cells is evident at tR = 0 (A), except in plate areas in which cells reach confluency (C). Under identical
experimental conditions, wt c-Src never exhibits a detectable nuclear location (E). (B) and (D) show the phase-contrast pictures of cells in
(A) and (C), respectively. The tiny arrows in (A) and (B) point out nucleoli from which the anti-Src staining is partly excluded. (F) shows
chromosomal DAPI staining of cells in (E).
Triton X-100 before fixation (Fig. 3G): a residual pericentriolar concentration of c-Src occasionally persisted (thin
arrows, Fig. 3G), but most often, the perinuclear pp60c-src
patches were no longer present. In addition, the previously
uniform labeling of c-Src at the inner cell surface was
replaced by a peculiar pattern of a multitude of rounded
patches or vesicles. The relatively large size of these
patches and double-labeling experiments with anti-Src and
anti-clathrin (the major coat protein of coated pits) antibodies indicated that these structures were not coated pits
(not shown). They could represent early endosomes. We
stress that pre-Tritonizing the wt c-Src overexpresser cells
did not reveal any nuclear c-Src staining.
Second round of G2 and mitosis after release from a
G1/S block
A second peak of G2 and mitotic cells appeared 27 to 29
h after release from the thymidine-aphidicolin doubleblock. At tR = 27 h, a significant population of late G2 cells
(up to 25%) were present. These were characterized by the
presence of centriole pairs brightly labeled with anti-tubulin antibodies (arrows, Fig. 4J), the appearance of a typically punctate DAPI staining pattern (Fig. 4G and K) and
prominent myr− c-Src nuclear staining (Fig. 4E and I). As
well as this population of cells that were clearly in late G2
or already in early prophase, a significant number of cells
(between 40 and 50%) were observed in which the cytoplasmic myr− c-Src was not uniformly distributed but was
diffusely concentrated around the nucleus (arrow, Fig. 4A)
or in which myr− c-Src appeared to accumulate within the
dense microtubule meshwork surrounding the nucleus
(arrowheads, Fig. 4A and B).
In the late G2 cells, the conspicuous mAb327 nuclear
staining was not seen in the early condensing chromosomes
and instead filled the interchromosomal space, which was
unstained by DAPI (small arrows, Fig. 4E, G, I and K).
Phase-contrast microscopy (Fig. 4D, H and L) indicated that
the nuclear accumulation of pp60c-src/myr− occurred before
clear evidence of nuclear membrane breakdown and before
chromosomes became clearly distinguishable in phase contrast. As shown by double-immunolabeling of the cells with
anti-Src and anti-lamin B antibodies (Fig. 4F), it also
occurred before nuclear lamina disassembly.
Treatment of these synchronized cells, starting at tR = 25
h, with 10 µg/ml of nocadozole for 90 min did not prevent
the translocation of pp60c-src/myr− to the nucleus in late G2
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T. David-Pfeuty, S. Bagrodia and D. Shalloway
Fig. 3. Double immunolabeling of
wt (E-H) and myr− (A-D) c-Src
overexpresser cells with anti-Src
(A, C, E, G) and anti-tubulin (B,
D, F, H), following release from a
thymidine-aphidicolin doubleblock (between 9 and 25 h). The
cells in (C, D, G, H) were briefly
pretritonized before fixation as
described in Materials and
Methods. The arrows in (A, C)
point out the nuclear concentration
of myr− c-Src and the arrowheads
in (A), the cytoplasmic diffuse
myr− c-Src distribution that is
absent in pre-Tritonized cells (C).
Thin arrows, arrowheads and open
arrow in (E, F) designate the
pericentriolar, perinuclear and
plasma membrane-associated
distributions, respectively, of wt
pp60c-src. The thin black arrows in
(G, H) show a residual
pericentriolar concentration of cSrc in pre-Tritonized c-Src
overexpresser cells, which
otherwise exhibit a peculiar
pattern of a multitude of rounded
patches at the level of their upper
cell surface.
or early prophase cells (arrowheads, Fig. 5C and D). This
suggests that the interphasic microtubules are not involved
in the late G2 phase-dependent translocation of myr− c-Src
from the cytoplasm to the nucleus. In addition, nocadozole
treatment had no effect on the cytoplasmic distribution of
myr− c-Src (cf. Fig. 5A and C). In contrast, disruption of
the interphasic microtubules by nocadozole treatment of wt
c-Src overexpresser cells (Fig. 5E-H) induced a redistribution of pp60c-src into patches that were scattered through
the cytoplasm (Fig. 5G) as previously reported.
Differential localization of myristoylated and
nonmyristoylated c-Src proteins during the
various phases of mitosis
A chronological sequence of the variations in wt and myr−
c-Src localization during progression through mitosis was
reconstructed from observations on selected cells (from cell
populations 27 to 29 h after double-block release), whose
positions in the cycle were identified by analysis of their
tubulin and DAPI staining patterns. We found that the strikingly contrasting localization patterns of overexpressed
Localization of wt and myr− c-Src
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T. David-Pfeuty, S. Bagrodia and D. Shalloway
Fig. 5. Double labeling of asynchronous wt (E-H) and myr− (A-D) c-Src overexpresser cells, untreated (A, B, E, F) or treated with
nocadozole (C, D, G, H) with anti-Src (A, C, E, G) and anti-tubulin (B, D, F, H). The big arrowheads point out late G2 or early prophase
cells before nuclear membrane breakdown. Nocadozole treatment does not at all perturb the subcellular distribution of myr− c-Src even in
mitotic cells (compare A and C) but it greatly affects the wt pp60c-src distribution (compare E and G).
pp60c-src and pp60c-src/myr− still persist during mitosis. At
the end of the G2 phase, immediately before nuclear membrane breakdown, pp60c-src-containing patches typically
condense around the two centriole pairs, which are now
symmetrically located with respect to the nucleus (arrows,
Fig. 6A and B). c-Src appears to be clearly excluded from
the nucleus at this time. This contrasts with myr− c-Src,
which is predominantly intranuclear. However, myr− c-Src
Localization of wt and myr− c-Src
621
Fig. 6. Triple labeling of wt (A-C, J-L) and myr− (D-I, M-O) c-Src overexpresser cells with anti-Src (A, D, G, J, M), anti-tubulin (B, E, H,
K, N) and DAPI (C, F, I, L, O) during different subphases of mitosis. Rows of panels show late G2 or early prophase (A-F), prometaphase
(G-O) cells with appropriate filters. The long arrows point to the location of the two centriole pairs in G2 and spindle poles in prophase
and prometaphase. The small arrows in G and I indicate interchromosomal space that is heavily stained by anti-Src antibody.
also starts, slightly but detectably, to accumulate at the level
of the two diplosomes, which are freed from the depolymerized interphasic microtubules (arrows, Fig. 6D-F).
In prometaphase, during the process of chromosome
rearrangement orchestrated by the complex dynamics of the
spindle apparatus, the wt pp60c-src-containing patches,
which were previously concentrated around the two centriole pairs, partly dissolve from the centriole pairs, which
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T. David-Pfeuty, S. Bagrodia and D. Shalloway
now serve as spindle poles (arrows, Fig. 6J and K). In contrast, association of pp60c-src/myr− with the spindle poles is
greatly increased (large arrows, Fig. 6G, H, M and N).
pp60c-src/myr− continues to fill the interchromosomal space
(thin arrows, Fig. 6G and I).
The features observed in prometaphase became even
more apparent in metaphase: wt pp60c-src was mostly
excluded from the space occupied by the components of
the spindle apparatus and metaphase plate (Fig. 7a-c and gi) although faint staining of the spindle fibers and spindle
poles was occasionally observed (thin arrows and arrowheads, respectively, Fig. 7a, b, g and h). In contrast, all the
components of the spindle apparatus, spindle poles and
spindle fibers (arrowheads and thin arrows, respectively,
Fig. 7d, e, j and k), the residual interchromosomal space
and the contour of the metaphase plate (Fig. 7d and j), were
heavily labeled by pp60c-src/myr−.
In anaphase, wt pp60c-src is diffusely distributed through
the cytoplasm (Fig. 7m) whereas pp60c-src/myr− remains preferentially concentrated at the spindle poles (small arrows,
Fig. 7p), along the interpolar microtubules, and in the midzone separating the two sets of highly condensed chromosomes (open arrow, Fig. 7p).
In early telophase (Fig. 8A-F) wt pp60c-src started becoming concentrated again in the centrosomal area (arrowheads,
Fig. 8A and B) and at the cell surface of the two parting
daughter cells (thin arrow, Fig. 8A); some pp60c-src was
still distributed diffusely through the cytoplasm (open
arrow, Fig. 8A). In contrast, the pp60c-src/myr−-associated
fluorescence reappeared strongly and uniformly throughout
the cytoplasm (open arrow, Fig. 8D).
In late telophase (Fig. 8G-L), the residual diffuse cytoplasmic pp60c-src-associated fluorescence vanished and the
two main areas of pp60c-src distribution in the separated
cells were at the inner cell surface and in the centrosomal
area (long arrows and black arrowhead, respectively, Fig.
8G and H). pp60c-src was also concentrated at intercellular
contact areas (white arrowhead, Fig. 8G). At the same stage,
the pp60c-src/myr− distribution had not yet returned to its
interphase distribution (predominently in the cytoplasm).
As long as the DAPI staining retained its punctate pattern,
myr− c-Src remained concentrated mostly in the nucleus
(Fig. 8J-L). It subsequently reverted to the distribution typical of interphase cells (lower cell, Fig. 8J-L).
DISCUSSION
The same anti-Src mAb, mAb327 (Lipsich et al., 1983), has
been used to compare the subcellular localization of myristoylated (wt) and nonmyristoylated (myr−) c-Src in NIH
3T3-derived overexpresser cells (both expressing similar
levels of wt and mutant chicken pp60c-src, ~15- to 20-fold
higher than the level of endogeneous mouse pp60c-src)
during the various phases of the cell cycle. Similar observations have been made with a second anti-Src mAb, GD11
(Parsons et al., 1984), and with a polyclonal anti-Src antibody, αp60 (Resh and Erikson, 1985). A summary of the
results obtained is presented in Fig. 9. We recall the main
observations: (1) in G1 and S phases, pp60c-src partitions
primarily between the inner face of the plasma membrane
and the centrosome (with somewhat higher pericentriolar
concentration) while pp60 c-src/myr− is predominantly distributed diffusely in the cytoplasm with a small amount in the
nucleus; (2) in G2 phase pp60 c-src leaves the plasma membrane and is redistributed diffusely throughout the cytoplasm and into two clusters of patches surrounding the two
separating centriole pairs; in contrast, pp60c-src/myr− translocates predominantly to the nucleus prior to any clear evidence of nuclear membrane breakdown or nuclear lamina
disassembly; (3) during early mitosis, up to anaphase,
pp60c-src is found primarily in diffuse and patchy structures
dispersed throughout the cytoplasm whereas pp60c-src/myr−
strongly associates with specific structures involved in
mitosis: that is, with the main constituents of the spindle
apparatus and the segregating chromosomes; (4) in
telophase pp60c-src gradually resumes its characteristic
interphase, partitioning between the plasma membrane and
the centrosome, but also remains concentrated in part at the
intercellular contact area between the two still-attached
daughter cells; apparently more time is required for
pp60c-src/myr− to recover its typical interphase distribution,
since myr− c-Src remains concentrated in the nucleus as
long as the two daughter cells are connected.
For technical reasons the studies described here were performed using cell lines expressing abnormally high levels
of the Src proteins and the possibility of artifacts must be
considered. For example, saturating the highest-affinity,
physiological sites of interaction could possibly result in
secondary association with unsaturated, lower-affinity subcellular sites. We believe however that such a risk is minimal and, in any case, needs to be assumed when one is
working with proteins (like c-Src) that are normally present
at very low levels that are undetectable by ordinary available techniques. Because of the possibility of artifacts, one
should be especially cautious if the overexpressed protein
apparently colocalizes and/or copurifies with a major cellular constituent such as a cytoskeletal protein like tubulin
or myosin or a plasma membrane-associated receptor. But
the finding that overexpressed Src apparently colocalizes
with minor cellular components such as the pericentriolar
material or a nuclear antigen is, to us, more likely to reflect
a true situation, which, without the artifice of overexpression, would have remained undiscovered. Confidence in
these results obtained with overexpressed Src is supported
to the extent that they overlap previous studies indicating
also that wt pp60c-src and naturally occurring nonmyristoylated forms of Src exhibit major localization sites at the
plasma membrane (Loeb et al., 1987) and in the cytoplasm
(Krueger et al., 1982, 1984), respectively. Furthermore,
variations between the expression levels of Src in individual cells provided an internal check that indicated the lack
of any significant effects of dose on subcellular distribution: even in cells in which the level of overexpression was
lowest (but still detectable), the anti-Src antibodies showed
that pp60c-src preferentially occupied a centrosomal pool
and was secondarily located at the plasma membrane, while
pp60c-src/myr− preferentially occupied a nuclear pool at the
end of G2/early prophase and was secondarily located in
the cytoplasm. We infer that this indicates the presence of
high-affinity, physiological sites of interaction for pp60c-src
in the centrosomal area and for pp60 c-src/myr− in the nucleus.
Localization of wt and myr− c-Src
623
Fig. 7. Triple or double labeling of wt (a-c, g-i, m-o) and myr− (d-f, j-l, p-q) c-Src overexpresser cells with anti-Src (a,d,g,j,m,p), antitubulin (b,e,h,k,n,q) and DAPI (c,f,i,l,o) in metaphase and anaphase. Rows are displays with different filters of metaphase (a-l) and
anaphase (m-r) cells. r, phase-contrast image of the anaphase cell in (p, q). The positions of the spindle poles are indicated by arrowheads
in metaphase cells (a,b,d,e,g,h,j and k) and by thin arrows in anaphase cells (n, p and q). The thin arrows (e, h and k) identify spindle
fibers. These fibers were only weakly stained by anti-Src antibody in wt c-Src overexpresser cells (thin arrow in g) but strongly stained in
myr− c-Src overexpresser cells (thin arrows in d and j). The open arrows in (n,p and q) show interpolar microtubules that are associated
with myr− c-Src (p) but not with wt c-Src (m) during anaphase.
624
T. David-Pfeuty, S. Bagrodia and D. Shalloway
Fig. 8. Triple labeling of wt (A-C, G-I) and myr− (D-F, J-L) c-Src overexpresser cells with anti-Src (A, D, G, J), anti-tubulin (B, E, H, K)
and DAPI (C, F, I, L) in early (A-F) and late (G-L) telophase. The black arrowheads in (A, B, G and H) point out newly clustering
centrosomal patches of pp60c-src; thin arrows in (A) and (G), identify plasma membrane-associated wt c-Src; open arrows identify diffuse
cytoplasmic c-Src (A) or myr− c-Src (D and J); larger arrows in (G) and (J) show nuclei either devoid of c-Src (G) or staining for myr− cSrc (J); the white arrowhead in (G) marks an intercellular contact area where pp60c-src has accumulated.
Also, the significant differences observed between the cell
cycle-dependent subcellular distributions of the wild-type
and nonmyristoylated forms suggest that the patterns
observed reflect specific associations and not merely nonspecific low-affinity sites of interaction.
Many cases are known where protein mutation leads to
mislocalization, particularly when the mutation affects a
region involved in the specification of localization (as for
the myristoylation site in pp60 src) or when it induces a conformational change that exposes cryptic localization
sequences (as for v-Sis, lacking its signal sequence; Lee et
al., 1987). These changes in protein localization very frequently correlate with changes in the biological activity of
the proteins, emphasizing the influence of the subcellular
environment on the physiological function of an enzyme.
These remarks are especially relevant to the Src field and
Localization of wt and myr− c-Src
625
−
Fig. 9. Summary of cell cycle-dependent localization of wt pp60c-src (s, left panel) and pp60c-src/myr (-, right panel). First row: the
interphase distribution of the two proteins is plasma membrane-associated and centrosomal for wt pp60c-src, and cytoplasmic diffuse and
−
weakly nuclear for pp60c-src/myr . Second row: in late G2, before nuclear membrane breakdown, wt pp60c-src condenses around the two
−
c-src/myr
centriole pairs and pp60
concentrates in the nucleus. Third and fourth rows: in metaphase and anaphase, wt pp60c-src exhibits a
−
diffuse or patchy distribution through the cytoplasm; pp60c-src/myr condenses on mitosis-specific structures. Fifth row: in telophase, wt
−
c-src
pp60
resumes very fast its interphasic distribution and concentrates in intercellular contact areas; pp60c-src/myr remains concentrated in
the nucleus in early G1 before recovering its typical interphasic (mainly cytoplasmic) distribution.
further justify our present investigation, since it has previously been shown that myristoylation is required for plasma
membrane localization of pp60src (Buss et al., 1986) and
that it augments the transforming potential of the viral and
mutated cellular enzymes (Cross et al., 1984; Pellman et
al., 1985; Schuh and Brugge, 1988) even though it does not
affect their intrinsic protein-tyrosine kinase-specific activities (Schultz et al., 1985; Kamps et al., 1986; Reynolds et
al., 1989). However, it has often been tacitly assumed that
abrogation of myristoylation simply results in loss of a
specific (plasma membrane) localization, not that it may
lead to a highly specific, alternative pattern of localization.
The detailed comparative immunofluorescence study presented here clearly illustrates the complexity of the factors
influencing the localization of pp60c-src. In addition to
inducing a gain of function (plasma membrane localization), myristoylation induces three losses of function. It: (1)
prevents pp60c-src translocation to the nucleus during
growth arrest; (2) prevents nuclear translocation during G2
in a normal cell cycle; and (3) strongly reduces the affin-
626
T. David-Pfeuty, S. Bagrodia and D. Shalloway
ity of pp60 c-src for components of the spindle apparatus and
for the interchromosomal space during mitosis. (Among a
dozen overexpressed c-src mutants studied, the myristoylation-defective one is the only one that induced a nuclear
translocation of the protein and an association with the
mitotic apparatus.) This diversity of effect is consistent with
the view, arising from studies of a variety of myristoylated
viral and cellular proteins (McIlhinney, 1990, for review),
that myristoylation may mediate protein-protein interactions rather than protein-membrane interactions. Even the
effect of myristoylation on plasma membrane association
may be mediated via effects on protein-protein interactions,
since pp60src must interact with saturable high-affinity binding sites for membrane binding (Resh, 1989; Goddard et
al., 1989; Resh and Ling, 1990).
It is noteworthy that wt pp60c-src has a strong affinity for
specific structures that are well developed during interphase
before nuclear envelope breakdown (i.e. the plasma membrane, the centrosome and (during G2) the two pericentriolar areas). Beginning at mitosis, when the interphasic
microtubules start depolymerizing, pp60 c-src is redistributed
into diffuse and patchy structures throughout the cytoplasm,
an effect that can be mimicked by artificial disruption of
the interphasic microtubules through nocadozole treatment.
These observations: first, indicate that both the maintenance
of pp60c-src association with the plasma membrane and its
accumulation in the centrosomal area depend on the
integrity of the interphasic microtubule network; and,
second, they also suggest that the redistribution of wt
pp60c-src starting at the end of G2 could simply result from
the natural change in the state of organization and the subsequent depolymerization of the interphasic microtubules.
During mitosis, wt pp60c-src does not display particularly
high affinity for any specific structures participating in
mitotic events. These observations are in direct contrast
with those for pp60c-src/myr−; its interphase distribution does
not depend at all on the presence of a well-developed microtubule network but, starting at late G2 phase and during
mitosis it exhibits a strong affinity for many components
that participate in mitotic events (spindle poles and fibers,
interchromosomal material).
pp60src has been linked to control of a wide variety of
events occurring throughout the cell, from the plasma
membrane to the nucleus (for review, Cooper, 1990).
While its varied activities might be mediated by multiple
static subpopulations that participate in (tyrosine) phosphorylation cascades, it is interesting to consider the possibility that c-Src participates in intracellular signalling as
a physically transported messenger between the plasma
membrane and the nucleus. We have previously shown that
wt pp60 c-src apparently associates with vesicles containing
ConA-receptor complexes (endosomes) throughout the
entire ConA-receptor-mediated endocytotic process
(David-Pfeuty and Nouvian-Dooghe, 1990). This led us to
suggest that the accumulation of pericentriolar and perinuclear pp60c-src-containing patches during interphase might
represent plasma membrane-derived vesicles or endosomes
in transit between the plasma membrane and the centrosome. This hypothesis is supported, on one hand, by the
finding reported here that a subpopulation of plasma membrane-associated wt pp60c-src appears to be a component
of vesicular structures that are insoluble in the presence of
nonionic detergents (possibly early endosomes) and, on the
other hand, by a biochemical fractionation study (Kaplan
et al., 1992) showing that a subpopulation of wt pp60c-src
cofractionates with late endosomes. The report (Linstedt et
al., 1992) that pp60c-src also associates specifically with
synaptic vesicles in the neuroendocrine PC12 cell line is
also consistent with such an hypothesis. We find that the
lack of myristoylation of pp60c-src/myr− blocks association
not only with the plasma membrane, but also with ConAinduced endocytotic vesicles and endosomes (unpublished
results). This is consistent with the observation that
pp60c-src/myr− does not accumulate like wt pp60c-src at the
centrosome in interphase cells. It also implies that
pp60c-src/myr− must be transported to the nucleus by an independent mechanism.
A central question raised by this work is whether the
behavior of overexpressed pp60c-src/myr− could possibly
reflect that of a naturally occurring subset of pp60c-src in
normal cells. Buss and Sefton (1985) have estimated that
as much as 16% of pp60v-src may actually not be myristoylated. If an equivalent percentage of pp60c-src is nonmyristoylated in normal cells, it would be undetectable by
conventional techniques. Overexpression of wt pp60c-src by
a factor of 10 to 20 would even be insufficient to raise such
a nonmyristoylated subpopulation above the threshold level
of detectability by immunofluorescence (which is a few fold
higher than the endogeneous level of pp60c-src); this may
account for the fact that we did not detect such a subpopulation in wt c-Src overexpresser cells. We do not know if
the molecules in this putative non-myristoylated population
possess an N-terminal methionine or if, as for wild-type
pp60a-40Vc-Srca+40V, it is removed by aminopeptidase
activity. While there is no reason to believe that the presence or absence of this residue (or the additional alanines
present in the pcLN mutant) plays any significant role or
that the localization site(s) that govern nuclear translocation are at the amino end of the molecule, the possibility
that small amino acid changes in this region could have
effects on localization cannot be excluded. Interestingly,
abundant nuclear concentration of myr− c-Src in late
G2/early prophase cells and within the mitotic apparatus
does not interfere with mitotic progression; nor is progression through the cell cycle prevented by the massive nuclear
accumulation of myr− c-Src that occurs in cells that have
been blocked at the G1/S transition by thymidine-aphidicolin treatment. These observations imply that a naturally
occurring subset of pp60c-src/myr− would probably not exert
an inhibitory effect on cell cycle and mitotic progression.
It is also possible that the behavior of pp60c-src/myr− could
mimic to some extent that of a subpopulation of wt
pp60c-src. Indeed allosteric modifications of pp60c-src, perhaps resulting from phosphorylations or dephosphorylations
within the amino-proximal region of the molecule, might
interfere with the protein-protein interaction required for
plasma membrane localization and could generate cytosolic enzymes capable of being transported to the nucleus.
This is consistent with the report that myristate is present
in soluble cytoplasmic as well as membrane-bound, pp60src
(Buss et al., 1984). Such hypothetical motions would probably be regulated in a more refined manner that would pre-
Localization of wt and myr− c-Src
vent the excessive nuclear accumulation observed here with
the constitutive mutant myr− c-Src. Within this model, no
predictions can be made regarding the issue of whether a
naturally occurring subset of c-Src proteins (whose behaviour would only be partially mimicked by mutant
pp60c-src/myr−) would exert an inhibitory or positive effect
on cell cycle and mitotic progression.
It is worthwhile to mention here two recent observations
that are relevant to our observations. First, Zhao et al.
(1992) have reported that in vitro calcium-induced keratinocyte differentiation occurs concomitant with a marked
increase in nuclear phosphotyrosine content and along with
a translocation of c-Src to the nucleus. Second, C. Willman
(personal communication) has shown that a naturally occurring variant of c-Fgr lacking the amino-terminal domain
arises by alternative internal translation initiation in
myeloid cells; this variant has a nuclear location in myeloid
cells and in stably transfected fibroblasts. These data support the hypothesis that naturally occurring forms of
pp60c-src and of other members of the Src family could
indeed move into the nucleus and exert a physiological
function in that location. Our results also raise the intriguing suggestion that myristoylation might be required to prevent wt pp60c-src from interacting with nuclear constituents
during G2 phase and with the mitotic apparatus during mitosis. Interestingly, another well-described myristoylated
enzyme is the catalytic subunit of cAMP-dependent protein
kinase II (cAMP-dPKII), which translocates to the nucleus
solely following cAMP-induced dissociation from the
Golgi-associated cAMP-dPKII regulatory subunit (Nigg et
al., 1985). Since many protein kinases (including Src)
exhibit little specificity in in vitro assays, catalyzing the
phosphorylation of a wide variety of substrates that apparently are never phosphorylated under physiological conditions, it is easy to conceive that the spatial and temporal
distributions of such enzymes need to be subjected to very
strict control in vivo in order to avoid unscheduled protein
phosphorylation that could be catastrophic for cell survival.
We thank Yolande Nouvian-Dooghe for her excellent technical assistance, Mesdames Irène Gaspard and Colette Pouget for
their expert artwork, Eric Bailly for his helpful advices, Sarah Parsons, Marilyn Resh, Françoise Danon and Paul-Henri Mangeat for
providing antisera and Joan Brugge for plasmid pRSVc-srcLN.
We also thank Madame Françoise Arnouilh for typing the manuscript. This study was supported by the Centre National pour la
Recherche Scientifique, the Curie Institute and the Association
pour la Recherche sur le Cancer (France) and by grants CA32317
and CA47333 and RCDA CA01139 from the National Institutes
of Health (USA).
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(Received 18 December 1992 - Accepted 14 March 1993)