(Received for publication, May 3, 1995; and in revised form, July 13, 1995)
From the
Recently we reported the localization of phosphoinositide
3-kinase (PI 3-kinase) by immunofluorescence to microtubule bundles and
the centrosome (Kapeller, R., Chakrabarti, R., Cantley, L., Fay, F.,
and Corvera, S.(1993) Mol. Cell. Biol. 13, 6052-6063).
[Abstract]
In complementary experiments we used the recombinant p85 subunit of PI
3-kinase to identify proteins that associate with phosphoinositide
3-kinase and found that phosphoinositide 3-kinase associates with
/
-tubulin. The association occurs in vivo but was
not significantly affected by growth factor stimulation. We localized
the region of p85 that interacts with
/
-tubulin to the
inter-SH2 domain. These results support the immunofluorescence data and
show that p85 directly associates with
/
-tubulin. We then
determined whether phosphoinositide 3-kinase associates with
-tubulin. We found a dramatic growth factor-dependent association
of phosphoinositide 3-kinase with
-tubulin. Phosphoinositide
3-kinase associates with
-tubulin in response to insulin and, to a
lesser extent, in response to platelet-derived growth factor. Neither
epidermal growth factor nor nerve growth factor treatment of cells
results in association of phosphoinositide 3-kinase and
-tubulin.
Phosphoinositide 3-kinase is also immunoprecipitated with antibodies to
pericentrin in response to insulin, indicating that phosphoinositide
3-kinase is recruited to the centrosome. Neither phosphoinositide
3-kinase activity, nor intact microtubules are necessary for the
association. Treatment of cells with 0.5 M NaCl dissociates
-tubulin from the centrosome and disrupts the association of
phosphoinositide 3-kinase with pericentrin, but not
-tubulin.
Recombinant p85 binds to
-tubulin from both insulin stimulated and
quiescent cells. These results suggest that the association of
phosphoinositide 3-kinase with
-tubulin is direct. These data
suggest that phosphoinositide 3-kinase may be involved in regulating
microtubule responses to insulin and platelet-derived growth factor.
Phosphoinositide 3-kinase (PI 3-kinase) ()is a
heterodimer of a 110-kDa catalytic subunit and an 85-kDa regulatory
subunit and has both phosphoinositide and protein kinase
activities(2, 3) . In response to a number of growth
factors PI 3-kinase is activated resulting in the production of
PtdIns-3,4-P
and PtdIns-3,4,5-P
(4) .
The downstream targets of PI 3-kinase are still not well understood.
Members of the novel group of protein kinases C are activated by
PtdIns-3,4,5-P
and these proteins are likely targets of PI
3-kinase signaling(5) . Studies using both the PI 3-kinase
inhibitor, wortmannin and platelet-derived growth factor (PDGF)
receptor mutants, have shown that PI 3-kinase is necessary for ruffling
in response to PDGF, insulin, and insulin-like growth factor-1 (6, 7, 8) . Mutants of the PDGF receptor that
do not bind PI 3-kinase are also defective in cell motility in an assay
of cell migration toward a PDGF gradient(9) . PI 3-kinase
clearly has effects on the actin cytoskeleton.
Recently we have found evidence suggesting an interaction of PI 3-kinase and microtubules(1) . Immunofluorescence studies using an antibody to the 85-kDa subunit of PI 3-kinase have shown that PI 3-kinase is concentrated in the perinuclear region, especially around the centriole. Stimulation of cells with PDGF increases the staining of p85 in the perinuclear region. PI 3-kinase also localizes to some microtubule bundles. The stimulation of PI 3-kinase localization to the perinuclear region suggests that PI 3-kinase, in a complex with growth factor receptors, moves along microtubules to reach the perinuclear area(1, 10) . Microtubule-dependent vesicular and organelle transport has also been described in response to serum(11, 12) . The PI 3-kinase localized to microtubules could be either a component of vesicles moving along microtubules or directly associated with microtubules. There is no explanation for localization of p85 to the centrosome.
-Tubulin
is a recently discovered form of tubulin which is localized primarily
to the centrosome in mammalian cells and is proposed to be the link
between the microtubule organizing center and the microtubule
polymers(13, 14) . It is able to nucleate microtubule
formation and is also able to bind to the centrosome. This central role
in microtubule formation makes
-tubulin an attractive candidate
for regulation of microtubule formation.
Although there is quite
suggestive evidence that microtubule-dependent processes occur in
response to growth factors (10, 11) and
pp60 and insulin-dependent phosphorylation of
- and
-tubulin have been described(15, 16) ,
it is not clear that significant changes in microtubules themselves
occur in response to growth factors. It might be expected that cells
responding to growth factors would alter microtubules to facilitate any
number of responses from shape change to transport. These sorts of
changes in microtubules are technically difficult to study in response
to growth factors and little data are available describing alterations
in microtubule dynamics or structure in response to growth factors.
In an effort to better understand the function of PI 3-kinase we
analyzed proteins that associated with recombinant p85. Surprisingly,
we found that p85 associates directly with /
-tubulin. These
data provide a mechanism for the previous observation that a fraction
of p85 localizes with microtubules by immunofluorescence. Since p85
bound to
/
-tubulin we also examined whether PI 3-kinase
associates with
-tubulin. We found that PI 3-kinase associates
with
-tubulin and that there is a dramatic increase in the
association in response to insulin and PDGF. The growth
factor-dependent association of PI 3-kinase with
-tubulin suggests
that microtubules themselves could be regulated in response to growth
factors and that PI 3-kinase might play a role in this regulation.
Figure 1:
PI 3-kinase and -tubulin
immunoprecipitate together from CHO-IR cell lysates. A, CHO-IR
cell lysates, from quiescent or insulin-stimulated cells, were
incubated with immobilzed GST or GST-85 and washed as described under
``Experimental Procedures.'' Tyrosine-phosphorylated peptides
were included at a concentration of 10 µM, to determine
whether the association involved the SH2 domains of p85. The proteins
were then separated by SDS-PAGE, transferred to nitrocellulose, and
blotted for
-tubulin. B, CHO-IR cells were treated or not
with insulin (100 nM) for 5 min at 37 °C. Cells were lysed
and immunoprecipitated with control Ig,
-tubulin, or p85
antibodies, as indicated. The immunoprecipitates were washed four times
with lysis buffer, separated by SDS-PAGE, and transferred to
nitrocellulose. Whole cell lysates (WCL) and the
immunoprecipitations were analyzed for the presence of the p85 subunit
of PI 3-kinase and
-tubulin by immunoblotting. p85 was detected by
alkaline phosphatase and
-tubulin was detected by ECL. C,
/
-tubulin, purified from rat brain, was incubated with GST or
GST-85 on GSH beads. The beads were washed, the proteins separated by
SDS-PAGE, transferred to nitrocellulose, and Western blotted for
-tubulin. A similar association experiment from CHO-IR lysate was
done simultaneously as a control.
To investigate whether tubulin and PI 3-kinase
associate in intact cells, p85 and -tubulin immunoprecipitates
from insulin-treated and control CHO-IR cells were examined for the
presence of p85
and
-tubulin by Western blotting. As shown in Fig. 1B,
-tubulin was detected in p85
immunoprecipitates from both insulin-stimulated and control CHO-IR
cells (lanes 3 and 4). Treatment of CHO-IR cells with
insulin, however, did not change the amount of
-tubulin that
immunoprecipitated with p85. The
-tubulin antibody also
immunoprecipitated p85 (Fig. 1B, lanes 5 and 6). Neither did insulin treatment affect the amount of p85
observed in
-tubulin immunoprecipitates (Fig. 1, lanes
5 and 6). An irrelevant antibody did not
immunoprecipitate either tubulin or p85 from CHO-IR cell lysates (Fig. 1, lanes 7 and 8), indicating that
association of tubulin with PI 3-kinase is specific. These results
indicate that tubulin and PI 3-kinase interact in vivo and
that this association is not changed by insulin. Based on Western
blotting, we estimate that 5-10% of cellular p85 associates with
/
-tubulin. Similar results were obtained when PI 3-kinase
activity, rather than Western blotting, was measured on the
immunoprecipitates (data not shown).
To determine whether the
interaction between p85 and -tubulin was direct, we incubated
GST-85-coated beads with tubulin purified from rat brain. Coomassie
Blue staining showed that tubulin was purified to homogeneity (not
shown). We also incubated GST- or GST-85-coated beads with untreated
CHO-IR cell lysates as a control. The precipitates were analyzed for
the presence of
-tubulin by Western blotting. GST-85 precipitated
-tubulin from the purified preparation, indicating that the
association between p85 and tubulin is direct (Fig. 1C).
Figure 2:
The inter-SH2 domain of the p85 subunit of
PI 3-kinase mediates the association with /
-tubulin. A, the constructs used to identify the region of p85 that
binds to tubulin. B, the GST fusion proteins containing
different domains of p85 were incubated for 30 min at 4 °C with
CHO-IR cell lysates with or without insulin stimulation. The beads were
washed and the precipitated proteins separated by SDS-PAGE followed by
transfer to nitrocellulose. The membrane was probed initially with the
-tubulin antibody (panels A and B). The membrane
was then stripped and probed with goat anti-mouse horseradish
peroxidase (HRP) antibody to be certain the membrane was fully
stripped (panels C and D). The membrane was then
probed with the
-tubulin antibody (panels E and F). C, the GST-85 fusion proteins were incubated with
tubulin purified from rat brain. The precipitates were then analyzed
for the presence of
- and
-tubulin as above. WCL,
whole cell lysates.
With each of these constructs
there is a small increase in the amount of tubulin that associates with
the p85 constructs after insulin stimulation. Since we have not been
able to reproducibly find a difference in the association of PI
3-kinase and /
-tubulin following insulin stimulation, as
judged by activity measurements on
/
-tubulin
immunoprecipitates, we are uncertain of the physiologic relevance of
this finding.
Since tubulin exits as a heterodimer of and
subunits we wanted to confirm that the
subunit was also
present. The membrane was stripped and reprobed with
-tubulin
antibody (Fig. 2B, panels E and F).
-Tubulin was also precipitated from CHO-IR cells with GST-85,
GST-N+C, and to some extent by the GST-CSH2, but not by other GST
fusion proteins containing other domains of p85. The inter-SH2 domain
of p85 is predicted to contain a coiled-coil (23) and tubulin
also has a coiled-coil structure. It is possible that interaction of
these two coiled-coil domains mediates the binding of p85 to tubulin.
Figure 3:
Association of PI 3-kinase with
-tubulin. A, CHO-IR cells were treated or not with
insulin (100 nM) for 5 min at 37 °C. Cells were lysed and
incubated with control Ig,
-tubulin, or p85 antibodies, as
indicated, for 3 h at 4 °C. The immunoprecipitates were washed four
times with lysis buffer and analyzed for the presence of PI 3-kinase
p85 and
-tubulin by immunoblotting. B, PI 3-kinase
activity associated with
-tubulin. CHO-IR cells were treated with
insulin (100 nM) at 37 °C for the indicated times. The
cells were then lysed and immunoprecipitated with
-tubulin
antibodies. The immunoprecipitates were washed and assayed for PI
3-kinase activity. The lipids were extracted, separated by TLC, and
visualize by autoradiography. PIP, phosphatidylinositol
phosphate; PIP
,
phosphatidylinositol-3,4,5-trisphosphate.
To confirm the Western blot findings we also immunoprecipitated
-tubulin from CHO-IR cells and assayed the immunoprecipitates for
PI 3-kinase activity and determined the time course of the association.
In quiescent cells we found minimal PI 3-kinase activity associated
with
-tubulin, but stimulation with 100 nM insulin
resulted in the appearance of PI 3-kinase in the immunoprecipitates (Fig. 3B). The associated activity peaked between 5 and
15 min after insulin stimulation and decayed over an hour. We found
maximal association of PI 3-kinase with
-tubulin at an insulin
concentration of 10 nM, indicating that the effect is mediated
at physiologic insulin concentrations (not shown). The increase in PI
3-kinase activity in
-tubulin immunoprecipitates could be
explained either by a stimulation of activity of PI 3-kinase
constitutively associated with
-tubulin or recruitment of PI
3-kinase in response to insulin. The Western blots demonstrate that PI
3-kinase is recruited to
-tubulin in response to insulin (Fig. 3A).
A comparison of the amount of activity
that immunoprecipitates with - or
-tubulin antibodies to
-tubulin antibodies showed that much less activity was associated
with the
- or
-tubulin immunoprecipitates. Since more p85
protein is present in
- or
-tubulin immunoprecipitates, this
indicates that the specific activity of PI 3-kinase associated with
-tubulin is higher.
Figure 4:
Association of PI 3-kinase with
-tubulin and pericentrin. CHO-IR cells were treated or not with
insulin as indicated. Cells were then lysed in the presence of 0.2 M or 0.5 M NaCl. The NaCl was then diluted to 200
mM and immunoprecipitates were done using either the
-tubulin or pericentrin antibodies. The immunoprecipitates were
washed and assayed for PI 3-kinase activity as described in the legend
to Fig. 3. PIP, phosphatidylinositol phosphate;
PIP
,
phosphatidylinositol-3,4,5-trisphosphate.
As a control for the effect of the 0.5 M NaCl and to determine whether PI 3-kinase associated with
-tubulin at the centrosome we determined the presence of PI
3-kinase activity in immunoprecipitates using an antibody to
pericentrin, another centrosomal protein(25) . We found that PI
3-kinase activity was present in the pericentrin immunoprecipitates
from insulin-stimulated, but not quiescent cells (Fig. 4B). This confirmed that PI 3-kinase is recruited
to the centrosome in response to insulin. These results do not exclude
the possibility that PI 3-kinase also associates with
-tubulin
that is not at the centrosome. Furthermore, pericentrin
immunoprecipitates from insulin-stimulated cells lysed in 0.5 M NaCl lack PI 3-kinase activity. Western blots of similar
immunoprecipitates show no difference in the amount of pericentrin
present (data not shown). These data suggest that PI 3-kinase is in a
tight complex with
-tubulin and pericentrin and remains associated
with
-tubulin even after salt disruption of
-tubulin binding
to the centrosome.
Figure 5:
The p85 subunit of PI 3-kinase associates
with -tubulin. GST-and GST-85 were incubated with cell lysates
from control or insulin-treated CHO-IR cells and the precipitation and
Western blotting was carried out described in the legend to Fig. 2. WCL, whole cell
lysates.
The association of
GST-85 with -tubulin from quiescent cells suggests that no
modification of
-tubulin is necessary for association with PI
3-kinase in response to insulin, but that PI 3-kinase is modified in
some manner that allows it to interact with
-tubulin. It is likely
that a domain of p85 is revealed in response to insulin that is able to
interact with
-tubulin. The presence of p110 associated with p85
in native PI 3-kinase may change the structure of p85 such that a
covalent modification or association with another protein is required
to unmask the domain. We attempted to identify the domain of p85 that
binds to
-tubulin, as we did for
/
-tubulin. We were not
able to precipitate
-tubulin with any of the subdomains of p85
that were used in the experiment shown in Fig. 2(not shown).
There are several possible explanations. More than one domain may be
necessary to bind
-tubulin or our constructs may have split the
domain necessary for interaction with
-tubulin. These results
indicate that
-tubulin associates with a different domain of p85
than
/
-tubulin.
Figure 6:
Neither PI 3-kinase activity nor intact
microtubules are required for recruitment of PI 3-kinase to
-tubulin. A, CHO-IR cells were treated with 25 µM LY294002 for 20 min and then stimulated with insulin for 5 min.
-Tubulin immunoprecipitates were done and assayed for PI 3-kinase
activity as described in the legend to Fig. 3. B,
CHO-IR cells were treated with 20 µg/ml nocodazole for 20 min and
then stimulated with insulin for 5 min and immunoprecipitated with the
-tubulin antibody as described in the legend to Fig. 3. PIP, phosphatidylinositol phosphate; PIP
,
phosphatidylinositol bisphosphate; PIP
,
phosphatidylinositol-3,4,5-trisphosphate.
Since the centrosome is the origin of microtubules we thought that
PI 3-kinase might move along microtubules to reach the centrosome and
-tubulin. This could occur as insulin receptors undergo
endocytosis and carry PI 3-kinase with them. We used nocodazole to
disrupt microtubules to determine whether intact microtubules were
necessary for the recruitment of PI 3-kinase to
-tubulin. We found
that there was no effect of nocodazole on the association of PI
3-kinase with
-tubulin (Fig. 6B). We also
determined by immunofluorescence staining of microtubules that a 20-min
treatment with nocodazole caused significant microtubule
depolymerization. This indicates that intact microtubules are not
necessary for PI 3-kinase to associate with
-tubulin and the
centrosome.
Figure 7:
Insulin and PDGF but not NGF or EGF
induce the association of PI 3-kinase with -tubulin. A,
A431 cells were treated with EGF, insulin, or nothing, as indicated.
The cells were then immunoprecipitated with
-tubulin antibodies
and assayed for PI 3-kinase activity as described in the legend to Fig. 3. B, NIH 3T3 cells expressing the human insulin
receptor were treated with insulin, PDGF, or nothing, as indicated.
-Tubulin was then immunoprecipitated and PI 3-kinase assayed. C, PC-12 cells were either treated with insulin, EGF, NGF, or
nothing and
-tubulin was immunoprecipitated and PI 3-kinase
assayed. PIP, phosphatidylinositol phosphate; PIP
,
phosphatidylinositol-3,4,5-trisphosphate.
We have found that PI 3-kinase, probably through the
inter-SH2 domain of p85, binds to /
-tubulin. This association
is direct, as indicated by detection of the complex formed from
recombinant p85 and purified tubulin. Although we saw a small effect of
insulin on the association of GST-85 constructs with
/
-tubulin, the physiologic relevance of this difference is
not clear. Immunofluorescence studies using antibodies to p85 suggested
that p85 localizes near microtubule bundles(1) . Our data
suggest that PI 3-kinase truly is localized to microtubules, through a
direct association with the
/
heterodimer.
It was previously proposed that PI 3-kinase moves with the PDGF receptor in stimulated cells along microtubules to localize near the microtubule organization center(1) . The direct association of PI 3-kinase with tubulin suggests a distinct function of PI 3-kinase bound to microtubules. There is little effect of growth factors on the association and if PI 3-kinase were moving with a vesicle one would expect a motor, not PI 3-kinase, to directly contact tubulin. For these reasons it does not seem likely that the PI 3-kinase associated with tubulin is part of a vesicle moving along the microtubule. We do not know the function of PI 3-kinase associated with microtubules. It could play a role in microtubule formation and stability or perhaps in regulating the budding or fusion of vesicles localized on microtubules.
The association of PI 3-kinase with /
-tubulin led us to
examine whether PI 3-kinase might also bind to
-tubulin. We found
that insulin and PDGF stimulate the association of PI 3-kinase with
-tubulin. EGF and NGF do not stimulate the association. PI
3-kinase is recruited to
-tubulin at the centrosome, but we cannot
exclude the possibility that PI 3-kinase is also recruited to
-tubulin at sites other than the centrosome. Recruitment of PI
3-kinase to
-tubulin does not seem to require either the enzymatic
activity of PI 3-kinase nor intact microtubules. We cannot be certain
that
-tubulin associates directly with PI 3-kinase since a
purified form of
-tubulin is not available. Since GST-85
associates with
-tubulin from quiescent as well as
insulin-stimulated cells, the effect of insulin that allows association
of PI 3-kinase and
-tubulin is probably mediated by a modification
of PI 3-kinase and not
-tubulin.
The immunofluorescence study
of p85 identified two areas of perinuclear localization(1) . A
more diffuse localization whose intensity increases in response to PDGF
and is blocked by nocodazole is consistent with a late endosomal
compartment(29) . Additionally p85 was found to localize to the
centrosome(1) . The localization was not affected by
nocodazole. The association of PI 3-kinase with -tubulin explains
the localization of p85 to the centrosome. It is not clear how p85 is
recruited to
-tubulin. It is not likely that PI 3-kinase is
transported with endosomes containing the receptor, since nocodazole
blocks the redistribution of p85 in response to PDGF(1) , but
does not block recruitment of PI 3-kinase to
-tubulin.
Insulin
is the primary stimulus that leads to the association of -tubulin
and PI 3-kinase. Insulin and other growth factors share many common
downstream signals. One unique aspect of insulin signaling is the
phosphorylation of IRS-1. We thought that IRS-1 might mediate the
association with
-tubulin and explain why insulin seems to be the
primary stimulus leading to association. We found no evidence for the
presence of IRS-1 in
-tubulin immunoprecipitates. Also, since
IRS-1 is not phosphorylated in response to PDGF it could not explain
the PDGF response. We are uncertain of the upstream signal that allows
PI 3-kinase to associate with
-tubulin in response to insulin.
The obvious question raised by these data is what does PI 3-kinase
do at the centrosome in response to insulin? These data suggest that
there are growth factor-dependent effects on microtubules. The
centrosome is known to orient in the direction of cell movement in
cells undergoing chemotaxis and it is possible that PI 3-kinase plays a
role in this orientation(30) . There is ample evidence that PI
3-kinase is necessary for PDGF-dependent chemotaxis(9) . PI
3-kinase has also been implicated in trafficking of the PDGF receptor
to the perinuclear area and the PI 3-kinase -tubulin interaction
could be important in this event(10) .