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(Received for publication, March 14, 1996, and in revised form, April 22, 1996)
From the Department of Medicine, Jack Bell Research Centre,
University of British Columbia, Vancouver,
British Columbia V6H 3Z6, Canada
ABSTRACT Phosphatidylinositol (PI) 3-kinase is activated
as a result of cytokine-induced association of the enzyme with specific
tyrosine-phosphorylated proteins. PI 3-kinase lipid products, PI
3,4-P2 and PI 3,4,5-P3, have been shown,
in vitro, to directly activate novel and atypical protein
kinase C (PKC) isozymes. However, the mechanism by which PI 3-kinase
may be involved in regulation of PKC isoforms in vivo is
presently unknown. We investigated a possible relationship by looking
for associations between these enzymes. We found that in a human
erythroleukemia cell line, as well as in rabbit platelets, PI 3-kinase
and PKC INTRODUCTION Cytokine stimulation of mitogenesis of hematopoietic cells is
known to occur through at least two signaling pathways, both of which
depend on tyrosine phosphorylation of key regulatory proteins; one is
the p21ras/mitogen-activated protein kinase
network and the other is the PI1 3-kinase
pathway. Following ligand binding, the common The involvement of p21ras in mitogenesis is well understood,
but the role of PI 3-kinase, which is thought to be essential as well
(15), is less well characterized. Recent evidence suggests that two
serine/threonine kinases, p70 S6 kinase (16, 17) and PKB (18), are
thought to be activated downstream of PI 3-kinase. In hemopoietic
cells, we showed previously that IL-3, IL-5, and GM-CSF as well as IL-4
and Steel factor all activate PI 3 kinase (14), and indeed, this signal
is important in the ability of cytokines to inhibit apoptosis (19).
An intriguing set of observations made in the past few years has
suggested that PI 3,4-P2 and PI 3,4,5-P3 can
activate several novel and atypical PKC isoforms (PKC In an effort to further delineate pathways operating downstream of the
related receptors for IL-3, IL-5, and GM-CSF, we have examined the role
of PKC isoforms. This is a complex task, as there are at least 11 distinct family members that may be regulated by several independent
mechanisms. In this report, we focus on the potential interaction of
one specific PKC isoform, PKC EXPERIMENTAL PROCEDURES TF-1 cells, a human erythroleukemia cell line (a kind
gift from Dr. T. Kitamura, DNAX, Palo Alto, CA), were maintained in
RPMI 1640 (Life Technologies, Inc.) with 10% fetal bovine serum
(Intergen) plus 5 ng/ml IL-5 (purified recombinant human IL-5 prepared
in a baculovirus expression system was kindly provided by Dr. D. Nicholson, Merck-Frosst, Kirkland, Quebec). In experiments in which
cells were stimulated with cytokines, human recombinant cytokines were
used; IL-3 and GM-CSF were from R&D Systems and IL-5 as described
above. RBL-2H3 cells (a kind gift from Dr. John Rivera, NIH) were
maintained in RPMI 1640 with 10% fetal bovine serum. Cells were
trypsinized and passaged every 3 days.
TF-1 cells,
grown in complete RPMI with IL-5, were placed in cytokine-free medium
with 1% serum 18 h prior to assay. Cells (5 × 106/500 µl) were washed three times in Hanks' balanced
salt solution buffered with 20 mM Hepes, pH 7.4, and then
preincubated in RPMI 1640 buffered with 20 mM Hepes, pH
7.4, at 37 °C for 30 min. Recombinant human GM-CSF (50 ng/ml), IL-3
(100 ng/ml), or IL-5 (500 ng/ml) were added for 15 min. Cells were
solubilized in 500 µl of solubilization buffer (50 mM
Tris-Cl, pH 7.7, 1% Triton X-100, 10% glycerol, 100 mM
NaCl, 2.5 mM EDTA, 10 mM NaF, 0.2 mM Na3VO4, 1 mM
Na2MoO4, 40 µg/ml phenylmethylsulfonyl
fluoride, 1 µM pepstatin, 0.5 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor), centrifuged to remove debris, and
then mixed with immunoprecipitating antibodies (anti-PKC Following immunoprecipitation as
described above, beads were used to assay for PI 3-kinase activity as
described (14, 27). Briefly, the beads were washed twice with
solubilization buffer and three times with 10 mM Tris-Cl
buffer, pH 7.4. Phosphatidylinositol (10 µg/sample) in 30 mM Hepes, pH 7.4, and 40 µl of kinase buffer (30 mM Hepes, pH 7.4, 30 mM MgCl2, 50 µM ATP, 200 µM adenosine,
[32P]ATP 10 µCi/µl) were added to the beads. The
reaction was stopped after 15 min with 1 N HCl.
Chloroform:methanol (1:1) was used to extract the lipid, and the
labeled PI-3-P was separated by thin layer chromatography using
oxalate-treated aluminum-backed silica gels (EM Scientific) and solvent
comprised of NH4OH:water:methanol:chloroform. Rabbit
platelets were prepared as described (28).
RBL-2H3 cells were
trypsinized and washed in RPMI with serum. Cells were passively
sensitized by incubation in the same medium with supernatant from a
monoclonal antibody line producing IgE specific for dinitrophenol (a
kind gift from Dr. John Rivera, NIH) at a dilution of 1:25. After
1 h at 37 °C, cells were washed twice in Tyrode's buffer, pH
7.4, with Ca2+ and Mg2+ added, and resuspended
in the same buffer at 5 × 106 cells/ml. Cross-linking of
the Fc RESULTS AND DISCUSSION The association of PKC isoforms
We next determined if this association was inducible by cytokine
stimulation. After 15 min of stimulation with GM-CSF, there was an
increased association of PI 3-kinase in anti-PKC To investigate whether the PI 3-kinase co-immunoprecipitated with
PKC
We next studied the role of PI 3-kinase activity in mediating the
association with PKC Recent reports have described tyrosine phosphorylation of PKC
To determine if PI 3-kinase associates with PKC isoforms in any other
signaling systems we next investigated rabbit platelets treated with
the potent agonist, PAF. PAF-treated platelets also showed an increase
in PI 3-kinase protein co-immunoprecipitated with PKC
We also characterized the serine/threonine kinase activity in anti-p85
immunoprecipitates and to date have found Ca2+-independent
protein serine/threonine kinase activity as evidenced by
phosphorylation of a Ser-containing peptide substrate corresponding to
the PKC The importance of PI 3-kinase in signal transduction has been pointed
out in numerous publications over the past few years. There have been
many proteins shown to associate with PI 3-kinase following stimulation
of cells, generally as a result of tyrosine-phosphorylated proteins
associating via the PI 3-kinase p85 SH2 domains. This type of
association is known to result in PI 3-kinase activation, independent
of the tyrosine phosphorylation of the p85 subunit itself (35). We
demonstrate here for the first time that at least two members of the
PKC family of enzymes, PKC In the TF-1 cells, IL-3, IL-5, and GM-CSF are each able to provide a
complete mitogenic stimulus, while only GM-CSF and, to a lesser extent,
IL-5 are able to cause increased PI 3-kinase/PKC FOOTNOTES Acknowledgments We thank David Fong for technical assistance
and Dr. Michael Gold for helpful discussions.
REFERENCES
Volume 271, Number 24,
Issue of June 14, 1996
pp. 14514-14518
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Specifically Associates with
Phosphatidylinositol 3-Kinase Following Cytokine Stimulation*
and
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
associate in a specific manner that is modulated by cell
activation. Granulocyte-macrophage colony-stimulating factor treatment
of cells caused increased association of PKC and PI 3-kinase as did
treatment of platelets with platelet-activating factor. Results using
two PI 3-kinase inhibitors, wortmannin and LY-294002, showed that the
former inhibited this association, while the latter did not, suggesting
that PI 3-kinase lipid products may not be a prerequisite for the PI
3-kinase/PKC association. Our results also suggest that tyrosine
phosphorylation of PKC is not involved in its association with PI
3-kinase.
subunit of receptors
for interleukin (IL)-3, IL-5, and granulocyte/macrophage
colony-stimulating factor (GM-CSF) becomes tyrosine-phosphorylated (1,
2). Several other signaling proteins including JAK-2 (3), STAT-5 (4),
p46, and p52 Shc (5, 6), p42/44 mitogen-activated protein kinases (7,
8), and SH-PTP2 (9) become tyrosine-phosphorylated as well. This
cascade of tyrosine phosphorylation is thought to be initiated by the
activation of the receptor-associated JAK-2 kinase and the subsequent
phosphorylation of the receptor (3), although many of the details of
the subsequent events have yet to be worked out. Tyrosine
phosphorylation of Shc, followed by SH2-mediated association with the
adaptor protein Grb-2 and the p21ras activator, m-SOS1, have
been shown to be involved in the activation of p21ras (5, 6,
10), and activation of PI 3-kinase in hemopoietic cells is a result of
cytokine-induced association of PI 3-kinase with specific
tyrosine-phosphorylated proteins (11, 12, 13, 14). A
tyrosine-phosphorylated 70-kDa substrate was shown to associate
with PI 3-kinase following treatment of cells with IL-3 or GM-CSF (14),
although this substrate was subsequently shown to be the tyrosine
phosphatase, SH-PTP2, and its association may be indirect in a complex
with Grb-2 (9).
, -, -
,
and -
) (20, 21). These results were obtained from in
vitro assays, but the mechanism whereby PKC isoforms may be
activated by PI 3-kinase in vivo is not known. The
activation of PKC activity by cytokines has been reported in a few
publications (22, 23), but it is not clear how this may occur. In the
case of cytokines such as IL-3, IL-5, and GM-CSF, there is no evidence
that a classical PI-phospholipase C pathway is operating, although
evidence for increased production of diacylglycerol derived from
phosphatidylcholine has been reported (24, 25, 26).
, with the PI 3-kinase enzyme. We find
that the two enzymes can be co-immunoprecipitated as shown both by
immunoblotting and enzyme activities. Furthermore, the association is
modulated by cytokines, particularly by GM-CSF, in a human hemopoietic
cell line. In another system, platelets activated by
platelet-activating factor (PAF), we can also demonstrate an increase
in the association between the two enzymes following activation of the
cells. In addition, increased tyrosine phosphorylation of PKC in
response to FcRI receptor activation in another cell type does not
lead to any change in association between PKC and PI 3-kinase.
Therefore, we have discovered an association between PI 3-kinase and
one specific PKC isoform that is probably independent of tyrosine
phosphorylation of PKC and is somehow modulated by specific
receptor-mediated events.
mouse
monoclonal (Transduction Laboratories; 2 µg/500 µl of
solubilization buffer), anti-PKC rabbit polyclonal IgG (Life
Technologies, Inc.; 4 µg/500 µl of cell lysate), or anti-p85 rabbit
polyclonal antiserum (raised against the p85 SH2-glutathione
S-transferase fusion protein; a kind gift from Dr. Melanie
Welham; 1 µl/500 µl of cell lysate) for 1 h on ice. Following
addition of protein A-Sepharose beads (Pharmacia Biotech Inc.), samples
were rotated at 4 °C for 1 h. The beads were washed 5 times
with cold solubilization buffer. For immunoblots, proteins were eluted
with SDS sample buffer containing 2-mercaptoethanol and boiled at
90 °C for 1 min prior to loading on 8.0% polyacrylamide gels using
an acrylamide:bis ratio of 118:1. Proteins were transferred onto
nitrocellulose using an LKB Novablot semi-dry transfer apparatus. The
nitrocellulose blots were preincubated with Tris-buffered saline (5%
bovine serum albumin, 1% ovalbumin, 0.05% NaN3) when
blotted with anti-phosphotyrosine antibody 4G10 (Upstate Biotechology
Inc., Lake Placid, NY), anti-p85, or anti-p110 (Santa Cruz
Biotechology), or they were blocked using 5% skim milk powder for
immunoblots with anti-PKC, -, -
, -, -µ, or - (Santa
Cruz Biotechology).
RI Cross-linking in RBL-2H3 Cells
RI receptor was achieved by addition of dinitrophenol-human
serum albumin (Sigma) at a final concentration of 400 ng/ml. At various
times, cells were centrifuged, then solubilized, and
PKC-immunoprecipitated as described above.
and with PI 3-kinase was
examined in the erythroleukemia cell line TF-1. Cell lysates from TF-1
cells, immunoprecipitated with antibodies to either PKC or PKC
and analyzed by immunoblotting with antibodies to the p85 or the p110
subunits of PI 3-kinase, showed that PI 3-kinase was being
co-immunoprecipitated (Fig. 1, A and
B, and Fig. 2). In reciprocal experiments,
immunoprecipitation with antibody to p85 co-immunoprecipitated PKC
and -, as detected by immunoblotting. Other PKC isoforms found in
TF-1 cells include , , µ, , and
.2 None of the latter PKC isoforms were
found to co-immunoprecipitate with PI 3-kinase, as determined by
immunoblotting following immunoprecipitation with anti-p85 antibody,
either in untreated cells or following stimulation with GM-CSF, IL-3,
or IL-5 (data not shown).
Fig. 1.
Anti-PKC immunoprecipitates from TF-1 cell
lysates contain PI 3-kinase. A, control samples indicate a
basal level of PI 3-kinase association with PKC. This association is
dramatically enhanced by stimulation with GM-CSF, slightly with IL-5,
and not at all with IL-3. B, the same blot washed (without
stripping) and reprobed with antibody to PI 3-kinase subunit p110,
revealing a similar pattern of PI 3-kinase association with PKC.
C, the same blot washed and reprobed with anti-PKC to
verify equal amounts of immunoprecipitated PKC in all samples. These
blots are representative of three independent experiments. Ippt,
immunoprecipitate.
Fig. 2.
TF-1 cell lysates immunoprecipitated with
anti-PKC antibody and analyzed by immunoblotting contain PI
3-kinase. Control samples indicate a basal level of PI 3-kinase
association with PKC. This association is not enhanced by cytokine
stimulation and is not affected to a significant extent by 100 nM WM pretreatment of cells. This blot is representative of
three independent experiments. Ippt,
immunoprecipitate.
immunoprecipitates.
This increased association was marginal in IL-5-treated cells but was
not evident in IL-3-treated cells (Fig. 1, A and
B). There was no detectable increase in PI 3-kinase protein
in any of the PKC immunoprecipitates (Fig. 2). The difference in the
effect using GM-CSF compared with IL-3 was very reproducible at this
time point, but further studies should be done to test additional
times.
or PKC remains active, anti-PKC and anti-PKC
immunoprecipitates were analyzed for PI 3-kinase activity in
vitro. GM-CSF stimulation of the cells led to increased PI
3-kinase activity associated with PKC compared with control samples
or IL-5- or IL-3-stimulated cells (Fig. 3A),
thereby correlating with the detection of PI 3-kinase in immunoblots.
Anti-PKC immunoprecipitates also contained active PI 3-kinase;
however, the PI 3-kinase activity was not affected by cytokine
treatment of cells.
Fig. 3.
Cell lysates immunoprecipitated with
anti-PKC contain active PI 3-kinase. A, immunoblot
analysis demonstrates a reduction in PI 3-kinase association with
PKC when cells are pretreated with WM specifically in the
IL-5-stimulated and GM-CSF-stimulated samples. B, the same
immunoblot washed and reprobed with anti-PKC to confirm an equal
amount of PKC in each sample. C, a basal level of PI
3-kinase activity is present in the immunoprecipitates in cells that
were starved of cytokine overnight. GM-CSF-stimulated cells show an
average of 2.25-fold increased activity from three experiments, based
on counts/min incorporated into phosphatidylinositol. WM pretreatment
of cells results in decreased PI 3-kinase activity in the
immunoprecipitates from control (50% decrease), IL-5-treated cells
(70% decrease), and GM-CSF-treated cells (70% decrease).
Ippt, immunoprecipitate.
, taking advantage of two potent inhibitors of
PI 3-kinase, wortmannin (WM) and LY-294002. Cells were pretreated with
100 nM WM or 50 µM LY-294002 prior to
cytokine stimulation, conditions that are known to inhibit the majority
of PI 3-kinase activity (29, 30).3 WM
decreased the association of PKC with PI 3-kinase in all cases (Fig.
3, A and B), but LY-294002 had no effect (data
not shown). The relative decreases were consistently more pronounced in
GM-CSF- and IL-5-treated cells than in IL-3-treated cells. WM is known
to bind covalently to the active site of the p110 subunit, while
LY-294002 is a competitive inhibitor. The contrasting results between
WM and LY-294002 suggest that the PI 3-kinase/PKC association is not
modulated by the lipid products of PI 3-kinase activity but may be
affected by alterations of the p110 subunit caused by WM.
Alternatively, we cannot rule out the possibility that WM is having its
effect on the PI 3-kinase/PKC association independently of its
effect on PI 3-kinase activity. Other inhibitory effects of WM have
been described (31), and in our laboratory we have recently shown that
WM has inhibitory effects on other kinases that are not seen with
concentrations of LY-294002 that cause an equivalent inhibition of PI
3-kinase activity.3
following stimulation with
12-O-tetradecanoylphorbol-13-acetate, carbachol, substance
P, or FcRI cross-linking (32, 33, 34). In TF-1 cells, a faint band is
detected in anti-phosphotyrosine blots of anti-PKC
immunoprecipitates, and this band comigrates with PKC (data not
shown). However, we have been unable to detect any differences in the
levels of tyrosine phosphorylation following cytokine stimulation.
Phosphoamino acid analysis of PKC will be necessary to confirm
whether PKC is being tyrosine-phosphorylated to any significant
extent. In another independent system, we have confirmed the results of
Haleem-Smith et al. (34), showing that cross-linking of the
FcRI receptor in RBL-2H3 cells leads to a large increase in tyrosine
phosphorylation of PKC (Fig. 4A), but
interestingly, no PI 3-kinase was present in PKC immunoprecipitates,
either before or after receptor activation (Fig. 4B).
Together, these results strongly suggest that the association between
PI 3-kinase and PKC that we have observed is independent of PKC
tyrosine phosphorylation and is therefore not likely to be mediated by
the SH2 domains of the PI 3-kinase p85 subunit.
Fig. 4.
Tyrosine phosphorylation of PKC does not
result in its association with PI 3-kinase. RBL-2H3 cells were
passively sensitized with specific IgE and then either left untreated
or stimulated by addition of antigen for 1 min or 10 min. A,
anti-phosphotyrosine immunoblots of PKC immunoprecipitates show that
cross-linking of the FcRI receptor results in increased tyrosine
phosphorylation in RBL-2H3 cells and increased tyrosine phosphorylation
of PKC. B, in the same immunoprecipitates, there
was no evidence for PI 3-kinase being associated, although the p85
subunit of PI 3-kinase could be easily detected in the whole cell
extracts. The whole cell extracts represent protein from 1 × 105 cells, and the immunoprecipitatesw ere from 5 × 106 cells. Ippt, immunoprecipitate;
Pre-IP, cell extract prior to immunoprecipitation.
, compared with
controls, as determined on immunoblots using anti-p85 antibodies (Fig.
5A). This association was again inhibited by
WM. The PI 3-kinase was active in the PKC immunoprecipitates as
determined by an in vitro PI 3-kinase assay (Fig.
5B).
Fig. 5.
Platelet lysates immunoprecipitated with
anti-PKC antibody also contain PI 3-kinase. A, immunoblot
analysis of anti-PKC immunoprecipitates of platelet lysates revealed
the presence of PI 3-kinase. Treatment of platelets with
platelet-activating factor increased the amount of PKC associated
with PI 3-kinase. Pretreatment of platelets for 10 min with 100 nM WM decreases this association. The identity of the band
below the PI 3-kinase p85 band is not known but may represent a
degradation product. B, PI 3-kinase activity is present in
the immunoprecipitates, and this activity is reduced by pretreatment of
platelets with WM. Ippt, immunoprecipitate.
pseudo-substrate site. There were no increases in kinase
activity in response to calcium, and the activity was at least
partially lipid-dependent. These features are consistent
with the characteristics of PKC and PKC activities. We are
investigating this further to determine the role PI 3-kinase may play
in regulation of the PKC activity, particularly that of PKC.
and PKC, also associate with PI
3-kinase, and in the case of PKC, the association is increased upon
stimulation of hemopoietic cells with GM-CSF or upon activation of
platelets. In our system, PKC appears to be constitutively
tyrosine-phosphorylated to a small extent, as seen on immunoblots.
However, we cannot discern differences in levels of tyrosine
phosphorylation with cytokine stimulation. Several PKC isoforms contain
the consensus sequence recognized by PI 3-kinase SH2 domains in the p85
subunit including PKC, which has YNYM (36), and PKC, which has
YEMM (37). However, the lack of increased tyrosine phosphorylation of
PKC following cytokine stimulation, along with the lack of PKC/PI
3-kinase association in a system in which PKC is heavily
tyrosine-phosphorylated suggests that we are observing an association
that is unlike previously demonstrated interactions with PI 3-kinase.
However, we do not know whether we are observing a direct interaction
or one that also involves other proteins.
association.
Coupled with the finding that PAF activation of platelets is able to
increase the association, the results suggest that the functional role
of this interaction may be unrelated to the mitogenic effect of
cytokines. There have been conflicting reports regarding the role of
PKC in mitogenesis (38, 39). Furthermore, as suggested by Myers
et al. (40), the association of PI 3-kinase with various
proteins may be unrelated to a role for that enzyme in mitogenesis. One
might also speculate that the PI 3-kinase/PKC association could be
related to the effects of GM-CSF and perhaps IL-5 on inducing
differentiation, effects that are distinct from those of IL-3. Further
studies will be required to delineate the exact function of this novel
association as well as other systems in which this type of regulation
may be occurring.
Recipient of a Heart and Stroke Foundation of Canada traineeship.
§
Recipient of a Medical Research Council of Canada/British Columbia
Lung Association Fellowship. To whom correspondence should be
addressed: Dept. of Medicine, Jack Bell Research Centre, University of
British Columbia, 2660 Oak St., Vancouver, B.C. V6H 3Z6, Canada. Tel.:
604-875-4707; Fax: 604-875-4497; E-mail, vince{at}brc.ubc.ca.
1
The abbreviations used are: PI,
phosphatidylinositol; IL, interleukin; GM-CSF, granulocyte-macrophage
colony-stimulating factor; PAF, platelet-activating factor; PKC,
protein kinase C; WM, wortmannin.
2
S. L. Ettinger and V. Duronio, unpublished
observations.
3
M. P. Scheid and V. Duronio, submitted for
publication.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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 S. M Ward, M.-F. Brennan, V. M Jackson, and K. M Sanders Role of PI3-kinase in the development of interstitial cells and pacemaking in murine gastrointestinal smooth muscle J. Physiol., May 1, 1999; 516(3): 835 - 846. [Abstract] [Full Text] [PDF]
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 Z.-W. Hu, X.-Y. Shi, R. Z. Lin, and B. B. Hoffman Contrasting Signaling Pathways of {alpha}1A- and {alpha}1B-Adrenergic Receptor Subtype Activation of Phosphatidylinositol 3-Kinase and Ras in Transfected NIH3T3 Cells Mol. Endocrinol., January 1, 1999; 13(1): 3 - 14. [Abstract] [Full Text]
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J. Canicio, E. Gallardo, I. Illa, X. Testar, M. Palacin, A. Zorzano, and P. Kaliman p70 S6 Kinase Activation Is Not Required for Insulin-Like Growth Factor-Induced Differentiation of Rat, Mouse, or Human Skeletal Muscle Cells Endocrinology, December 1, 1998; 139(12): 5042 - 5049. [Abstract] [Full Text] [PDF]
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A. Graness, A. Adomeit, R. Heinze, R. Wetzker, and C. Liebmann A Novel Mitogenic Signaling Pathway of Bradykinin in the Human Colon Carcinoma Cell Line SW-480 Involves Sequential Activation of a Gq/11 Protein, Phosphatidylinositol 3-Kinase beta , and Protein Kinase Cepsilon J. Biol. Chem., November 27, 1998; 273(48): 32016 - 32022. [Abstract] [Full Text] [PDF]
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 W. Li, Y.-X. Jiang, J. Zhang, L. Soon, L. Flechner, V. Kapoor, J. H. Pierce, and L.-H. Wang Protein Kinase C-delta Is an Important Signaling Molecule in Insulin-Like Growth Factor I Receptor-Mediated Cell Transformation Mol. Cell. Biol., October 1, 1998; 18(10): 5888 - 5898. [Abstract] [Full Text]
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 J. F. Kuemmerle and T. L. Bushman IGF-I stimulates intestinal muscle cell growth by activating distinct PI 3-kinase and MAP kinase pathways Am J Physiol Gastrointest Liver Physiol, July 1, 1998; 275(1): G151 - G158. [Abstract] [Full Text] [PDF]
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K. E. Davis, D. J. Straff, E. A. Weinstein, P. G. Bannerman, D. M. Correale, J. D. Rothstein, and M. B. Robinson Multiple Signaling Pathways Regulate Cell Surface Expression and Activity of the Excitatory Amino Acid Carrier 1 Subtype of Glu Transporter in C6 Glioma J. Neurosci., April 1, 1998; 18(7): 2475 - 2485. [Abstract] [Full Text] [PDF]
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N. M. Conus, B. A. Hemmings, and R. B. Pearson Differential Regulation by Calcium Reveals Distinct Signaling Requirements for the Activation of Akt and p70S6k J. Biol. Chem., February 20, 1998; 273(8): 4776 - 4782. [Abstract] [Full Text] [PDF]
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Y. Hong, D. Dumenil, B. van der Loo, F. Goncalves, W. Vainchenker, and J. D. Erusalimsky Protein Kinase C Mediates the Mitogenic Action of Thrombopoietin in c-Mpl-Expressing UT-7 Cells Blood, February 1, 1998; 91(3): 813 - 822. [Abstract] [Full Text] [PDF]
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P. Kaliman, J. Canicio, P. R. Shepherd, C. A. Beeton, X. Testar, M. Palacín, and A. Zorzano Insulin-like Growth Factors Require Phosphatidylinositol 3-Kinase to Signal Myogenesis: Dominant Negative p85 Expression Blocks Differentiation of L6E9 Muscle Cells Mol. Endocrinol., January 1, 1998; 12(1): 66 - 77. [Abstract] [Full Text]
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K. Inukai, M. Funaki, T. Ogihara, H. Katagiri, A. Kanda, M. Anai, Y. Fukushima, T. Hosaka, M. Suzuki, B.-C. Shin, et al. p85alpha Gene Generates Three Isoforms of Regulatory Subunit for Phosphatidylinositol 3-Kinase (PI 3-Kinase), p50alpha , p55alpha , and p85alpha , with Different PI 3-Kinase Activity Elevating Responses to Insulin J. Biol. Chem., March 21, 1997; 272(12): 7873 - 7882. [Abstract] [Full Text] [PDF]
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C. Bahr, A. Rohwer, L. Stempka, G. Rincke, F. Marks, and M. Gschwendt DIK, a Novel Protein Kinase That Interacts with Protein Kinase Cdelta . CLONING, CHARACTERIZATION, AND GENE ANALYSIS J. Biol. Chem., November 10, 2000; 275(46): 36350 - 36357. [Abstract] [Full Text] [PDF]
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