(Received for publication, April 19, 1995; and in revised form, August 9, 1995)
From the
AG-18, an inhibitor of protein-tyrosine kinases, was employed to
study the role of tyrosine-phosphorylated proteins in insulin- and
phorbol ester-induced signaling cascades. When incubated with Chinese
hamster ovary cells overexpressing the insulin receptor, AG-18
reversibly inhibited insulin-induced tyrosine phosphorylation of
insulin receptor substrate-1, with minimal effects either on receptor
autophosphorylation or on phosphorylation of Shc64. Under these
conditions, AG-18 inhibited insulin-stimulated phosphorylation of the
ribosomal protein S6, while no inhibition of insulin-induced activation
of mitogen-activated protein kinase (MAPK) kinase or MAPK was detected.
In contrast, 12-O-tetradecanoylphorbol-13-acetate
(TPA)-induced activation of MAPK kinase and MAPK and phosphorylation of
S6 were inhibited by AG-18. This correlated with inhibition of
TPA-stimulated tyrosine phosphorylation of several proteins, the most
prominent ones being pp114 and pp120. We conclude that
Tyr-phosphorylated insulin receptor substrate-1 is the main upstream
regulator of insulin-induced S6 phosphorylation by
p70, whereas MAPK signaling seems to be
activated in these cells primarily through the adaptor molecule Shc. In
contrast, TPA-induced S6 phosphorylation is mediated by the
MAPK/p90
cascade. A key element of this
TPA-stimulated signaling pathway is an AG-18-sensitive protein-tyrosine
kinase.
Insulin binding to its receptor leads to complex changes in
Ser/Thr phosphorylation of multiple intracellular proteins(1) .
Enhanced Ser/Thr phosphorylation of the ribosomal protein S6 is one of
the early biological responses to insulin and involves two major
insulin-stimulated S6 kinases, p90(2) and p70
(3) .
Activation of p90
involves formation of a
complex between Tyr-phosphorylated forms of two major substrates of the
insulin receptor kinase, insulin receptor substrate-1 (IRS-1) (
)(4) and Shc(5) , with the SH2 domain of
Grb2. The insulin signal is further propagated via a sequential
activation of m-Sos/Ras (6, 7, 8, 9) and the MAPK
(extracellular signal-regulated kinase) cascade, including
Raf-1(10) , MAPKK (MEK) (11) , and MAPK (extracellular
signal-regulated kinase) (12) (see Refs. 13 and 14 for review).
MAPK then phosphorylates and activates several regulatory
proteins(13) , including the serine/threonine kinase
p90
(15) , to finally regulate several
cellular processes as proliferation, differentiation, and
morphology(13, 14) .
p70 is
not activated by MAPK and appears to lie on a separate signaling
pathway(16) . One of its upstream activators is IRS-1, whose
phosphorylation by insulin receptor kinase creates a binding site for
the SH2 domains of the p85 regulatory subunit of phosphatidylinositol
3-kinase (PI3K)(17, 18) . The association between p85
and IRS-1 results in activation of PI3K (17, 18) via a
mechanism independent of the direct activation of PI3K by
Ras(19) . Activation of PI3K then stimulates p70
by an as yet unknown mechanism(20, 21) .
A large variety of extracellular signals, aside from insulin, lead
to ribosomal S6 phosphorylation. One example is the tumor-promoting
phorbol ester (TPA) that activates the Ca- and
phospholipid-dependent protein kinase C(22) . Although protein
kinase C has been implicated as playing an important role in
insulin-induced activation of the MAPK cascade(23) , other
studies suggest that insulin (24) and insulin-like growth
factor I (25) activate the MAPK cascade independent of protein
kinase C.
To study the relative contribution of MAPK/p90 and PI3K/p70
to insulin- and
TPA-induced stimulation of S6 phosphorylation, we made use of
tyrphostins, synthetic competitive inhibitors of several tyrosine
kinases (see (26) for review) that inhibit insulin receptor
kinase activity in vitro(27) and block
insulin-induced lipogenesis and anti-lipolysis in fat
cells(28) . Employing Chinese hamster ovary (CHO) cells that
overexpress the wild-type insulin receptor gene (CHO.T)(29) ,
we found that AG-18 effectively inhibits insulin-induced IRS-1
phosphorylation as well as S6 kinase activity. On the other hand,
insulin induction of the MAPK cascade is not affected by AG-18. The
phorbol ester TPA also stimulates S6 kinase activity that is inhibited
by AG-18, but unlike the insulin stimulation, this inhibition is
correlated with the inhibition of the MAPK cascade. These results
implicate a bifurcation in insulin signaling where IRS-1 mediates, to a
large extent, S6 phosphorylation via p70
, while
Shc may mediate a signaling pathway leading to MAPK/p90
activation. In contrast, the TPA effect on S6
phosphorylation seems to be transmitted via the
MAPK/p90
pathway. Most important, a
protein-tyrosine kinase, whose activity is inhibited by AG-18, is one
of the elements linking protein kinase C to the MAPK cascade.
Figure 1:
Inhibition of
insulin receptor kinase-catalyzed phosphorylation of poly(Glu,Tyr)
(4:1) by AG-18. Phosphorylation was carried out as described under
``Experimental Procedures'' with 0.2 (), 0.5 (
),
and 1.0 (
) mg/ml poly(Glu,Tyr) (4:1) and in the presence of the
indicated concentrations of AG-18. The results are presented as Dixon
plots. Each point is an average of duplicate measurements that did not
vary by >10%.
As shown in Fig. 2(A and B) and
consistent with previous studies(38, 39) , incubation
of CHO.T cells with insulin induced tyrosine phosphorylation of two
major proteins: the 95-kDa -subunit of the insulin receptor
(insulin receptor kinase) and one of its major cellular targets,
IRS-1(4, 18, 38, 40, 41) .
Additional proteins that underwent enhanced Tyr phosphorylation in
response to insulin were pp60/pp62 (42, 43, 44) and two (out of the three)
isoforms of Shc (Shc64 and Shc54), whose identity was verified by
immunoprecipitation from cell extracts with Shc antibodies (data not
shown).
Figure 2: Inhibition of protein tyrosine phosphorylation in CHO.T cells by AG-18. Confluent CHO.T cells were incubated for 16 h in serum-free F-12 medium with the indicated concentrations of AG-18. At the end of incubation, buffer or 0.1 µM insulin was added for a 10-min incubation period. Supernatants, prepared from the cell extracts, were resolved by 10% SDS-PAGE and immunoblotted with anti-Tyr(P) antibodies. The upper part of B, where the insulin receptor (IR) and IRS-1 are overexposed, is reproduced at a lower exposure in A.
Incubation of the cells for 16 h with increasing
concentrations of AG-18 resulted in a dose-dependent inhibition of
insulin-stimulated phosphorylation of several proteins (Fig. 2, A and B). This inhibition could not be attributed to
an inhibitory effect of AG-18 on insulin binding since incubation of
CHO.T cells with this drug had no effect on the number of insulin
receptors expressed on the cell surface nor did it affect the affinity
of insulin for its receptors in these cells (data not shown). AG-18
inhibited insulin-induced Tyr phosphorylation of IRS-1, with a
half-maximal effect at 50 µM. Inhibition of pp60/pp62
phosphorylation was also readily detected (Fig. 2, A and B). In contrast, autophosphorylation of pp95, the
-subunit of the insulin receptor, as well as Tyr phosphorylation
of Shc64 were largely unaffected, whereas phosphorylation of Shc54 was
only partially inhibited. Failure of AG-18 to inhibit Shc
phosphorylation was also reflected by the inability of the drug to
inhibit insulin-induced complex formation of Shc and Grb2. This was
demonstrated when insulin-treated cells were preincubated for 16 h
either in the presence or absence of 150 µM AG-18. Under
these conditions, similar amounts of Grb2 (2.3 versus 1.9% of
the total) were found in Shc immunoprecipitates.
Interestingly, AG-18 was a more potent inhibitor when applied to intact cells (Fig. 2) compared with its inhibitory effects on substrate phosphorylation of the insulin receptor kinase in a cell-free system (Fig. 1). These differences could be accounted for by AG-18 accumulation within the cell, making its effective intracellular concentration higher than that applied extracellularly. Alternatively, the native conformation of the insulin receptor kinase maintained in vivo could be more susceptible to the inhibitory effects of the drug.
Figure 3:
Effect of AG-18 on insulin-induced
phosphorylation of ribosomal protein S6. Left, confluent CHO.T
cells were incubated for 16 h in serum-free medium with the indicated
concentrations of AG-18. At the end of incubation, buffer () or
0.1 µM insulin was added for 10 (
), 20 (
), or
60 (
) min. Cells were then washed three times with ice-cold PBS
and frozen in liquid nitrogen. Cell extracts were prepared, and S6
kinase activity was determined. The intensity of the band corresponding
to S6 was quantitated by densitometry and is presented in arbitrary
units. Right, an autoradiogram of the effects of AG-18 on S6
phosphorylation following a 60-min incubation with insulin is
presented.
Figure 4: Effect of AG-18 on TPA-induced phosphorylation of S6. Confluent CHO.T cells were incubated for 16 h in serum-free medium with the indicated concentrations of AG-18. At the end of incubation, buffer or 0.4 µg/ml TPA was added. Following a 30-min incubation, cells were washed three times with ice-cold PBS and frozen in liquid nitrogen. S6 phosphorylation was determined as described under ``Experimental Procedures.''
Figure 5: Effect of AG-18 on insulin- and TPA-induced activation of MAPK. Confluent CHO.T cells were incubated for 16 h in serum-free medium with the indicated concentrations of AG-18. At the end of incubation, 0.1 µM insulin (A) or 0.4 µg/ml TPA (B) was added for a 10- or 30-min incubation period. Cells were then washed three times with ice-cold PBS; cell extracts were prepared; and MAPK activity was assayed as described under ``Experimental Procedures.''
Figure 6: Effect of AG-18 on insulin- and TPA-induced activation of MAPKK. Confluent CHO.T cells were incubated for 16 h in serum-free medium with the indicated concentrations of AG-18. At the end of incubation, 0.1 µM insulin (A) or 0.4 µg/ml TPA (B) was added for a 10- or 30-min incubation period. Cells were then washed three times with ice-cold PBS; cell extracts were prepared; and MAPKK activity was assayed as described under ``Experimental Procedures.''
Figure 7:
Effect of AG-18 on TPA-induced protein
tyrosine phosphorylation. Upper, confluent CHO.T cells were
incubated for 16 h in serum-free medium with the indicated
concentrations of AG-18. At the end of incubation, 0.4 µg/ml TPA
was added for a 30-min incubation period. Cells were then washed three
times with ice-cold PBS, and cell extracts were prepared, resolved by
10% SDS-PAGE, and immunoblotted with anti-Tyr(P) antibodies. Lower, the intensity of the bands corresponding to pp120
() and pp114 (
) was quantitated by densitometry, and the
percent inhibition induced by AG-18 was calculated. The effects of
AG-18 on TPA-induced S6 phosphorylation (
) and MAPK activity
(
) are also presented for comparison.
A protein-tyrosine kinase inhibitor from the tyrphostin
family (AG-18) was used to distinguish between the pathways leading to
the activation of p70 and the activation of the
MAPK/p90
cascade. AG-18 effectively inhibits
insulin-induced activation of S6 kinase while having no inhibitory
effect on insulin-induced activation of the MAPK cascade. These results
indicate that activation of MAPK per se is not sufficient for
stimulation of S6 phosphorylation and suggest that insulin-induced
activation of S6 kinase(s) may occur through an alternative pathway. In
this respect, our results complement studies demonstrating that MAPK
activation by the insulin receptor is not required for insulin-induced
metabolic processes such as glucose transport or glycogen synthase in
3T3-L1 adipocytes (49) .
Inhibition of S6 phosphorylation by
AG-18 largely parallels the inhibitory effects of AG-18 on
insulin-induced phosphorylation of IRS-1 and is compatible with the
notion that IRS-1 phosphorylation mediates many insulin
responses(41, 50, 51) , including the
stimulation of S6 phosphorylation. The latter presumably involves
activation of PI3K (17, 18) and subsequent activation
of p70(20, 21, 52) , which
occurs independent of activation of p21
and the MAPK
cascade (53) . Conversely, we have shown that inhibition of
IRS-1 and S6 phosphorylation occurs without inhibition of the MAPK
cascade. Although we cannot rule out the possibility that AG-18 fails
to inhibit phosphorylation of IRS-1 at Tyr
, which is part
of the Grb2-binding site(54) , our findings support the view
that there are alternative pathways for insulin-induced activation of
the MAPK cascade, independent of IRS-1(55, 56) . This
conclusion is supported by the observation that association of Grb2/Sos
with IRS-1 plays little if any role in MAPK activation in L6
myoblasts(57) . A likely candidate to stimulate the MAPK
cascade is Shc, which serves as a downstream effector of the insulin
receptor (5) and acts to activate the Ras/MAPK
pathway(8, 55, 58, 59, 60) .
Indeed, AG-18 failed to inhibit insulin-induced Tyr phosphorylation of
Shc64, and phosphorylation of Shc54 was only partially inhibited.
Similarly, AG-18 failed to inhibit insulin-induced complex formation
between Shc and Grb2. Hence, although our results clearly support the
involvement of Shc in MAPK stimulation, different Shc isoforms might
play different roles in insulin signal transduction, and further
studies are required to address this possibility.
Activation of
p90 as a result of Shc phosphorylation, together with
activation of the MAPK cascade, could account for the residual S6
phosphorylation observed in the presence of 150 µM AG-18
in insulin-treated cells. The fact that this residual S6
phosphorylation is rather low (
20%) suggests, however, that the
predominant mode of insulin-activated S6 phosphorylation (at least in
CHO cells) occurs through the IRS-1/PI3K/p70
signaling
pathway. Taken together, our findings are consistent with a model (Fig. S1) in which IRS-1 mediates insulin-induced activation of
p70
, whereas the signals leading to the activation of the
MAPK/p90
cascade are transmitted via the adaptor molecule
Shc.
Figure S1: Scheme 1Tentative model illustrating the signaling pathways induced by insulin and TPA that stimulate S6 phosphorylation. Many proteins known to take part in other aspects of insulin and TPA-mediated responses were eliminated for simplicity. The expected sites of action of AG-18 are indicated. PM, plasma membrane; IR, insulin receptor; PKC, protein kinase C; PTK, protein-tyrosine kinase, Erk, extracellular signal-regulated kinase.
Tyrphostins were previously shown to inhibit insulin-stimulated lipogenesis in fat cells, while they failed to inhibit the anti-lipolytic effect of the hormone(28) . These differences could be accounted for by the different potency of tyrphostins to inhibit phosphorylation of insulin receptor kinase substrates that could mediate these processes (IRS-1 and Shc). Accordingly, we suggest that IRS-1 is more prone to inhibition by these competitive inhibitors because it is less abundant and/or has a lower affinity toward insulin receptor kinase when compared with other insulin receptor substrates (e.g. Shc) that mediate activation of the MAPK cascade. This assumption is supported by recent findings(56) , where cells expressing insulin receptor mutants (of Tyr autophosphorylation sites within the kinase region) maintained insulin-induced phosphorylation of Shc, whereas phosphorylation of IRS-1 was largely reduced. Alternatively, some of the insulin-induced Tyr-phosphorylated proteins (e.g. Shc64) could serve as substrates for intermediary protein-tyrosine kinases rather than as substrates for the insulin receptor kinase itself. These intermediary protein-tyrosine kinases could undergo activation upon insulin receptor autophosphorylation, which is not inhibited by AG-18 (see above). Activation could involve, for example, binding of SH2 domains of these putative intermediary protein-tyrosine kinases to unique Tyr(P) residues within the cytoplasmic portion of the insulin receptor.
A different picture
emerges when the effects of TPA on S6 phosphorylation are studied. A
good concordance exists between the inhibitory effects of AG-18 on
TPA-induced MAPKK and MAPK activity and S6 phosphorylation, which
suggests that protein kinase C induces S6 phosphorylation
preferentially through the MAPK signaling pathway. Moreover, the
difference in the effects of AG-18 on insulin- versus TPA-activated MAPK suggests that, in these cells, insulin-induced
activation of the MAPK cascade occurs via a protein kinase
C-independent pathway. Hence, p70 appears to be mainly
responsible for insulin-induced S6 phosphorylation(20) , while
p90
could mediate the effects of TPA(61) .
Since AG-18 is a very poor inhibitor of Ser/Thr protein kinases, including protein kinase C(62) , the inhibitory effects of AG-18 suggest that one or more AG-18-sensitive proteintyrosine kinases mediate the effects of protein kinase C on the MAPK pathway and S6 phosphorylation. This conclusion is supported by the facts that (i) TPA induces protein tyrosine phosphorylation in CHO.T cells, and (ii) AG-18 inhibits both TPA-stimulated tyrosine phosphorylation and TPA-stimulated MAPK activity with a similar dose-response curve. Although we cannot rule out the possibility that insulin and protein kinase C activate different isoforms of the dual specificity MAPKK, our results are most consistent with the hypothesis that the TPA-activated protein-tyrosine kinase presumably differs from the dual specificity kinase, MAPKK. This conclusion is primarily based on the fact that insulin-induced activation of MAPKK is insensitive to the presence of AG-18.
Several studies implicate protein kinase C, the direct
effector of TPA, as a mediator of protein tyrosine phosphorylation
events. In rat basophilic leukemia cells, Tyr phosphorylation of a
110-kDa protein occurs secondary to calcium influx and protein kinase C
activation (63) . Activation of protein kinase C and/or the
induction of calcium influx was implicated in immunoglobulin E
receptor-induced Tyr phosphorylation of focal adhesion-associated
tyrosine kinase (pp125) in fibronectin-adherent rat
basophilic leukemia cells(64, 65) . Similarly, protein
kinase C was shown to mediate carbachol-stimulated tyrosine
phosphorylation in human SH-SY5Y neuroblastoma cells(66) . Our
results suggest that a protein kinase C-stimulated protein-tyrosine
kinase should be present upstream of MAPKK in the protein kinase C
signaling pathway, leading to the activation of p90
.
The nature of the TPA-activated protein-tyrosine kinase is presently unknown, but among its potential substrates, we find pp114 and pp120, whose inhibited phosphorylation correlates with inhibition of MAPK activity. Hence, we can formulate a tentative signaling cascade (Fig. S1) in which a TPA-activated protein-tyrosine kinase stimulates the common Grb2/Sos and the Ras signaling pathway (8, 55, 58, 59, 60) and in such a way leads to activation of MAPK and S6 phosphorylation(13) . Further studies are required, however, to figure out the role of pp114/pp120 and to determine whether the TPA-activated protein-tyrosine kinase indeed utilizes the Grb2/Sos/Ras signaling elements to induce activation of the MAPK cascade.