(Received for publication, June 22, 1995; and in revised form, August 4, 1995)
From the
Insulin stimulation of 3T3-L1 adipocytes results in rapid activation of the insulin receptor tyrosine kinase followed by autophosphorylation of the receptor and phosphorylation of insulin receptor substrate 1 (IRS-1), its major substrate. The insulin receptor resides mostly at the cell surface of 3T3-L1 adipocytes under basal conditions, while about two-thirds of IRS-1 fractionates with intracellular membranes and one-third fractionates with cytosol. To test whether insulin receptor internalization is required for optimal tyrosine phosphorylation of IRS-1, 3T3-L1 adipocytes and CHO-T cells were incubated at 4 °C which inhibits receptor endocytosis but not its tyrosine kinase activity. Under these conditions, tyrosine phosphorylation of IRS-1 in the low density microsome fraction in response to insulin was as intense as that observed at 37 °C, indicating that endocytosis of insulin receptors is not necessary for tyrosine phosphorylation of IRS-1 to occur. Surprisingly, at 37 °C, insulin action on 3T3-L1 adipocytes progressively decreased the amount of IRS-1 protein associated with the low density microsome fraction and increased that in the cytosol. This redistribution of IRS-1 from the low density microsome fraction to the cytosol in response to insulin was accompanied by decreased electrophoretic mobility of IRS-1 on SDS-polyacrylamide gel electrophoresis. Incubation of adipocytes at 4 °C blocked the appearance of tyrosine-phosphorylated IRS-1 in the cytosol. Taken together, these data indicate that insulin receptors phosphorylate IRS-1 at the cell surface, perhaps in coated pits which are included in the low density microsome fraction. The results also suggest a desensitization mechanism in which the tyrosine-phosphorylated membrane-bound IRS-1, associated with signaling molecules such as phosphatidylinositol 3-kinase, is released into the cytoplasm in concert with its serine/threonine phosphorylation.
Intrinsic tyrosine kinase activity of the cell surface insulin
receptor is required to mediate its biological actions suggesting the
importance of its protein substrates in signal transduction. One such
cellular substrate is the insulin receptor substrate 1 (IRS-1) ()(for recent reviews, see (1, 2, 3) ). The amino acid sequences deduced
from cloned rat, mouse, and human IRS-1 cDNAs (4, 5, 6) indicate the presence of multiple
potential sites of phosphorylation by both tyrosine and
serine/threonine protein kinases. In the intact cell, insulin
stimulation causes rapid tyrosine phosphorylation of IRS-1 at multiple
sites(7) . These phosphotyrosine-containing sequences within
IRS-1 serve as binding sites for a number of proteins containing Src
homology 2 (SH2) domains(1, 2, 3) . Proteins
recruited to IRS-1 in this manner include the p85 regulatory subunit of
phosphatidylinositol 3-kinase (PI 3-kinase)(8, 9) ,
the tyrosine phosphatase Syp(10) , and the adaptor proteins
Grb2 (11, 12) and Nck(13) . The enzymatic
activities of both PI 3-kinase (8) and Syp (14) are
markedly increased upon their binding to IRS-1. Thus, IRS-1 is thought
to serve as a docking protein which facilitates the actions of various
signaling proteins in initiating their biological responses.
Recent evidence directly implicates IRS-1 and PI 3-kinase in one of the major metabolic actions of insulin, glucose transport. The binding of p85 to IRS-1 is required for activation of the p110 catalytic subunit of PI 3-kinase which is responsible for catalyzing the phosphorylation of phosphoinositides at the D-3 position of the inositol ring(8, 15) . PI 3-kinase also possesses a serine kinase activity (16, 17) which phosphorylates both p85 and IRS-1(18) . Insulin causes stimulation of PI 3-kinase activity in isolated rat adipocytes(19) , 3T3-L1 adipocytes (20) , and cells overexpressing the insulin receptor(21, 22, 23) . Specific inhibitors of both the lipid and serine kinase activities of PI 3-kinase abolish insulin-stimulated glucose transport and the translocation of GLUT4 in insulin-responsive cells(18, 24, 25) . Furthermore, the introduction of IRS-1 antisense constructs into GLUT4-transfected fat cells decreases the sensitivity to insulin of GLUT4 glucose transporter movement to the cell surface, and this effect can be overcome by co-expressing IRS-1 in these cells(26) .
Studies of the insulin receptor indicate that critical structural
element(s) exist in its cytoplasmic domain to propagate signaling to
components in the pathway leading to glucose transport. Specifically,
point mutations of Tyr-960 in the NPXY motif located within
the juxtamembrane sequence of the receptor which do not alter receptor
kinase activity or the rate of I-insulin endocytosis
abolish the phosphorylation of IRS-1, the activation of PI 3-kinase,
and insulin-stimulated glucose transport in cells expressing these
mutant
receptors(27, 28, 29, 30, 31, 32) .
Interestingly, a direct interaction between the amino terminus of IRS-1
and the region of the insulin receptor containing phosphorylated
Tyr-960 has been shown utilizing the yeast two-hybrid
system(33) . However, it is unclear how IRS-1 interacts with
the insulin receptor, an integral plasma membrane protein, in intact
cells.
The distribution of IRS-1 in rat fat cells was recently shown
to be mostly cytosolic, but approximately 20% of the IRS-1 was
associated with intracellular membranes(34) . The IRS-1 in both
these subcellular fractions was tyrosine-phosphorylated following
insulin stimulation. The authors of this work proposed that
internalized, activated insulin receptors are responsible for the
phosphorylation of the IRS-1 located in the intracellular membranes. A
previous study indicated that basal PI 3-kinase activity in rat fat
cells appears to be cytosolic while essentially all of the
insulin-stimulated PI 3-kinase activity, presumably in IRS-1PI
3-kinase complexes, exists in microsomal membranes(19) , a
heterogeneous mixture of Golgi membranes and endosomes. It was further
determined that the activated complexes of IRS-1 and PI 3-kinase were
present in vesicles distinct from those containing the
insulin-regulated glucose transporters GLUT4 and those containing
internalized insulin receptors(35) . Hence, these studies
suggest that activated IRS-1
PI 3-kinase complexes are
specifically localized to intracellular membranes, but it remains
unclear as to where these complexes are initially formed and how these
complexes are targeted to this site.
The present studies were designed to determine whether insulin receptors must first internalize into endosomes in order for IRS-1 to become tyrosine-phosphorylated. We took advantage of the fact that, at low temperature, endocytosis of the insulin receptor is inhibited (36, 37) while its tyrosine kinase activity remains functional. Our results demonstrate that IRS-1 in 3T3-L1 adipocytes undergoes insulin-dependent tyrosine phosphorylation at both 4 °C and 37 °C, indicating that receptor internalization is not required for its phosphorylation. We also show that, under basal conditions, the majority of IRS-1 in 3T3-L1 adipocytes is present in the low density microsome fraction, and that following insulin stimulation, tyrosine-phosphorylated IRS-1 is released from these membranes into the cytosol. Furthermore, this release is accompanied by a change in the electrophoretic mobility of IRS-1, suggesting that serine/threonine phosphorylation of IRS-1 is occurring. These data are consistent with the hypothesis that in 3T3-L1 adipocytes, IRS-1 undergoes directed intracellular routing which is regulated by insulin.
CHO-T cells were grown in
15-cm dishes and were serum-starved for 12-18 h in
F12 media supplemented with 0.5% BSA. Cell fractions from CHO-T cells
were then prepared as above except that the first homogenization step
consisted of 10 strokes with a motor-driven Teflon/glass homogenizer in
24 ml of ice-cold Buffer A.
Figure 1:
Insulin causes tyrosine
phosphorylation of IRS-1 present in the low density microsome fraction
of 3T3-L1 adipocytes at both 4 °C and 37 °C. Plasma membrane (PM), low density microsome (LDM), and cytosolic
fractions were prepared from 3T3-L1 adipocytes treated with (+) or
without(-) 1 µM insulin for 15 or 60 min at 4 °C
or 37 °C by the methods described under ``Experimental
Procedures.'' Protein (25 µg) from each fraction was resolved
by SDS-PAGE on 7.5% gels and electrophoretically transferred to
nitrocellulose for 8 h at 150 mA. The filters were blocked and
subsequently incubated with 0.5 µg/ml 4G10 anti-phosphotyrosine
antibody as described under ``Experimental Procedures.''
Bands corresponding to IRS-1 (160 kDa) and the subunit of the
insulin receptor (IR, 95 kDa) are designated with an arrowhead. The immunoblots presented are representative of
five experiments.
Figure 2: Tyrosine phosphorylation of IRS-1 in low density microsome and cytosol of 3T3-L1 adipocytes treated with insulin at 4 °C and 37 °C. The data shown in Fig. 1for IRS-1 were quantitated using a scanning densitometer. The data were normalized by adjusting the numerical value obtained from densitometric scanning to the total protein from each subcellular fraction prepared from an equal number of cells and are depicted as arbitrary units.
As demonstrated in Fig. 1, levels of tyrosine-phosphorylated IRS-1 present in the plasma membrane fractions at both temperatures following insulin stimulation are substantially (approximately 20-fold) lower than levels present in the low density microsome fractions under identical conditions. Thus, the results of the plasma membrane fractions are not displayed graphically in Fig. 2.
Figure 3:
Insulin receptors in 3T3-L1 adipocytes are
tyrosine-phosphorylated at both 4 °C and 37 °C in response to
insulin. Plasma membrane (PM) and low density microsomes (LDM) were prepared from 3T3-L1 adipocytes treated with
(+) or without(-) 1 µM insulin for 15 or 60 min
at 4 °C or 37 °C by the methods described under
``Experimental Procedures.'' Protein (125 µg) from each
fraction was solubilized in HEPES buffer containing 1% Brij and
incubated with 5 µg of anti-insulin receptor IgG.
Samples were incubated with goat anti-mouse Sepharose and then washed
several times. The immune pellets were solubilized in sample buffer
containing reductant, resolved by SDS-PAGE on 7.5% gels, and were then
electrophoretically transferred to nitrocellulose for 8 h at 150 mA.
The filters were blocked and subsequently incubated with 0.5 µg/ml
4G10 anti-phosphotyrosine as described under ``Experimental
Procedures.'' The band corresponding to the
subunit of the
insulin receptor (IR, 95 kDa) is designated with an arrowhead.
Figure 4: Insulin receptor tyrosine phosphorylation in the plasma membranes and low density microsomes of 3T3-L1 adipocytes treated with insulin at 4 °C and 37 °C. The data shown in Fig. 3for insulin receptor were quantitated using a scanning densitometer. The data were normalized by adjusting the numerical value obtained from densitometric scanning to the total protein from each subcellular fraction prepared from an equal number of cells and are depicted as arbitrary units.
Tyrosine phosphorylation of the insulin receptor in the plasma membrane fraction increases in a time-dependent manner at both 4 °C and 37 °C. There are 4-fold more tyrosine-phosphorylated insulin receptors in the plasma membrane fraction at 37 °C relative to 4 °C following a 15-min insulin stimulation suggesting that tyrosine phosphorylation of receptors is slower at 4 °C. Tyrosine phosphorylation of insulin receptors in the plasma membrane fraction increases slightly with increased exposure to insulin at 37 °C, while the increase in tyrosine phosphorylation of insulin receptors in the plasma membrane fraction at 4 °C is greater. This is most likely due to the fact that, at 37 °C, tyrosine-phosphorylated insulin receptors are being internalized in a time-dependent fashion, and hence their numbers would be expected to decrease in the plasma membrane fraction and increase in the low density microsome fraction. This is reflected in the data shown in the bottom panel of Fig. 4. Levels of tyrosine-phosphorylated insulin receptor in the low density microsome fraction from cells stimulated at 37 °C are slightly increased over time. Our data indicate the presence of tyrosine-phosphorylated insulin receptors in the low density microsome fraction at both 37 °C and at 4 °C.
To confirm that insulin receptors are not significantly internalized following insulin stimulation at 4 °C, we performed immunofluorescence microscopy using CHO-T cells. We first tested whether insulin stimulation of CHO-T cells at 4 °C would result in the tyrosine phosphorylation of IRS-1 present in the low density microsome fraction as observed in 3T3-L1 adipocytes. Confluent CHO-T cells were stimulated with insulin at 4 °C or 37 °C for 15 min and 60 min, and subcellular fractions were prepared and immunoblotted with anti-phosphotyrosine antibody. As the results presented in Fig. 5demonstrate, tyrosine-phosphorylated IRS-1 is present in the low density microsome fractions prepared from cells stimulated with insulin at both 4 °C and 37 °C. Additionally, similar to the effect observed in 3T3-L1 adipocytes, a substantial amount of tyrosine-phosphorylated IRS-1 is present in the cytosol at 37 °C following insulin stimulation. Thus, our findings show that CHO-T cells are similar to 3T3-L1 adipocytes in that IRS-1 associated with the low density microsome fraction can be tyrosine-phosphorylated in an insulin-dependent manner at 4 °C.
Figure 5: Insulin causes tyrosine phosphorylation of IRS-1 present in the low density microsome fraction of CHO-T cells at both 4 °C and 37 °C. Low density microsome (LDM) and cytosolic fractions were prepared from CHO-T cells treated with (+) or without(-) 1 µM insulin for 15 or 60 min at 4 °C or 37 °C by the methods described under ``Experimental Procedures.'' Low density microsome protein (25 µg) and cytosolic protein (75 µg) were resolved by SDS-PAGE on 7.5% gels and were electrophoretically transferred to nitrocellulose for 8 h at 150 mA. The filters were blocked and subsequently incubated with 0.5 µg/ml 4G10 anti-phosphotyrosine antibody as described under ``Experimental Procedures.'' Bands corresponding to IRS-1 (160 kDa) are designated with an arrowhead. The immunoblots presented are representative of two experiments.
Figure 6: Internalization of tyrosine-phosphorylated insulin receptors is inhibited at 4 °C in CHO-T cells. CHO-T cells grown on coverslips were treated with (+) or without(-) 1 µM insulin for 60 min at 4 °C or 37 °C by the methods described under ``Experimental Procedures.'' Cells were fixed, permeabilized, and blocked as described under ``Experimental Procedures.'' Cells were incubated with 20 µg/ml 4G10 anti-phosphotyrosine, 1.5 µg/ml anti-insulin receptor IgG1, or 20 µg/ml mouse IgG as negative control, washed, and then incubated with fluorescein isothiocyanate- (ANTI-IR) or rhodamine- (ANTI-P-TYR) conjugated anti-mouse. Cells were washed, postfixed, and mounted. Top panel: A, nonimmune mouse IgG, 4 °C; B, anti-phosphotyrosine, -ins, 4 °C; C, anti-phosphotyrosine, +ins, 4 °C; D, nonimmune mouse IgG, 37 °C; E, anti-phosphotyrosine, -ins, 37 °C; F, anti-phosphotyrosine, +ins, 37 °C. Bottom panel: G, nonimmune mouse IgG, 4 °C; H, anti-insulin receptor, -ins, 4 °C; I, anti-insulin receptor, +ins, 4 °C; J, nonimmune mouse IgG, 37 °C; K, anti-insulin receptor, -ins, 37 °C; L, anti-insulin receptor, +ins, 37 °C.
Figure 7: Insulin causes a temperature-dependent release of IRS-1 protein from the low density microsome fraction of 3T3-L1 adipocytes into the cytosol. Plasma membrane (PM), low density microsome (LDM), and cytosolic fractions were prepared from 3T3-L1 adipocytes treated with (+) or without(-) 1 µM insulin for 15 or 60 min at 4 °C or 37 °C by the methods described under ``Experimental Procedures.'' Protein (25 µg) from each fraction was resolved by SDS-PAGE on 7.5% gels and electrophoretically transferred to nitrocellulose for 8 h at 150 mA. The filters were blocked and subsequently incubated with 2 µg/ml anti-IRS-1 as described under ``Experimental Procedures.'' Bands corresponding to IRS-1 (160 kDa) are designated with arrowheads. The immunoblots presented are representative of three experiments.
The concentration dependence of insulin in inducing the release of IRS-1 protein from the low density microsome fraction into the cytosol is shown in Fig. 9. 3T3-L1 adipocytes were stimulated with insulin at the indicated concentrations at 37 °C, subcellular fractions were prepared and immunoblotted with anti-IRS-1. The IRS-1 bands were scanned, and the data were normalized per cell and presented graphically. The data demonstrate that with increasing insulin concentration, there is an increasing loss of IRS-1 protein from the low density microsome fraction with a corresponding increase in cytosolic IRS-1. It appears that 10 nM insulin results in maximal loss of IRS-1 protein from the low density microsome fraction since stimulation with higher concentrations of insulin did not further increase the effect although increases in cytosolic IRS-1 have still not reached a plateau by 1 µM insulin. The percentages of IRS-1 protein present in the cytosolic and low density microsome fractions under basal conditions and following 1 µM insulin stimulation in this experiment were similar to those observed previously (see Fig. 8).
Figure 9: Effect of insulin concentration on the release of IRS-1 protein from the low density microsome fraction of 3T3-L1 adipocytes into the cytosol. Low density microsome (LDM) and cytosolic fractions were prepared from 3T3-L1 adipocytes treated with insulin at the indicated concentrations for 60 min at 37 °C by the methods described under ``Experimental Procedures.'' Equal amounts of protein from each fraction were resolved by SDS-PAGE on 7.5% gels and were electrophoretically transferred to nitrocellulose for 8 h at 150 mA. The filters were blocked and subsequently incubated with 2 µg/ml anti-IRS-1 as described under ``Experimental Procedures.'' Bands corresponding to IRS-1 were quantitated using a scanning densitometer. The data were normalized by adjusting the numerical value obtained from densitometric scanning to the total protein from each subcellular fraction prepared from an equal number of cells and are depicted as arbitrary units.
Figure 8: Time course of the temperature-dependent release of IRS-1 protein into the cytosol of 3T3-L1 adipocytes. The data shown in Fig. 7for IRS-1 were quantitated using a scanning densitometer. The data were normalized by adjusting the numerical value obtained from densitometric scanning to the total protein from each subcellular fraction prepared from an equal number of cells, and are displayed as the percent IRS-1 present in each subcellular fraction.
Figure 10: PI 3-kinase is associated with tyrosine-phosphorylated IRS-1 in the low density microsome fraction of 3T3-L1 adipocytes at both 4 °C and 37 °C. Low density microsomes (LDM) and cytosols were prepared from 3T3-L1 adipocytes treated with 1 µM insulin for 15 min at 4 °C or 37 °C. 125 µg of total protein from each fraction were immunoprecipitated with anti-IRS-1 and then subjected to a PI 3-kinase assay. Spots corresponding to PI 3-phosphate on the thin layer plate were quantitated using a Betascope. Data were normalized by adjusting the counts/min to the total protein from each subcellular fraction prepared from an equal number of cells.
Our present approach to determine whether insulin receptor internalization is required for tyrosine phosphorylation of IRS-1 in intact 3T3-L1 adipocytes and CHO-T cells depends upon the premise that insulin receptors cannot internalize at low temperature while their tyrosine kinase activity is functional. Immunofluorescence microscopy presented here (Fig. 6) directly documents that activated insulin receptors remain largely at the cell surface at 4 °C, consistent with previously published reports that insulin receptor internalization is inhibited at low temperature(36, 37) . Thus, our results demonstrating that IRS-1 undergoes similar levels of insulin-dependent tyrosine phosphorylation at 4 °C versus 37 °C ( Fig. 1and Fig. 2for 3T3-L1 adipocytes and Fig. 5for CHO-T cells) show that insulin receptor internalization is not necessary for IRS-1 tyrosine phosphorylation in these cells. A previous study suggested that internalization of activated insulin receptors is required for the tyrosine phosphorylation of IRS-1 in microsomal membranes(34) . This conclusion was based on the observations that there are 5-6-fold more tyrosine-phosphorylated insulin receptors in low density microsomes relative to plasma membranes shortly after exposure to insulin, and that both the kinase activity associated with these receptors and the phosphorylation state of IRS-1 in the low density microsomes paralleled the phosphotyrosine content of the receptors. These correlations were quite striking. However, we have clearly demonstrated in two different cell types that, under conditions where insulin receptor internalization is inhibited (4 °C), insulin elicits levels of tyrosine-phosphorylated IRS-1 that are identical with those observed under physiological conditions (37 °C) where receptor internalization does occur.
If insulin receptor
internalization is not required for tyrosine phosphorylation of IRS-1
in the low density microsome fraction, which is thought to consist of
intracellular membranes, what is the mechanism for such
phosphorylation? Results presented in Fig. 3and Fig. 4demonstrate that there are tyrosine-phosphorylated insulin
receptors present in the low density microsome fraction following
insulin stimulation of adipocytes at 4 °C even though
internalization of receptors is inhibited. Results using
autoradiographic electron microscopy have demonstrated that at low
temperature, insulin-bound receptor complexes redistribute at the cell
surface and cluster within coated pits (43) although this
occurs more slowly than at 37 °C. Clathrin-coated vesicles are
present in the low density microsome fraction of adipocytes. ()Therefore, a likely explanation for the presence of
tyrosine-phosphorylated receptors in the low density microsome fraction
at low temperature is that clathrin-coated vesicles are included in
this fraction. These considerations lead us to propose that a pool of
the membrane-bound IRS-1 protein is associated with coated pits, and
that tyrosine phosphorylation of IRS-1 by the insulin receptor can
occur in these structures. Consistent with this concept, we found that
significant amounts of IRS-1 protein in the low density microsome
fraction of 3T3-L1 adipocytes are resistant to Triton X-100 extraction,
as is clathrin (data not shown). As depicted in the model of Fig. 11, IRS-1 phosphorylation in coated pits may then be
followed by its endocytosis and its delivery to endosomal sites where
PI 3-kinase or other associated proteins may act. This hypothesis is
not inconsistent with the possibility that IRS-1 may also be
phosphorylated by internalized insulin receptors in intracellular
membrane compartments at 37 °C as suggested by Kublaoui et
al.(34) .
Figure 11: Model of insulin-regulated IRS-1 recycling in the cell. Membrane-bound IRS-1 is hypothesized to be tyrosine-phosphorylated by the insulin receptor in coated pits at the cell surface. Tyrosine-phosphorylated IRS-1 associates with PI 3-kinase and is internalized on vesicles where it delivers PI 3-kinase, and perhaps other signaling components, to specific sites in the endosomes. Serine/threonine phosphorylation of IRS-1, caused by PI 3-kinase or another protein kinase(s), is hypothesized to cause the release of IRS-1 from its binding element in the membrane into the cytosol, where it is dephosphorylated. Dephosphorylated IRS-1 in the cytosol then associates with its binding element in coated pits where it can be tyrosine-phosphorylated by the insulin receptor.
The mechanism by which IRS-1 might interact
with membranes is unknown. Activated insulin receptors cannot account
for the putative IRS-1 binding component in membranes because IRS-1 is
associated with low density microsome in the absence of insulin ( Fig. 7and Fig. 8). IRS-1 contains a structural domain,
the pleckstrin homology (PH) domain, that is thought to represent an
interaction motif(44) . The PH domain in -adrenergic
receptor kinase has been shown to bind heterotrimeric G protein
subunits at the plasma membrane, thus recruiting
-adrenergic receptor kinase to the cell
surface(45, 46) . Similarly, the region containing the
PH domain in oxysterol-binding protein is responsible for its binding
to Golgi membranes(44) . The PH domains of several proteins
have been shown to interact selectively with phosphatidylinositol
derivatives(47) . An important hypothesis for future
investigation is whether the PH domain in IRS-1 is required for its
association with membranes in the low density microsome fraction.
The present results also demonstrate a striking regulatory action of insulin on IRS-1 binding to low density membranes. In control 3T3-L1 adipocytes, approximately two-thirds of the IRS-1 protein is associated with intracellular membranes, while approximately one-third is cytosolic ( Fig. 7and Fig. 8). Insulin stimulation at 37 °C causes a substantial decrease in the levels of IRS-1 protein in the low density microsome fraction with a concomitant increase in IRS-1 protein in the cytosol. The insulin concentration required to elicit this release of IRS-1 protein into the cytosol is within the range of concentrations required to observe biological effects such as glucose transport regulation in these cells (Fig. 9). In addition, this effect is observed in 3T3-L1 adipocytes as early as 3 min following stimulation with 100 nM insulin (data not shown) which correlates temporally with insulin-induced translocation of GLUT4 glucose transporters to the plasma membrane(34) . Taken together, these results strongly suggest that this novel effect of insulin that causes dissociation of IRS-1 from low density membranes is of physiological importance. A likely role for this phenomenon is desensitization of the insulin effect. Thus, as depicted in the model of Fig. 11, subsequent to delivery of tyrosine-phosphorylated, PI 3-kinase-associated IRS-1 to specific sites within intracellular membranes, IRS-1 is released for recycling back to its location of tyrosine phosphorylation. Such a mechanism might operate in concert with dephosphorylation of phosphotyrosine sites on IRS-1 by tyrosine phosphatases. Interestingly, we find PI 3-kinase-associated IRS-1 in the cytosol following insulin treatment (Fig. 10), indicating that IRS-1 release from membranes occurs prior to its complete dephosphorylation of tyrosine phosphates.
Some apparent diversity of
results has been reported with regard to the amount of
tyrosine-phosphorylated IRS-1 that is present in the cytosol of
adipocytes. A previous study (35) suggested that in primary rat
adipocytes activation of PI 3-kinase correlated closely with the extent
of tyrosine-phosphorylated IRS-1 present in low density microsomes, but
no PI 3-kinase activity bound to IRS-1 in cytosol was observed.
However, another study showed clearly detectable
tyrosine-phosphorylated IRS-1 in the cytosolic fraction of rat
adipocytes after incubation with insulin(34) . Our findings in
3T3-L1 adipocytes are consistent with this latter report. Insulin
stimulation of these cultured adipocytes at physiological temperature
caused a time-dependent diminution of tyrosine-phosphorylated IRS-1 in
the low density microsome fraction, and the simultaneous appearance of
tyrosine-phosphorylated IRS-1 in the cytosol ( Fig. 1and Fig. 2). Insulin-stimulated PI 3-kinase activity associated with
the low density microsome fractions correlates well with the levels of
tyrosine-phosphorylated IRS-1 present in this fraction (Fig. 10). We also observed PI 3-kinase activity associated with
tyrosine-phosphorylated IRS-1 in the cytosol of 3T3-L1 adipocytes
following insulin stimulation. Although we have no direct data
indicating whether the cytosolic IRS-1PI 3-kinase complexes in
the cytosol are required for biological actions of insulin, our data
strongly suggest that these cytosolic complexes are derived from
released membrane-bound components.
As depicted in the model of Fig. 11, our findings are consistent with the hypothesis that phosphorylation of IRS-1 on serine/threonine residues is involved in the mechanism of IRS-1 release from intracellular membranes. Thus, following insulin stimulation at 37 °C in both 3T3-L1 adipocytes and in CHO-T cells, the tyrosine-phosphorylated IRS-1 present in the low density microsome fraction exhibits decreased electrophoretic mobility ( Fig. 1and Fig. 5). This decreased electrophoretic mobility of IRS-1 coincides with the decreased amount of IRS-1 associated with the low density microsome fraction and the appearance of tyrosine-phosphorylated IRS-1 in the cytosol. Moreover, both the release of IRS-1 into the cytosol and the electrophoretic mobility shift in IRS-1 due to insulin are abolished at low temperature ( Fig. 7and Fig. 8). The electrophoretic mobility shift in IRS-1 at 37 °C may not be explained by tyrosine phosphorylation of IRS-1 because this also occurs at 4 °C. IRS-1 is known to be phosphorylated on serine within its PH domain in response to insulin (48) . Additionally, PI 3-kinase has been shown to be a dual specificity enzyme in that it possesses both lipid and serine kinase activities (16, 17) , and IRS-1 was demonstrated to be an insulin-dependent substrate for this serine kinase activity of PI 3-kinase in intact cells(18) . We hypothesize that insulin may regulate the intracellular localization of IRS-1 by causing it to be phosphorylated by PI 3-kinase and subsequently released from intracellular membranes into the cytosol. Alternatively, perhaps other serine/threonine protein kinases are involved in IRS-1 phosphorylation in response to insulin. Further work is required to determine whether serine/threonine phosphorylation of IRS-1 is required for the release mechanism and which protein kinase(s) may be involved.
It is noteworthy that in contrast to results obtained with 3T3-L1 adipocytes, increased tyrosine-phosphorylated IRS-1 was detected in the cytosol of CHO-T cells stimulated with insulin at 4 °C (Fig. 5). However, this cytosolic tyrosine-phosphorylated IRS-1 in CHO-T cells does not exhibit decreased electrophoretic mobility, as does that prepared from 3T3-L1 or CHO-T cells stimulated with insulin at 37 °C ( Fig. 1and Fig. 5). One possibility is that tyrosine-phosphorylated IRS-1 in CHO-T cells binds more weakly to intracellular membranes relative to 3T3-L1 adipocytes.
In summary, the results presented here provide two new insights into the dynamics of IRS-1 function in insulin receptor signaling. First, based on the marked tyrosine phosphorylation of IRS-1 in response to insulin at low temperature, we can conclude that insulin receptor endocytosis is not required for initiating the IRS-1 signaling pathway. This is consistent with studies showing that in certain cell types, mutant insulin receptors which fail to undergo internalization are still effective in mediating downstream biological effects(49) . Secondly, based on the striking redistribution of IRS-1 protein in subcellular fractions in response to insulin, we suggest that IRS-1 cycles between membrane-bound and cytosolic locations in an insulin-regulated manner. This recycling may reflect an important physiological mechanism for the release of IRS-1 signaling complexes from targeted membrane sites where regulatory events may occur.