Insulin Stimulates PKCzeta -mediated Phosphorylation of Insulin Receptor Substrate-1 (IRS-1)

A SELF-ATTENUATED MECHANISM TO NEGATIVELY REGULATE THE FUNCTION OF IRS PROTEINS*

Yan-Fang Liu, Keren Paz, Avia Herschkovitz, Addy AltDagger , Tamar TennenbaumDagger , Sanford R. SampsonDagger , Motoi Ohba§, Toshio Kuroki§, Derek LeRoith, and Yehiel Zick||

From the Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel, the Dagger  Faculty of Life Sciences, Gonda-Goldschmied Center, Bar-Ilan University, Ramat-Gan 52900, Israel, the § Institute of Molecular Oncology, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan, and the  Molecular and Cellular Endocrinology Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, August 10, 2000, and in revised form, January 2, 2001




    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Incubation of rat hepatoma Fao cells with insulin leads to a transient rise in Tyr phosphorylation of insulin receptor substrate (IRS) proteins. This is followed by elevation in their P-Ser/Thr content, and their dissociation from the insulin receptor (IR). Wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, abolished the increase in the P-Ser/Thr content of IRS-1, its dissociation from the IR, and the decrease in its P-Tyr content following 60 min of insulin treatment, indicating that the Ser kinases that negatively regulate IRS-1 function are downstream effectors of PI3K. PKCzeta fulfills this criterion, being an insulin-activated downstream effector of PI3K. Overexpression of PKCzeta in Fao cells, by infection of the cells with adenovirus-based PKCzeta construct, had no effect on its own, but it accelerated the rate of insulin-stimulated dissociation of IR·IRS-1 complexes and the rate of Tyr dephosphorylation of IRS-1. The insulin-stimulated negative regulatory role of PKCzeta was specific and could not be mimic by infecting Fao cells with adenoviral constructs encoding for PKC alpha , delta , or eta . Because the reduction in P-Tyr content of IRS-1 was accompanied by a reduced association of IRS-1 with p85, the regulatory subunit of PI3K, it suggests that this negative regulatory process induced by PKCzeta , has a built-in attenuation signal. Hence, insulin triggers a sequential cascade in which PI3K-mediated activation of PKCzeta inhibits IRS-1 functions, reduces complex formation between IRS-1 and PI3K, and inhibits further activation of PKCzeta itself. These findings implicate PKCzeta as a key element in a multistep negative feedback control mechanism of IRS-1 functions.




    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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The insulin receptor mediates insulin action through the phosphorylation of substrate proteins on Tyr residues (1-3). The major substrates of the insulin receptor kinase are Shc (4) and the IRS1 family of proteins, IRS-1 (5), IRS-2 (6), IRS-3 (7), and IRS-4 (8). IRS proteins contain a conserved PH (pleckstrin homology) domain (9, 10) located at the amino terminus, adjacent to a phosphotyrosine binding (PTB) domain. The PTB domain is present in a number of signaling molecules (11) and shares 75% sequence identity between IRS-1 and IRS-2 (12). This domain interacts with the NPXY motif of the juxtamembrane region of IR and promotes IR-IRS-1 interaction (13-15). The carboxyl-terminal region of IRS proteins is poorly conserved. It contains multiple tyrosine phosphorylation motifs that serve as docking sites for SH2 (Src homology-2) domain-containing proteins like the p85alpha regulatory subunit of PI3K, Grb2, Nck, Crk, Fyn, and SHP-2, which mediate the metabolic and growth-promoting functions of insulin (1-3).

IRS proteins contain more than 70 potential Ser/Thr phosphorylation sites for kinases like PKA (cAMP-dependent protein kinase), PKC, and MAPK (5, 6, 16). The phosphorylation of Ser/Thr residues of IRS proteins has a dual function, serving either for a positive or negative modulation of insulin signal transduction. Phosphorylation of Ser residues within the PTB domain of IRS-1 by insulin-stimulated PKB protects IRS proteins from the rapid action of protein tyrosine phosphatases and enables the Ser-phosphorylated IRS proteins to maintain their Tyr-phosphorylated active conformation, implicating PKB as a positive regulator of IRS-1 functions (17). In contrast, a wortmannin-sensitive Ser/Thr kinase, different from PKB, phosphorylates IRS proteins and acts as a negative feedback control regulator that turns off insulin signals by inducing the dissociation of IRS proteins from IR (17). These observations raise the question as to which kinases act as negative modulators of IRS proteins function.

Several Ser/Thr kinases located downstream of PI3K are potential candidates to fulfill this role. These include the mammalian target of rapamycin (mTOR) (18) and p70S6 kinase (19), which are activated by phosphoinositide-dependent kinase-1 (PDK-1) (20). Other candidates are members of the PKC family. Indeed, 12-O-tetradecanoylphorbol 13-acetate, a potent activator of various PKC isoforms, effectively inhibits both IRS-1 interactions with the juxtamembrane region of the insulin receptor and insulin's ability to phosphorylate IRS proteins, thus implicating diacylglycerol-activated PKCs as potential regulators of IR-IRS interactions (21-23).

Atypical PKCs, exemplified by PKCzeta , are downstream effectors of PI3K (24) and PDKs (25) and act as mediators of insulin action. These proteins do not contain PH domains, and the exact mechanism of their activation is unknown. Atypical PKCs bind phosphatidylinositol 1,4,5-trisphosphate with reasonably high affinities, which results in their activation (26). Hence, phosphatidylinositol 1,4,5-trisphosphate may promote PKC signaling by colocalizing the enzyme in close proximity to its substrates. PKCzeta can be activated by insulin, and this activation is blocked by inhibitors of PI3K and the expression of dominant negative p85 (27, 28). Phosphorylation of PKCzeta is required for its full activation; two of the regulatory phosphorylation sites on PKCzeta are targets for phosphorylation by PDK-1 and PDK-2 (25), thus representing another mechanism by which activation of PI3K affects the activity of PKCzeta .

In the present study we provide evidence that overexpression of PKCzeta , but not other PKC isoforms potentiates the insulin-stimulated dissociation of IRS-1 from the insulin receptor, accelerates the rate of Tyr dephosphorylation of IRS-1, and as a result, reduces complex formation between IRS-1 and PI3K. These findings implicate PKCzeta as an insulin-stimulated and a PI3K-dependent kinase that down-regulates IRS-1 functions by a tightly regulated process. Agents that induce insulin resistance, such as TNF, a known activator of PKCzeta (29, 30) could take advantage of this mechanism. Activation of PKCzeta by TNF in an insulin-independent manner (29, 30) could account for the enhanced Ser phosphorylation of IRS proteins and their dissociation from IR, which takes place when cells are exposed to TNF (21), thus providing us with a possible molecular mechanism for the induction of an insulin-resistant state.


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ABSTRACT
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EXPERIMENTAL PROCEDURES
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Materials-- Recombinant human insulin was a gift from Novo-Nordisc (Copenhagen, Denmark). Wortmannin, wheat-germ agglutinin coupled to agarose beads, protease inhibitor mixture, protein A-Sepharose CL-4B, and monoclonal anti-phospho-MAPK antibodies were purchased from Sigma. Rapamycin, PD98059, Go6983, bisindolylmaleimide I (GF109203X), and 12-(2-cyanoethyl)-6,7,2,13,-terahydro-13-methyl-5-oxo-(5H)-indolo[2,3-alpha ]-pyrrolo[3,4-c]carbazole (Go6976) were purchased from Calbiochem. Monoclonal PY-20 antibodies and polyclonal anti-IR beta -subunit antibodies were obtained from Transduction Laboratories (Lexington, KY). Polyclonal IRS-1 antibody (YR-1) was prepared as described (31). Polyclonal anti-PKCzeta /lambda was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Polyclonal anti-p85 antibodies were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). The anti-phospho-PKB (Ser473) was purchased from New England Bio-Labs, Inc. (Beverly, MA).

Treatment of Cells-- Rat hepatoma Fao cells were grown in RPMI medium supplemented with 10% fetal calf serum as described (21, 32). H-35 rat hepatoma cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. At 80% confluence, the cells were deprived of serum for 16 h before each experiment. The medium was aspirated, and the cells were incubated with the indicated inhibitors in serum-free medium for different time periods at 37 °C. Cells were then incubated with or without 100 nM insulin for 1 or 60 min at 37 °C. Cells were washed three times with ice-cold phosphate-buffered saline and harvested in buffer A (25 mM Tris-HCl, 2 mM sodium orthovanadate, 0.5 mM EGTA, 10 mM NaF, 10 mM sodium pyrophosphate, 80 mM beta -glycerophosphate, 25 mM NaCl, protease inhibitor mixture 1:1000, pH 7.4). Following three cycles of freezing and thawing, the cell extracts were centrifuged at 12,000 × g for 20 min at 4 °C, and the supernatants were collected. Samples were resolved by means of SDS-PAGE and immunoblotted with the indicated antibodies.

Adenovirus Constructs-- Recombinant adenoviruses were constructed in three steps essentially as described (33). The cDNA encoding mouse wild-type PKCzeta , a kinase-inactive form of this enzyme (PKCzeta -KD, generated by replacing K282 (AAG) at the ATP binding site by R(AGG)), as well as cDNA encoding PKCalpha , eta , and delta  were inserted into a cassette cosmid. The cassette cosmid for constructing recombinant Ad of the E1 substitution type, pAdex1, was an 11-kilobase pair charomid vector bearing an Ad5 genome spanning 0 to 99.3 map units with deletions of E1 (map units 1.3-9.3) and E3 (map units 79.6-84.8), in which a unique SwaI site was created by linker insertion at the E1 deletion. The expression unit was excised with the appropriate restriction enzymes, blunt-ended with Klenow fragment of DNA polymerase I, and purified by gel electrophoresis. Thereafter the fragment was ligated with SwaI-linearized pAxCAwt (33). After overnight ligation, the DNA sample was digested with SwaI to exclude empty religated cosmids lacking a coding sequence, and an aliquot was packaged in vitro using Gigapack (Stratagene, La Jolla, CA). Colonies were obtained after plating the transduced Escherichia coli DH5alpha ; the majority of the clones contained the desired insert. Ad5-dIX, which has an E3 deletion (map units 79.6-84.8) was used as the parent virus for recombinant Ad construction. The DNA-terminal protein complex of the parent Ad was prepared and purified utilizing a CsCl density gradient with guanidine hydrochloride. The EcoT22I-digested adenovirus DNA-terminal protein complex was mixed with the cassette cosmid bearing the desired expression unit, and human embryonic kidney 293 cells were transfected with the mixed DNA by the calcium phosphate method using a CellPhect Transfection kit (Amersham Pharmacia Biotech). One day later, the cells were dispensed in 96-well plates in 10-fold serial dilutions and mixed with untransfected 293 cells. After being maintained in culture for 10-15 days, virus-containing supernatants were isolated and propagated further to assess restriction analysis and expression of inserted genes.

Infection of Fao Cells with Adenovirus-PKC-- Fao cells in 90-mm plates were cultured in RPMI medium supplemented with 10% fetal calf serum till 60% confluence. The medium was aspirated, and 150-µl aliquots of PKCzeta , alpha , eta , or delta  recombinant adenoviruses were added to the plate in 3 ml of RPMI medium containing 10% fetal calf serum for 2 h at 37 °C. The cultures were supplemented with 8 ml of RPMI medium containing 10% fetal calf serum and were further incubated with the adenoviral constructs for 16 h. The cultures were then washed twice with RPMI, trypsinized, and divided into three samples. 48 h post-infection, cells were starved for 16 h, further treated as indicated above, and then harvested in buffer A.

Binding of IRS-1 to Immobilized IR-- Insulin receptors were extracted from Fao cells at 70% confluence with buffer B (50 mM Tris-HCl, 1% Nonidet P-40, 0.25% deoxycholate, 1 mM sodium orthovanadate, 1 mM EGTA, 1 mM NaF, 150 mM NaCl, and protease inhibitor mixture 1:1000, pH 7.4). Cell extracts were centrifuged at 12,000 × g for 20 min, and the supernatant, which contained the soluble IR, was collected. Immobilization of IR on wheat germ agglutinin-coupled agarose beads was carried out as described previously (34). In brief, wheat germ agglutinin-coupled beads were equilibrated in buffer B, and aliquots (1 mg) of the solubilized IR were incubated with 30 µl of packed wheat germ agglutinin-beads for 1 h at 4 °C. The immobilized IR was washed with buffer A prior to use. Fao cells at 80% confluence grown in 90-mm dishes were treated as indicated, washed three times with phosphate-buffered saline, and harvested in 400 µl of buffer A. Following three cycles of freezing and thawing, the cell extracts were centrifuged at 12,000 × g for 30 min at 4 °C, and the supernatants were collected. Aliquots (1.0 mg) were incubated for 2 h with 30 µl of immobilized IR while shaking at 4 °C. The beads were washed three times with buffer A and boiled in 50 µl of Laemmli "sample buffer" (35). Samples were resolved by means of SDS-PAGE and immunoblotted with anti-IRS-1 or anti-IR beta  subunit antibodies.

Immunoprecipitation-- Fao cells were solubilized at 4 °C in buffer A. Cell extracts were then centrifuged at 12,000 × g for 15 min at 4 °C, and the supernatants were collected. Aliquots (1.0 mg) were incubated for 2 h at 4 °C with polyclonal IRS-1 antibody coupled to 15 µl of packed protein A-Sepharose beads. Immunocomplexes were washed three times with buffer A, resolved by means of SDS-PAGE, and immunoblotted with the indicated antibodies.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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IRS-1 Serves as a Substrate for an Insulin-stimulated Wortmannin-sensitive Ser/Thr Kinase-- Consistent with our previous studies (17), incubation of Fao cells with 10-7 M insulin rapidly stimulated Tyr phosphorylation of the IRS proteins (Fig. 1, A, a and B, e). Phosphorylation was maximal 1 min following insulin treatment and declined by 60 min incubation with the hormone, accompanied by a decrease in the electrophoretic mobility of IRS-1 and a marked (>40%) reduction in its ability to interact in vitro with the insulin receptor (Fig. 1B, f). Preincubation of the cells with wortmannin, a potent inhibitor of PI3K, eliminated both the mobility shift and the reduction in the P-Tyr content of IRS-1, whereas preincubation of the cells with rapamycin, which inhibits the activation of p70 S6 kinase (36), partially reversed the long-term effects of insulin. Similarly, wortmannin effectively prevented the dissociation of IR·IRS-1 complexes that occurs following a 60-min treatment with insulin. In contrast, PD-98059, a specific MEK (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase) inhibitor (37), had no such protective effect, although it effectively inhibited insulin-stimulated activation of MAPK (Fig. 1A, d). Notably, both wortmannin and, to a lesser extent, rapamycin prevented insulin-stimulated Ser phosphorylation of IRS-1, but only wortmannin inhibited the activation of PKB, indicating that the insulin-stimulated IRS-1 kinases that inhibit its Tyr phosphorylation in response to insulin differ from PKB, the positive regulator of IRS proteins function (17).



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Fig. 1.   Effects of different inhibitors on Tyr phosphorylation of IRS-1 and its interaction with IR. Fao cells were incubated with or without the indicated inhibitors for 60 min at 37 °C. Cells were then further incubated with 100 nM insulin for the indicated times. A, cytosolic extracts were prepared, and samples (1 mg) were subjected to immunoprecipitation (IP) with IRS-1 antibodies. The immunocomplexes were resolved by means of 7.5% SDS-PAGE and immunoblotted with anti-PY antibody (a). In parallel, samples (100 µg) of total cell extracts were resolved by means of 7.5% SDS-PAGE and immunoblotted (IB) with anti-IRS-1 (b), anti-phospho-PKB (c), and anti-phospho-MAPK (d) antibodies. B, cell extracts (1 mg) were bound to immobilized IR as described under "Experimental Procedures." Bound IRS-1 was detected following immunoblotting with anti-IRS-1 antibodies (f). In parallel, cell extracts (100 µg) were resolved by means of 7.5% SDS-PAGE and immunoblotted with anti-P-Tyr (e) or anti-IRS-1 antibodies (g). The bar graphs represent quantitation of the results (mean ± S.D., n = 3-5). *, significantly different from the measurement at 1 min (p < 0.001); **, significantly different from the measurements taken at 1 min (p < 0.05) and at 60 min (p < 0.01); ***, significantly different from the measurement at 1 min (p < 0.025); #, significantly different from the measurement at 1 min (p < 0.001). The p values are based upon a paired t test.

Insulin-induced Ser Phosphorylation of IRS-1 Impairs Its Interaction with the p85 Regulatory Subunit with PI3K-- To determine whether insulin-stimulated Ser phosphorylation of IRS-1 also affects its ability to couple with downstream effectors, binding of IRS-1 to p85alpha , the regulatory subunit of PI3K, was studied. Consistent with numerous studies (cf. Refs. 1-3), insulin-stimulated Tyr phosphorylation of IRS-1 resulted in the effective coupling of IRS-1 to p85alpha (Fig. 2). Furthermore, the reduced level of Tyr phosphorylated IRS-1 following 60 min of insulin treatment was accompanied by a significant reduction in the amount of p85-associated IRS-1. Accordingly, pretreatment with wortmannin increased both the P-Tyr content of IRS-1 and its ability to interact with p85. In contrast, the PKC inhibitors Go6976 (inhibitor of PKCalpha , beta I, and µ) and GF109203X (inhibitor of PKCalpha , beta I, beta II, gamma , delta , and epsilon ) failed to exert a protective effect. The PKC inhibitor Go6983 (inhibitor of PKCalpha , beta , gamma , delta , and zeta ) exerted a partial inhibitory effect, indicating that PKCzeta , which is inhibited only by this inhibitor, could mediate insulin-stimulated Ser phosphorylation of IRS-1.



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Fig. 2.   Effects of PKC inhibitors on Tyr phosphorylation of IRS-1 and its association with PI3K. Fao cells were incubated with the indicated inhibitors for 60 min. Cells were then further incubated with 100 nM insulin for the indicated time at 37 °C, and cytosolic extracts were prepared. Samples (1 mg) were subjected to immunoprecipitation (IP) with IRS-1 antibodies (a and b). Immunocomplexes were resolved by means of 7.5% SDS-PAGE and immunoblotted (IB) with anti-PY (a) or anti-p85 antibodies (b). In parallel, samples (100 µg) of total cell extracts were resolved by means of 7.5% SDS-PAGE and immunoblotted with anti-IRS-1 (lane c), anti-phospho-PKB (d), or anti-phospho-MAPK (lane e) antibodies. The bar graphs represent quantitation of the results mean ± S.D., n = 3-5). *, significantly different from both the measurement taken at 1 min (p < 0.050 and the measurement taken at 60 min (p < 0.003); **, significantly different from the measurement at 1 min (p < 0.05); #, significantly different from the measurement at 1 min (p < 0.01); ##, different from the measurement 60 min (p = 0.07). The p values are based upon a paired t test.

PKCzeta Mediates Ser Phosphorylation of IRS-1-- To determine whether PKCzeta is directly involved in the negative regulation of IRS-1 protein functions, PKCzeta was overexpressed in Fao cells by infection with an adenoviruses vector. As shown in Fig. 3 (inset) PKCzeta is endogenously expressed in Fao cells, whereas infection with an adenovirus-based PKCzeta construct markedly elevated the cellular content of PKCzeta , assayed 48 h post-infection. Infection of Fao cells with adenoviral constructs bearing beta - galactosidase (Fig. 4B) or PKCzeta (Fig. 3b) did not affect the cellular content of IRS-1 nor did it affect the extent of Tyr phosphorylation of IRS-1 following a 1-min insulin treatment (Figs. 3 and 4). However, the P-Tyr content of IRS-1, assayed 60 min following insulin stimulation, was significantly reduced in cells overexpressing PKCzeta when compared with noninfected cells. The infection of Fao cells with an adenoviral construct expressing a kinase-inactive form of PKCzeta (K282R) failed to mimic the inhibitory effects of the wild-type PKCzeta on the insulin-stimulated Tyr phosphorylation of IRS-1 following 60 min of insulin treatment (Fig. 4A), indicating that only an active PKCzeta mediates the inhibitory effects. Wortmannin effectively inhibited the reduction in P-Tyr content of IRS-1 following 60 min of insulin treatment both in the nontreated as well as the PKCzeta -overexpressing cells, indicating that activation of the overexpressed PKCzeta is controlled by the endogenous PI3K activity and is independent of MAPK activation. Similarly, overexpression of PKCzeta did not affect the insulin-stimulated activation of PKB (Fig. 3c), indicating that PKB is not a downstream effector of PKCzeta . Overexpression of PKCzeta significantly potentiated insulin-stimulated MAPK activation (Fig. 3e), but MAPK could not be considered an insulin-stimulated IRS-1 kinase because its inhibition by PD98059 did not affect the P-Tyr content of IRS-1, assayed 60 min following insulin stimulation (Fig. 1).



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Fig. 3.   Effects of overexpression of PKCzeta on insulin-stimulated Tyr phosphorylation of IRS-1. Fao cells remained uninfected (None) or were transiently infected with Ad-PKCzeta . 48 h post-infection, cells were incubated with or without 100 nM wortmannin for 60 min and were then further incubated with 100 nM insulin for the indicated time at 37 °C. Cell extracts (1 mg) were subjected to immunoprecipitation (IP) with IRS-1 antibody (a and b). Immunocomplexes were resolved by means of 7.5% SDS-PAGE and immunoblotted with anti-PY (a) or anti-IRS-1 (b) antibodies. In parallel, total cell extract (100 µg) were resolved by means of 7.5% SDS-PAGE and immunoblotted with anti-PKCzeta (inset), anti-phospho-PKB (c), or anti-phospho-MAPK (alpha ) antibodies. The bar graphs represent quantitation of the results (mean ± S.D., n = 3-5). *, significantly different from the measurement taken at 1 min (p < 0.001) and the measurement taken at 60 min (p < 0.001) in noninfected cells. The p values are based upon paired t test. WT, wild type.



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Fig. 4.   Effects of overexpression of a kinase-inactive PKCzeta (K282R) on insulin-stimulated Tyr phosphorylation of IRS-1. Fao cells were remained uninfected (None) or were transiently infected with Ad-beta -galactosidase overexpressing beta -galactosidase (B) or Ad-PKCzeta -KD overexpressing a kinase-inactive PKCzeta (K282R) (A). 48 h post-infection, cells were incubated with 100 nM insulin for the indicated time at 37 °C. Cell extracts (1 mg) were subjected to immunoprecipitation (IP) with IRS-1 antibody (a, b, and d). Immunocomplexes were resolved by means of 7.5% SDS-PAGE and immunoblotted with anti-PY (a and d) or anti-IRS-1 (b) antibodies. In parallel, total cell extract (100 µg) were resolved by means of 7.5% SDS-PAGE and immunoblotted with anti-PKCzeta (c and f) or anti-IRS-1 (e) antibodies. The results of a representative experiment carried out six times (B) or two times (A) are shown.

Overexpression of PKCzeta Inhibits Complex Formation between IR and IRS-1 and the Subsequent Association of IRS-1 with PI3K-- The consequences of the reduction in P-Tyr content of IRS-1 as a result of PKCzeta overexpression were evaluated next. As shown in Fig. 5a overexpression of PKCzeta , accelerated the dissociation of IR·IRS-1 complexes observed following 60 min of insulin treatment (Fig. 5a, lane 3 versus 9). The marked reduction in complex formation was evident when we compared the amount of the Ser/Thr-phosphorylated IRS-1 bound to IR (the upper IRS-1 band in Fig. 5a, lanes 3 and 9) with the total Ser/Thr-phosphorylated IRS-1 protein (Fig. 5c, lanes 3 and 9). Notably, the nonphosphorylated (faster migrating) form of IRS-1, which is practically undetected in the total IRS-1 blots of cells treated with insulin for 60 min (compare Fig. 5c, lane 2 versus 3 or lane 8 versus 9) is concentrated upon binding to IR, and becomes visible when IRS-1-bound to IR is analyzed (Fig. 5a, upper versus lower bands in lanes 3, 5, 6, 9, 11, and 12). Here again, pretreatment with the PKC inhibitors Go6976 and GF109203X was ineffective, whereas Go6983, which inhibits several PKC isoforms including PKCzeta , partially restored complex formation (Fig. 5a, lane 3 versus 5) but only in the noninfected cells. The reduction in the IR·IRS-1 complex formation and the subsequent reduction in the P-Tyr content of IRS-1 (at 60 min; Figs. 3 and 6), resulted in reduced association between IRS-1 and its downstream effector, PI3K (Fig. 6); wortmannin but not PD98059 could reverse this effect. Of note, insulin-stimulated activation of IRS-1 kinases in cells overexpressing PKCzeta was also regulated by mTOR or its downstream effectors, because rapamycin partially reversed the inhibitory effects of the overexpressed PKCzeta on Tyr phosphorylation of IRS-1 and its association of p85 (Fig. 6).



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Fig. 5.   Effects of overexpression of PKCzeta on the interaction of IRS-1 with IR. Fao cells remained uninfected or were transiently infected with Ad-PKCzeta . 48 h post-infection, cells were incubated with the indicated inhibitors for 60 min and were then further incubated with 100 nM insulin for the indicated time at 37 °C. Cell extracts were prepared in buffer A, and samples (1 mg) were bound to immobilized IR as described under "Experimental Procedures." IRS·1-IR complexes were resolved by means of 7.5% SDS-PAGE and immunoblotted with anti-IRS-1 (a) and anti IR (b) antibodies. In parallel, samples of total cell extracts (100 µg) were resolved by means of 7.5% SDS-PAGE and immunoblotted with anti-IRS-1 (c) or anti-PKCzeta (d) antibodies, respectively. The bar graphs represent quantitation of the results (mean ± S.D., n = 3-5). *, significantly different (p < 0.001) from the measurement taken at 1 min (lanes 2 and 8) and the measurements taken at 60 min (lane 3) in noninfected cells (p < 0.002); **, significantly different (p < 0.02) from the measurement taken at 60 min in noninfected cells (lane 3); ***, not significantly different (p = 0.21) from the measurement taken at 60 min in PKCzeta -infected cells (lane 9). The p values are based upon a paired t test.



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Fig. 6.   Effects of overexpression of PKCzeta on insulin-stimulated association of IRS-1 with PI3K. Fao cells remained uninfected or were transiently infected with Ad-PKCzeta . 48 h post-infection, cells were incubated with the indicated inhibitors for 60 min and were then further incubated with 100 nM insulin for the indicated time at 37 °C. Cell extracts were prepared in buffer A, and samples (1 mg) were subjected to immunoprecipitation (IP) with IRS-1 antibodies (a-c). Immunocomplexes were resolved by means of 7.5% SDS-PAGE and immunoblotted (IB) with anti-PY (a), anti-IRS-1 (b), or anti p85 antibodies (c). In parallel, samples (100 µg) of total cell extracts were resolved by means of 7.5% SDS-PAGE and immunoblotted anti p85 antibodies (d). The bar graphs represent quantitation of the results (mean ± S.D., n = 3-5). *, significantly different (p < 0.03) from the measurement taken at 60 min (lane 9) in PKCzeta -infected cells; **, significantly different (p < 0.004) from the measurement taken at 60 min in noninfected cells (lane 3); ***, significantly different (p < 0.04) from the measurement taken at 1 min (lane 8) and the measurements taken at 60 min (lane 9) in PKCzeta -infected cells (p < 0.08); # significantly different (p < 0.006) from the measurement taken at 1 min (lane 2) and the measurement taken at 60 min (lane 3) (p < 0.04) in noninfected cells. The p values are based upon a paired t test.

The Specificity of the Effects of PKCzeta -- The specificity of the effects of PKCzeta was evaluated next. As shown in Fig. 7, overexpression of PKCalpha , PKCdelta , or PKCeta in Fao cells, introduced by infection with an adenoviral vector, failed to reproduce the effects of PKCzeta and did not accelerate the rate of Tyr dephosphorylation of IRS-1 following 60 min of insulin treatment. These PKC isoforms failed to induce MAPK activation in response to insulin. These results indicate that the negative feedback control on IRS protein function is selectively mediated by PKC isoforms that are downstream effectors of PI3K along the insulin signaling pathway.



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Fig. 7.   Effects of overexpression of different PKC isoforms on insulin-stimulated Tyr phosphorylation of IRS-1. Fao cells were remained uninfected or were infected with Ad-PKCalpha (A), delta  (B), or eta  (C) as described under "Experimental Procedures." 48 h post-infection, cells were incubated for 60 min with or without 100 nM wortmannin as indicated. Cells were then further incubated with 100 nM insulin for the indicated time at 37 °C. Cytosolic extracts were prepared, and samples (1 mg) were subjected to immunoprecipitation (IP) with IRS-1 antibody (a, b, e, f, i, and j). Immunocomplexes were resolved by means of 7.5% SDS-PAGE and immunoblotted with anti-PY (a, e, and i) or anti-IRS-1 (b, f, and j) antibodies. In parallel, samples (100 µg) of total cell extracts were resolved by means of 7.5% SDS-PAGE and immunoblotted with anti-PKCalpha (c), anti-PKCdelta (g), or anti-phospho-MAPK (d and h) antibodies.



    DISCUSSION
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ABSTRACT
INTRODUCTION
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REFERENCES

Atypical PKC isotypes PKCzeta and PKClambda are important elements in insulin signal transduction. They are activated by insulin through a PI3K-dependent mechanism (27, 38), and insulin-stimulated glucose transport depends upon the activation of one or both of these enzymes (27, 28, 38, 39). In the present study we provide evidence that PKCzeta also plays a major regulatory role in a negative feedback control mechanism induced by insulin to terminate its own signaling pathways. This mechanism involves PKCzeta -mediated Ser/Thr phosphorylation of IRS-1 that leads to its dissociation from the insulin receptor. The dissociated IRS protein fails to undergo further Tyr phosphorylation by the insulin receptor kinase, while being subjected to the action of protein Tyr phosphatases that reduce its P-Tyr content. The resulting Tyr-dephosphorylated IRS-1 is unable to recruit downstream effectors like PI3K, and the insulin signal is therefore terminated.

Several lines of evidence support such a mechanism. First, we could demonstrate that of several inhibitors tested, wortmannin, a PI3K inhibitor, effectively inhibits the dissociation of IRS-1 from the insulin receptor and the subsequent reduction in its P-Tyr content observed following a 60-min insulin treatment. Hence, a wortmannin-sensitive Ser/Thr kinase presumably acts as the feedback control regulator that turns off insulin signals. PKCzeta , an insulin-stimulated Ser/Thr kinase downstream of PI3K, could fulfill this role, and indeed Go6983, an inhibitor of several PKC isoforms including PKCzeta , partially prevented the reduction in P-Tyr content of IRS-1 following a 60-min insulin treatment. Second, overexpression of PKCzeta by infection of Fao cells with an adenoviral-based expression vector markedly potentiated the dissociation of IR·IRS-1 complexes and the resulting reduction in P-Tyr content of IRS-1. The overexpressed PKCzeta , like its endogenous counterpart, remained under the control of the insulin signaling pathway, since its effects were inhibited in the presence of wortmannin. Furthermore, overexpression of PKCzeta did not impair the acute (1 min) effects of insulin on Tyr phosphorylation of IRS-1, indicating that the basal activity of the overexpressed PKCzeta is rather low.

The effects of PKCzeta on Ser phosphorylation of IRS-1 seems to be quite unique in the sense that other PKC isoforms such as PKCalpha , delta , and eta , when overexpressed in Fao cells, fail to mimic the effects of PKCzeta following insulin stimulation. Similarly, a kinase-inactive form of PKCzeta fails to mimic the inhibitory effects of its wild-type counterpart when transiently overexpressed in Fao or H-35 cells. Still, these observations do not exclude the possibility that other PKC isoforms could phosphorylate the IRS proteins in an insulin-independent manner. In fact, we have shown (21) that 12-O-tetradecanoylphorbol 13-acetate, a potent activator of conventional and novel PKC isoforms, effectively inhibits both IRS-1 interactions with the juxtamembrane region of the insulin receptor and insulin's ability to phosphorylate IRS proteins (21). Similarly, the mutation of Ser612 of IRS-1, a potential MAPK phosphorylation site, eliminates the ability of 12-O-tetradecanoylphorbol 13-acetate to induce IR-IRS dissociation, indicating that diacylglycerol-activated PKCs (40) could act as potential regulators of IR-IRS interactions (22, 23). It should be noted, however, that although overexpression of PKCzeta potentiates insulin-mediated activation of MAPK, the latter does not seem to mediate PKCzeta effects on IRS-1 phosphorylation because PD98059, which effectively inhibits activation of MAPK does not inhibit Ser/Thr phosphorylation of IRS-1 in response to insulin treatment.

Although IRS proteins contain several PKC phosphorylation sites, it is presently unclear whether PKCzeta phosphorylates IRS-1 directly or whether its effects are mediated by a downstream effector of PKCzeta . Recent studies suggest that IRS-1 serves as an in vitro substrate for PKCzeta (41). Furthermore, endogenous IRS-1 coprecipitates with endogenous PKCzeta , and this association is increased 2-fold upon insulin stimulation (41). These findings suggest that PKCzeta could function as a direct IRS-1 kinase. However, several Ser/Thr kinases that are downstream effectors of PKCzeta could also fulfill this role. A potential candidate is the IkB kinase beta  (IKKbeta ), which binds PKCalpha as well as the atypical PKCzeta both in vitro and in vivo, and serves as an in vitro substrate for PKCzeta (42). Overexpression of PKCzeta positively modulates IKKbeta activity, whereas the transfection of a dominant negative mutant of PKCzeta severely impairs the activation of IKKbeta in TNF-stimulated cells (42).

The p70 S6 kinase (19) is also a potential candidate in view of the fact that rapamycin, which inhibits the insulin-stimulated activation of p70S6K (43), partially prevents the reduction in P-Tyr content of IRS-1 following 60 min of insulin treatment. Indeed, mTOR-mediated phosphorylation on Ser632, Ser662, and Ser731 of IRS-1 was shown to inhibit its insulin-stimulated Tyr phosphorylation and its ability to bind PI3K (23). Furthermore, p70 S6 kinase is activated by PKCzeta and participates in a PI3K-regulated signaling complex in some (44) but not all cellular models (30). Recently, the c-Jun NH2-terminal kinase (JNK) was shown to promote insulin resistance during association with IRS-1 and phosphorylation of Ser307. However it still needs to be determined whether JNK is a downstream effector of PI3K. Although earlier studies suggest that it is (45), recent findings indicate that phosphatidylinositol 3-kinase is not part of the insulin signaling pathway that leads to JNK activation (46).

Our current work and previous studies (17, 21) indicate that Ser/Thr phosphorylation of IRS protein following insulin stimulation has a dual role, either to enhance or to terminate the insulin signal. Ser residues of the PTB domain of IRS-1, located within consensus PKB phosphorylation sites, presumably function as positive effectors of insulin signaling (17). Once phosphorylated by PKBalpha , they serve to protect IRS proteins from the rapid action of protein Tyr phosphatases. In such a way, PKBalpha acts to propagate and accelerate insulin signaling by phosphorylating downstream effectors and phosphorylating IRS proteins, thus generating a positive feedback loop for insulin action. Insulin also activates PKCzeta , which mediates phosphorylation of yet unidentified Ser/Thr residues within the IRS protein. Phosphorylation of these sites is part of the negative feedback control mechanism induced by insulin, which leads to the dissociation of the IR·IRS complexes and results in the termination of insulin signal. Agents that induce insulin resistance, such as TNF, take advantage of this mechanism by stimulating Ser phosphorylation of IRS proteins and the dissociation of IR·IRS complexes (21). Interestingly, TNF induces the activation of PKCzeta in an insulin-independent manner (29, 30), implicating PKCzeta itself or its downstream Ser/Thr kinases (i.e. IKKbeta ) as potential TNF-stimulated IRS-1 kinases. Both Ser/Thr kinases, which phosphorylate IRS-1, PKB the positive regulator, and PKCzeta the negative regulator, are downstream effectors of PI3K. This finding suggests that their action should be orchestrated in a way that enables sustained activation of IRS-1, as a result of its phosphorylation by PKB, prior to the activation of PKCzeta , the action of which is expected to terminate insulin signal transduction. Our studies further indicate that the negative feedback control mechanism induced by PKCzeta has a built-in self-attenuation signal. Accordingly, insulin-stimulated and PI3K-mediated activation of PKCzeta inhibits IRS-1 functions and reduces complex formation between IRS-1 and PI3K, which then inhibits further activation of PKCzeta . It is presently unclear how PKCzeta promotes insulin action and glucose transport (27, 28, 38, 39) while acting as a negative feedback regulator of insulin signal transduction. Most likely, the positive and negative regulatory roles of PKCzeta are subjected to a tight spatio-temporal control along the insulin signal transduction pathway, such that the positive effects are turned on and terminated before the negative regulatory functions are being activated. Further studies are required to unravel the mechanisms that govern this intricate regulatory process.


    ACKNOWLEDGEMENT

We thank Dr. Ronit Sagi-Eisenberg for helpful comments and discussions.


    FOOTNOTES

* This work was supported by research grants from the Israel Ministry of Health, the Juvenile Diabetes Foundation International (1-1998-228), the Israel Science Foundation (founded by the Israel Academy of Sciences and Humanities), and the Minerva Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| Incumbent of the Marte R. Gomez professorial chair. To whom correspondence should be addressed. Tel: 972-8-9342-380; Fax: 972-8-9344-125; E-mail: yehiel.zick@weizmann.ac.il.

Published, JBC Papers in Press, January 29, 2001, DOI 10.1074/jbc.M007281200


    ABBREVIATIONS

The abbreviations used are: IRS, insulin receptor substrate; IR, insulin receptor; PTB, phosphotyrosine binding; PKB, protein kinase B; PKC, protein kinase C; PI3K, phosphatidylinositol 3-kinase; PDK, phosphoinositide-dependent kinase; PKCzeta -KD, a kinase-inactive PKCzeta ; MAPK, mitogen-activated protein kinase; mTOR, mammalian target of rapamycin; TNF, tumor necrosis factor alpha ; PAGE, polyacrylamide gel electrophoresis; JNK, c-Jun NH2-terminal kinase; Ad, adenovirus.


    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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