Interplay and Effects of Temporal Changes in the Phosphorylation State of Serine-302, -307, and -318...

Interplay and Effects of Temporal Changes in thePhosphorylation State of Serine-302, -307, and -318of Insulin Receptor Substrate-1 on Insulin Action inSkeletal Muscle Cells

Cora Weigert, Matthias Kron, Hubert Kalbacher, Ann Kathrin Pohl, Heike Runge, Hans-Ulrich Haring,Erwin Schleicher, and Rainer Lehmann

Division of Clinical Chemistry and Pathobiochemistry (C.W., M.K., A.K.P., H.R., E.S., R.L.),Department of Internal Medicine 4 (H.-U.H.), University Hospital Tuebingen; and InterfacultaryInstitute of Biochemistry (H.K.), University of Tuebingen, D-72076 Tuebingen, Germany

Transduction of the insulin signal is mediated bymultisite Tyr and Ser/Thr phosphorylation of theinsulin receptor substrates (IRSs). Previous studieson the function of single-site phosphorylation, par-ticularly phosphorylation of Ser-302, -307, and -318of IRS-1, showed attenuating as well as enhancingeffects on insulin action. In this study we investi-gated a possible cross talk of these opposedlyacting serine residues in insulin-stimulated skele-tal muscle cells by monitoring phosphorylation ki-netics, and applying loss of function, gain of func-tion, and combination mutants of IRS-1. Thephosphorylation at Ser-302 was rapid and tran-sient, followed first by Ser-318 phosphorylationand later by phosphorylation of Ser-307, which re-mained elevated for 120 min. Mutation of Ser-302to alanine clearly reduced the subsequent proteinkinase C-�-mediated Ser-318 phosphorylation. TheSer-307 phosphorylation was independent of Ser-302 and/or Ser-318 phosphorylation status. Thefunctional consequences of these phosphorylation

patterns were studied by the expression of IRS-1mutants. The E302A307E318 mutant simulating theearly phosphorylation pattern resulted in a significantincrease in Akt and glycogen synthase kinase 3phosphorylation. Furthermore, glucose uptake wasenhanced. Because the down-regulation of the insu-lin signal was not affected, this phosphorylation pat-tern seems to be involved in the enhancement butnot in the termination of the insulin signal. This en-hancing effect was completely absent when Ser-302was unphosphorylated and Ser-307 was phosphory-lated as simulated by the A302E307E318 mutant.Phospho-Ser-318, sequentially phosphorylated atleast by protein kinase C-� and a mammalian targetof rapamycin/raptor-dependent kinase, was part ofthe positive as well as of the subsequent negativephosphorylation pattern. Thus we conclude that in-sulin stimulation temporally generates differentphosphorylation statuses of the same residues thatexert different functions in insulin signaling. (Molec-ular Endocrinology 22: 2729–2740, 2008)

PHOSPHORYLATION OF DISTINCT residues is animportant posttranslational modification to regu-

late the function of proteins, including enzymatic ac-tivity and creation of docking sites for other proteins,thereby transducing signals from transmembrane re-ceptors to intracellular target molecules. The insulinreceptor substrates (IRSs) are key players in the signalingnetwork of tyrosine kinase receptors. The phosphoryla-tion of IRS by kinases modifies their property as multi-docking site proteins and consequently the diversifica-tion of the incoming signal, thereby achieving a specific

and adaptive cellular response (1–5). The best studiedIRS, IRS-1, contains 34 tyrosine residues and 244 serine/threonine residues (6). Whereas tyrosine phosphorylationof IRS-1 by the tyrosine kinase activity of the insulinreceptor is the crucial event for the signal transduction todownstream targets, serine/threonine phosphorylationappears to be responsible for the precise regulation andis discussed as a major mechanism for the termination ofthe insulin signal (1, 2). Unbalanced chronic stimulationof IRS-1 serine kinases leads to hyperphosphorylation ofIRS-1 and is a major pathophysiological mechanism inthe development of insulin resistance and subsequentlyof type 2 diabetes (5, 7).

During the last decade, several serine/threonine res-idues in IRS-1 have been identified as functionallyrelevant phosphorylation sites, and the knowledgeabout the identity and regulation of the IRS-1 kinasesinvolved is accumulating. Most of these data fit withthe model of a generalized mechanism for reducedinsulin action, important for the physiological determi-nation of insulin action but also responsible for the

First Published Online October 16, 2008Abbreviations: FCS, Fetal calf serum; GSK, glycogen syn-

thase kinase; IRS, insulin receptor substrate; JNK, c-jun-N-terminal kinase; mTOR, mammalian target of rapamycin;PKC, protein kinase C; siRNA, small interfering RNA; wt, wildtype.

Molecular Endocrinology is published monthly by TheEndocrine Society (http://www.endo-society.org), theforemost professional society serving the endocrinecommunity.

0888-8809/08/$15.00/0 Molecular Endocrinology 22(12):2729–2740Printed in U.S.A. Copyright © 2008 by The Endocrine Society

doi: 10.1210/me.2008-0102

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induction of insulin resistance. These sites (corre-sponding to rat IRS-1 sequence) include Ser-24 (8, 9),-267 (10), -302 (11), -307 (12–18), -318 (19, 20), -332(21), -357 (22), -408 (23), -522 (24), -612 (15, 25–27),-632 (15, 25, 28), -662 (25, 27), -731 (25), -789 (29, 30),and -1099/1100 (31, 32). The IRS-1 kinases responsi-ble for phosphorylation of the serine residues of IRS-1and for attenuation of insulin signaling are protein ki-nase C (PKC)-� (9), PKC-� (22), PCK-� (31, 33), c-jun-N-terminal kinase (JNK) (12, 13), inhibitor of NF �Bkinase (14, 34, 35), MAPKs (15), mammalian target ofrapamycin (mTOR) (15–17,36) and its downstream ki-nase S6 kinase-1 (28, 32, 37), glycogen synthase ki-nase (GSK)-3 (21), and salt-inducible kinase-2 (30).However, serine phosphorylation of IRS-1 appearsalso to be involved in the positive regulation of insulinsignal transduction (38). The phosphorylation of Ser-302 (39–41), -318 (42, 43), -325 (44), -629 (not presentin rodents) (45), -789 (46), and -1216 (47, 48) has beenassociated with improved insulin signaling. Althoughmany sites in IRS-1 have been identified, the investi-gation of the complex regulation and the role of IRS-1serine phosphorylation patterns in the modulation ofthe activation and inhibition of insulin signal transduc-tion are still challenging, reflected, for instance, by theinconsistent data reporting stimulatory as well as in-hibitory functions for Ser-302 (11, 39–41), -318 (19, 42,43), and -789 (29, 30, 46) in insulin signal transduction.

Very recently, capillary HPLC-electrospray tandemmass spectrometry has been introduced for globalassessment of the in vivo phosphorylation pattern ofIRS-1 obtained from biopsies of human vastus lateralismuscle (49, 50). Phosphorylation of 22 Ser/Thr sitesfrom Ser-302, -307, -318 up to Ser-1223 (rat IRS-1sequence) was detected (50). After 2 h of insulin stim-ulation a significant change in phosphorylation inten-sity could be demonstrated for several sites includinga 2.6-fold increase in Ser-307 phosphorylation (50).

However, these elegant studies did not provide in-sight into the functional role of these phosphorylationsites either singly or in combination, nor did they revealwhether or not the phosphorylation of each site is anindependent event. Therefore, it was the aim of ourstudy to investigate whether distinct phosphorylationsites are independently phosphorylated and whetherfunctional consequences are related to or indepen-dent of the combination of Ser phosphorylation. Wefocused our studies on the possible interdependenceof the phosphorylation of Ser-302, -307, and -318because the present reports describing the individualfunction of these residues indicated a role for Ser-307in the attenuation of insulin signaling (12–18), whereasthere was contrasting evidence on whether phospho-Ser-302 and -318 have a positive (39–41,43) or nega-tive effect (9, 16, 36–39) on insulin signal transduction.

We found sequential and interdependent changes inthe insulin-dependent phosphorylation state of Ser-302, -307, and -318 of IRS-1 in skeletal muscle cells,which was temporally mediated by different kinases.The phosphorylation of Ser-302 is an initial event and

appeared to be involved in the subsequent phosphor-ylation of Ser-318. Phospho-Ser-302/-318 exerts apronounced positive effect on the early insulin actionfollowed by the attenuation of insulin action whenSer-307 and Ser-318 are phosphorylated and Ser-302has become dephosphorylated. These novel pieces ofinformation may also reconcile conflicting findingsabout the role of Ser-302 and Ser-318.

RESULTS

Ser-302, -307, and -318 Differ from Each Other inthe Insulin-Induced Phosphorylation Kinetics

The phosphorylation of Ser-302, -307, and -318 atdefined time points during insulin treatment has beendemonstrated in cell culture, and in mice and humanstudies, but the phosphorylation kinetics are largelyunknown. Therefore, we compared the phosphoryla-tion states of the three sites of endogenous IRS-1 at 5,10, 30, 60, and 120 min after insulin stimulation in L6myotubes. We observed characteristic kinetics foreach individual residue (Fig. 1, A–C): phosphorylationof Ser-302 was an early event with a maximal intensityafter 10 min of insulin stimulation and a clear reductionafter 30, 60, and 120 min. The phosphorylation ofSer-307 showed different kinetics with a maximumafter 60 min of insulin stimulation. The early increase inthe phosphorylation of Ser-318 was comparable tothat of Ser-302; however, the high phosphorylationsignal lasted until late phase of insulin stimulation.Thus, the data suggest a sequential phosphorylationof the three serine residues in the order Ser-302, -318,and -307 with a clear and continuous reduction ofphospho-Ser-302 after 60 and 120 min far below theintensity of baseline values.

The insulin-induced tyrosine phosphorylation ofIRS-1 showed a maximum after 5 min and was signif-icantly decreased after 30 min with no further changeafter 60 and 120 min (Fig. 1, D and E). This led us tospeculate that the early phosphorylated serine resi-dues at position 302 and 318 might be involved inpositive insulin signaling.

To study the interplay and potential interdepen-dency of the opposing serine sites 302, 307, and 318,as well as their effects on signal transduction, IRS-1mutants with glutamate (E) simulating the phosphory-lated and alanine (A) imitating the unphosphorylatedserine residue were generated for further experimentsto simulate different phosphorylation states based onthe results from the kinetic study, as shown in Fig. 1F.

Role of Phospho-Ser-302 in the SubsequentPhosphorylation of Ser-318

Next we aimed to prove the hypothesis of a sequentialphosphorylation, i.e. whether the phosphorylation ofSer-302 is a prerequisite for the phosphorylation of

2730 Mol Endocrinol, December 2008, 22(12):2729–2740 Weigert et al. • Serine Phosphorylations of IRS-1



Ser-318 and subsequently, whether phosphorylationof Ser-318 regulates the phosphorylation of Ser-307.

First we studied whether Ser-302 phosphorylationtriggers phosphorylation of Ser-318. The IRS-1 A302/307

mutant simulates the absence of phospho-Ser-302and phospho-Ser-307. We used a double mutant, be-cause in this early phase of insulin signaling Ser-307 isnot phosphorylated (Fig. 1), and expression of the IRS-1A307 single mutant had only a marginal effect on thephosphorylation of Ser-318 (data not shown). In C2C12cells expressing IRS-1 A302/307 mutant, no insulin-in-duced phosphorylation on Ser-318 was observed after 5min of stimulation (Fig. 2A). These results suggest thatthe phosphorylation of Ser-302 may be involved in thesubsequent rapid phosphorylation of Ser-318.

Recently we have shown that phosphorylation ofSer-318 is mediated, at least in part, by PKC-� andthat insulin induces the association of IRS-1 andPKC-� in a time-dependent manner (42). The IRS-1/PKC-�-association is found after 5 min of stimulationbut is lost after 10 and 60 min (42). Therefore, wespeculated that phosphorylation of Ser-302 enhancesthis interaction. Using C2C12 cells expressing IRS-1E302A307A318, which simulates the basal phosphoryla-

tion state with the phosphorylation only on Ser-302(Fig. 1F), we observed a significant increase in theassociation of PKC-� to this IRS-1 mutant after 5 minof insulin stimulation (Fig. 2B). Because we have pre-viously shown that expression of the IRS-1 A318 singlemutant does not change the association to PKC-�compared with IRS-1 wild-type (wt) expressing cells(42), we concluded that the imitation of a permanentSer-302 phosphorylation could lead to an increased in-teraction of PKC-� with IRS-1. Moreover, mutation ofSer-302 to alanine reduced both basal and insulin-stim-ulated PKC-�/IRS-1 association (Fig. 2C). These datasuggest a potential role of phospho-Ser-302 in the sub-sequent PKC-�-mediated phosphorylation of IRS-1.

PKC-�, mTOR/Raptor-Dependent Kinase, andJNK Are Involved in the Insulin-Induced,Sequential Phosphorylation of Ser-302, -307,and -318

The results shown in Fig. 2 suggest a relevant role ofthe initial phosphorylation of Ser-302 for the binding ofPKC-� and the subsequent PKC-�-mediated phos-phorylation of Ser-318. However, the ongoing phos-

Fig. 1. Kinetics of Insulin-Induced Serine Phosphorylation of IRS-1A, Immunoblot analysis of phosphorylation of Ser-302, -307, -318, and IRS-1 protein in myotube cell extracts (Ser-307 and

-318) and of immunoprecipitated IRS-1 (Ser-302) after stimulation with 10 nM insulin for 0–120 min as indicated. Due to the lowersensitivity of the anti-phospho-Ser-302 antibody, immunoprecipitation of IRS-1 was performed to study this phosphorylation. B,Densitometric quantification of the immunoblots (n � 4, mean � SEM). Relative phosphorylation of the indicated serine comparedwith maximal insulin-stimulated phosphorylation of this site set as 100%. C, Representative immunoblot of the phosphorylationof Ser-307, -318, and IRS-1 protein of immunoprecipitated IRS-1 after stimulation with 10 nM insulin for 0–120 min as indicated.D, Immunoblot analysis of tyrosine phosphorylation of immunoprecipitated IRS-1 and (E) densitometric quantification (n � 5;mean � SEM). Values of unstimulated cells were set as 1. F. Scheme of the serine phosphorylation pattern induced by insulin andthe corresponding IRS-1 mutants used for the following studies. Ins, Insulin; IP, immunoprecipitation.

Weigert et al. • Serine Phosphorylations of IRS-1 Mol Endocrinol, December 2008, 22(12):2729–2740 2731



phorylation of Ser-318 after 10 and 60 min of insulinstimulation seems not to depend on the phosphoryla-tion at Ser-302 (Fig. 2A), which may be a hint that morethan one kinase is involved in the phosphorylation ofSer-318. We followed this aspect using small interfer-ing RNA (siRNA) approaches in C2C12 cells. First weknocked down PKC-�/� (the atypical PKC isoformsexpressed in mice; shortly referred to as PKC-�). In-terestingly, the phosphorylation of Ser-318 after 5 minof insulin stimulation was completely prevented andtended to be reduced after 10 min (P � 0.06), but wasnot impaired after 60 min (Fig. 3A), indicating thatPKC-� is responsible for the early, but not for theongoing, insulin-induced phosphorylation of Ser-318.Because the lasting phosphorylation state of the stud-ied IRS-1 region is dominated by Ser-307 and Ser-318(Fig. 1) and the insulin-mediated phosphorylation ofSer-307 is mediated by JNK and mTOR (12, 13, 15),we first studied the potential role of these kinases inthe phosphorylation of Ser-318.

First we performed experiments using SP 600125,an inhibitor of JNK, and rapamycin, an inhibitor of thekinase mTOR (Fig. 3, B and C). We could not detect aparticipation of JNK in the insulin-induced Ser-318phosphorylation in C2C12 cells at any time point stud-ied (Fig. 3B). The effectiveness of the JNK inhibitorwas indicated by the complete inhibition of the insulin-induced phosphorylation of Ser-307 and the reductionof the phosphorylation of the JNK substrate c-Jun (Fig.3C). The use of rapamycin, shown in Fig. 3B, led to acomplete inhibition of the phosphorylation of Ser-318.After that, we performed knock-down experiments ofJNK1/2 as well as raptor using siRNA oligonucleotides(Fig. 3D). Knockdown of JNK1 and JNK2 could notprevent the phosphorylation of Ser-318 (Fig. 3D). Con-trary to that, the knockdown of raptor prevented thephosphorylation of Ser-318 (Fig. 3D). Therefore wespeculated whether a mTOR/raptor-dependent kinaseis involved in the phosphorylation of Ser-318 at latertime points, because both treatment of cells with rapa-

Fig. 2. Role of Phospho-Ser-302 in the Subsequent Ser-318 PhosphorylationA, C2C12 cells transfected with empty vector (con), IRS-1 wt, or IRS-1 A302/307 were stimulated with 10 nM insulin as indicated.

A representative immunoblot for phospho-Ser-318 and the reprobe for IRS-1 are shown. In the lower part, densitometricquantification of the immunoblots is shown (n � 4; mean � SEM); *, P � 0.05 compared with IRS-1 wt transfected cells stimulatedwith insulin for 5 min. (B and C) C2C12 cells transfected with IRS-1 wt or IRS-1 E302A307A318 (B) and IRS-1 wt or IRS-1 A302 (C)were stimulated with 10 nM insulin for 5 min. PKC-�/� was immunoprecipitated and coprecipitated IRS-1 was visualized byimmunoblotting. IRS-1 protein expression in the extracts is shown in the upper panel. The lower part of the figure shows thedensitometric quantification (n � 5; mean � SEM); *, P � 0.05 vs. IRS-1 wt transfected cells stimulated with insulin for 5 min; #,P � 0.05 vs. unstimulated IRS-1 wt transfected cells. con, Control; IB, immunoblot; ins, insulin; IP, immunoprecipitation; rel.,relative.




mycin and knockdown of raptor using siRNA oligonu-cleotides prevented the phosphorylation of Ser-318 atall time points studied (Fig. 3, B and D). The data alsosuggest that a mTOR/raptor-dependent pathway may

be relevant to initiate the early phosphorylation of Ser-318. Because recent reports showed a mTOR path-way-dependent phosphorylation of Ser-302 upon in-sulin stimulation (11, 39), we addressed this issue inour C2C12 skeletal muscle cell culture model usingrapamycin to inhibit the phosphorylation of Ser-302,but no effect could be detected (data not shown).

The Subsequent Phosphorylation of Ser-307 IsIndependent of the Precursory Phosphorylationof Ser-302 and -318

Because phosphorylation of Ser-302 and -318 pre-ceded the phosphorylation of Ser-307 we investigatedthe potential effect of both sites on the later phosphor-ylation in C2C12 cells. The mutation of both serineresidues had no effect on the insulin-induced phosphor-ylation of Ser-307 (Fig. 4A). Furthermore, based on thekinetics study showing an early decrease of phospho-Ser-302 (Fig. 1), we investigated the effect of phosphor-ylation of Ser-318 simulated by the E318 mutation. How-ever, the expression of this mutant did not change thephosphorylation of Ser-307 (Fig. 4B). Because theknock-down of PKC-�, which prevents the early phos-phorylation of Ser-318 (Fig. 2A) had also no effect on theinsulin-induced phosphorylation of Ser-307 (data not

Fig. 3. Insulin-Induced Phosphorylation of Ser-318 is FirstMediated by PKC-� and Later on by a Raptor-DependentPathway

A, C2C12 cells transfected with siRNA oligonucleotidestargeting PKC-�/� were stimulated with 10 nM insulin as in-dicated. Shown are representative immunoblots for phos-pho-Ser-318 and PKC-�/�. Membranes were reprobed forIRS-1 protein. In the lower part, densitometric quantificationof the immunoblots is shown (n � 4, mean � SEM); *, P � 0.05compared with control transfected cells stimulated with in-sulin for 5 min. B and C, C2C12 cells were preincubated with10 �M JNK inhibitor (JNKI) SP 600125 (B and C) or 25 nM

rapamayin (B) for 30 min before stimulation with 10 nM insulin.Representative immunoblots for phospho-Ser-318 and thereprobe for IRS-1 (B), phospho-Ser-307, phospho-Ser-73,and the corresponding reprobes for IRS-1 and c-Jun protein(C) are shown. D, C2C12 cells transfected with siRNA oligo-nucleotides targeting JNK1 and -2 or raptor were stimulatedwith 10 nM insulin as indicated. Shown are representativeimmunoblots for phospho-Ser-318, JNK1 and -2, and raptor.Lanes from each blot shown here are from one gel but havebeen loaded in a different order. con, Control; si-, smallinterfering; ins, insulin.

Fig. 4. Phosphorylation of Ser-318 Is Not Necessary forPhosphorylation of Ser-307

A and B, C2C12 cells transfected with IRS-1 wt, IRS-1A302/318, or IRS-1 E318 were stimulated with 10 nM insulin asindicated. Shown are representative immunoblots for phos-pho-Ser-307 and the corresponding reprobe for IRS-1 pro-tein and in the lower part the densitometric quantification(n � 4, mean � SEM). ins, Insulin; rel., relative.




shown), we suggest that the combined phosphorylationof Ser-302/-318, as well as phosphor-Ser-318 alone, areno prerequisite for the phosphorylation of Ser-307; i.e.Ser-307 phosphorylation is independently regulated.

The Early Phosphorylation of Ser-302and Ser-318 Enhances Insulin Action

The studies on the insulin-stimulated kinetics of thephosphorylation of the three serine residues revealedan early phosphorylation pattern after 5 min of stimu-lation, including phosphorylation of Ser-302 and -318(simulated with IRS-1 E302A307E318), and a late phos-phorylation pattern including phosphorylation of Ser-307 and Ser-318 (simulated with IRS-1 A302E307E318)(Fig. 1F). The kinetic of the tyrosine phosphorylation ofIRS-1 suggests an involvement of the early phosphor-ylation of Ser-302/-318 in the enhancement of insulinsignaling (Fig. 1, D and E). Expression of IRS-1E302A307E318 to investigate the function of phos-phoSer-302/-318 on insulin signal transduction led toa considerable increase in the phosphorylation of Ser-473 of Akt after 10 min of insulin stimulation in C2C12cells, indicating enhanced activation of this kinase(Fig. 5, A and B). The phosphorylation of the down-stream kinase GSK-3 was also increased (Fig. 5A). Ofnote, the reduction of Akt phosphorylation after 60 minof insulin treatment was not significantly different fromIRS-1 wt transfected cells, indicating the precedingrole of phospho-Ser-302/-318 in the activation of thesignaling with no detectable influence on the termina-tion of insulin action (Fig. 5B). In contrast, C2C12 cellsexpressing the IRS-1 A302E307E318 mutant, which rep-resents the phosphorylation state of this IRS-1 regionin the ongoing insulin action, exhibit a slightly reducedphosphorylation of Akt and GSK-3 after 10 min ofinsulin stimulation. Thus, the early phosphorylation ofIRS-1 on Ser-302 and Ser-318 enhances insulin sig-naling, and this effect is absent when Ser-307 insteadof Ser-302 is phosphorylated. The effect on insulin-stimulated Akt phosphorylation was further studiedusing the single-alanine mutants IRS-1 A302, A307, andA318 (Fig. 5, C and D). The complete inhibition ofSer-307 phosphorylation using IRS-1 A307 led to asignificant increase in Akt phosphorylation at all timepoints studied, underlining the important inhibitoryfunction of this site. Expression of IRS-1 A318 reducedthe Akt phosphorylation after 5 min of insulin stimula-tion, demonstrating the positive function of phospho-

Fig. 5. The Early Phosphorylation Pattern Enhances InsulinAction

A, C2C12 cells were transfected with IRS-1 wt, IRS-1E302A307E318, or IRS-1 A302E307E318 and stimulated with 10nM insulin as indicated. Cell extracts were immunoblotted forphospho-Ser-473 of Akt, phospho-Ser-9/21 of GSK-3, andIRS-1. Membranes were reprobed for Akt and GSK-3 protein.Only phosphorylation of Ser-21 of GSK-3� was detected,whereas both GSK-3� and -� were expressed (data notshown). B, Densitometric quantification of the phosphoryla-tion of Ser-473 of Akt (n � 4, mean � SEM); *, P � 0.05 IRS-1E302A307E318 vs. IRS-1 wt; #, P � 0.05 IRS-1 A302E307E318 vs.IRS-1 wt after 10 min of insulin stimulation. C, C2C12 cellswere transfected with IRS-1 wt, IRS-1 A302, A307, or A318 andstimulated with 10 nM insulin as indicated. Cell extractswere immunoblotted for phospho-Ser-473 of Akt and IRS-1.

Membranes were reprobed for Akt protein. D, Densitometricquantification of the phosphorylation of Ser-473 of Akt (n � 4,mean � SEM); *, P � 0.05 IRS-1 A307 vs. IRS-1 wt; #, P � 0.05IRS-1 A318 vs. IRS-1 wt after 5, 10, or 60 min of insulinstimulation. E, Immunoprecipitation of IRS-1 wt transfectedC2C12 cells using immunopurified anti-phospho-Ser-318 an-tibodies. Immunoprecipitates were immunoblotted for phos-pho-Ser-307 and IRS-1. IB, Immunoblot; ins, insulin; IP,immunoprecipitation.




Ser-318 on acute insulin action. This has also beenreported in a recent study (42). The expression of thesingle Ala-302 mutant had no significant effect oninsulin-stimulated Akt phosphorylation.

From these data with the single-alanine mutants itwas somehow unexpected that expression of theIRS-1 A302E307E318 mutant did not result in a strongdown-regulation of insulin action. Earlier studies onthe function of the single phosphorylation of eachresidue clearly implicate the involvement of Ser-307and -318 in the attenuation of insulin action performedby loss of function studies using serine to alaninemutants (12, 13, 16, 19, 42, 51). This finding raises thequestion of whether both sites may occur in the phos-phorylated state on the same molecule. This crucial pointwas investigated in a next step by the immunoprecipita-tion of the fraction of IRS-1, which is phosphorylated onSer-318 upon insulin stimulation. Applying an immuno-purified phospho-Ser-318 antibody we detected com-parable amounts of IRS-1 after stimulation with insulinfor 5, 10, and 60 min, but not in unstimulated cells (Fig.5E), as expected from the kinetics shown in Fig. 1B.Moreover, we also found the time-dependent phosphor-ylation of Ser-307 in this immunoprecipitated, i.e. Ser-318 phosphorylated IRS-1 (Fig. 5E).

The Early Serine Phosphorylation of IRS-1Enhances Glucose Uptake

The regulation of insulin action was further investi-gated as insulin-stimulated glucose uptake in L6 myo-blasts expressing the IRS-1 triple mutants after 5 minof insulin stimulation (Fig. 6A). Simulating the earlySer-302/-318 phosphorylation by the E302A307E318

mutant led to an increased insulin-dependent glucoseuptake. Of note, in the unstimulated state the glucoseuptake was also significantly enhanced in cells ex-pressing the E302A307E318 mutant. Expression of IRS-1A302E307E318 did not reduce glucose uptake in com-parison with IRS-1 wt expressing cells (Fig. 6A). Thiscould indicate that the phosphorylation pattern has noinfluence on the acute insulin-stimulated glucose up-take. We also studied the effect of the single-alaninemutants IRS-1 A302, A307, and A318 (Fig. 6B). We ob-served a significant reduction of glucose uptake inA302 and A318 expressing cells after 5 min of insulinstimulation. These data support the positive functionof phospho-Ser-302 and phospho-Ser-318 in earlyinsulin action. Moreover, expression of IRS-1 E302 in-creased basal and insulin-stimulated glucose uptake(Fig. 6C), similar to the results obtained in IRS-1E302A307E318 expressing cells (Fig. 6A). No difference inthe insulin-stimulated glucose uptake was detected inIRS-1 A307 expressing cells in comparison with IRS-1 wt(Fig. 6B), despite its pronounced effect on Akt phosphor-ylation (Fig. 5, C and D). Because A307 represents theunphosphorylated Ser-307 residue, and phosphorylationof Ser-307 was almost absent after 5 min of insulinstimulation (Fig. 1), this result suggests that phosphory-

lation of Ser-307 is not involved in the early regulation ofinsulin-dependent glucose uptake.

DISCUSSION

During the last decade more than 20 serine/threonineresidues in IRS-1 have been identified as functionallyrelevant phosphorylation sites. Data from 16 sites fitwith the model of a generalized mechanism for re-duced insulin action responsible for the induction of

Fig. 6. The Early Serine Phosphorylation of IRS-1 EnhancesGlucose Uptake

A, Glucose uptake in L6 Glut4myc cells transfected withIRS-1 wt, IRS-1 E302A307E318, or IRS-1 A302E307E318 after 5min of insulin stimulation. Basal glucose uptake of IRS-1 wttransfected cells was set as 100%. *, P � 0.05 vs. IRS-1 wt.B, Glucose uptake after 5 min of insulin stimulation in L6Glut4myc cells transfected with IRS-1 wt, IRS-1 A302, IRS-1A307, and IRS-1 A318; *, P � 0.05 A302 and A318 vs. insulin-stimulated IRS-1 wt. C, Glucose uptake in L6 Glut4myc cellstransfected with IRS-1 wt, IRS-1 A302, and IRS-1 E302 after 5min of insulin stimulation. *, P � 0.05 A302 vs. basal orinsulin-stimulated IRS-1 wt; #, P � 0.05 E302 vs. basal orinsulin-stimulated IRS-1 wt (n � 4, mean � SEM). ins, Insulin.




insulin resistance (8–17,19–32). In contrast, the phos-phorylation of just three serines of IRS-1, namely Ser-325 (44), -629 (not present in rodents) (45), and -1216(48), had recently been associated with improved in-sulin signaling. Of note, for an additional three sites(Ser-302, -318, and -789) inconsistent data of stimu-latory as well as inhibitory functions on the insulinaction have been reported (11, 19, 29, 30, 39–43, 46).These reports prompted us to speculate that, depend-ing on the duration of insulin stimulation, temporallydifferent phosphorylation statuses of the same resi-dues in defined regions of IRS-1 might exist, whichexert different functions on signal transduction. Toprove this hypothesis, we investigated the cross talk ofthe insulin-dependent, temporal phosphorylation ofpotentially opposedly acting serine residues of IRS-1in skeletal muscle cells and the implications for insulinsignal transduction, by selecting Ser-307 as a negativeacting site and the adjacent Ser-302 and -318.

Applying phosphorylation kinetic studies, loss offunction, gain of function, and combination mutants ofIRS-1, we demonstrated that each site exhibits a char-acteristic phosphorylation kinetics, that a sequential,interdependent phosphorylation exists, and that theinterplay rather than the phosphorylation status of sin-gle-serine residues regulates insulin action. Phosphor-ylation of Ser-302 and -318 was found to be an earlyevent and was accompanied by tyrosine phosphory-lation of IRS-1. At later time points, coincident withreduction in tyrosine phosphorylation, phosphoryla-tion of Ser-307 and -318 was maximal.

The controversial data on the regulation of insulinsignaling by phosphorylated Ser-302 and -318, dem-onstrating positive (39–42) as well as negative effects(11, 19, 20) could be reconciled. We found that therapid phosphorylation of Ser-302 is an initial event andappeared to be present to some extent even withoutinsulin stimulation. Following a maximum after 10 minof insulin stimulation, which is similar to recent reportsachieved in 32D cells (39) and primary human adipo-cytes (52), the intensity of the Ser-302 phosphorylationdecreased during the ongoing insulin action belowbaseline values. Moreover, the data suggest a poten-tial role of phospho-Ser-302 in the rapid association ofinsulin-activated PKC-� with IRS-1, which, in turn,phosphorylated Ser-318. The positive function ofphosphor-Ser-302/-318 on insulin signal transductionwas reflected by a considerable increase in the phos-phorylation of Ser-473 of Akt after 10 min of insulinaction, indicating enhanced activation of this kinaseparalleled by a decrease in the activation of the inhib-itory downstream kinase GSK-3. The preceding role ofphosphor-Ser-302/-318 in the activation of insulin sig-naling was underlined further by a similar reduction ofAkt phosphorylation after 60 min of insulin treatmentirrespectively if the cells were transfected with IRS-1wt or the E302A307E318 mutant, which simulates thepermanent phosphorylation of Ser-302 and Ser-318.The investigation of the insulin-stimulated glucose up-take resulted in additional evidence for the positive

action of the combined phospho-Ser-302/-318. Ofnote, in the unstimulated state the glucose uptake wasalso significantly enhanced simulating the early Ser-302 or Ser-318 phosphorylation by the expression ofthe E302 or the E302A307E318 mutant in comparisonwith cells expressing IRS-1 wt.

Several observations implicated a possible role ofSer-302 phosphorylation in the subsequent phosphor-ylation of Ser-318. When the phosphorylation of Ser-302 was prevented by the substitution with alanine,the early, PKC-�-dependent phosphorylation of Ser-318 was blocked. Furthermore, the association ofIRS-1 and PKC-� was increased when simulating thephosphorylation of Ser-302 by mutation to glutamateand reduced by mutation to alanine. The rapid phos-phorylation of Ser-302 and Ser-318 within minutesafter the addition of insulin and the activation of down-stream signaling as well as glucose uptake suggestedthe involvement of these sites in a positive modulationof insulin action, underlining that serine phosphoryla-tion of IRS-1 is not only a major mechanism for theattenuation of insulin action (8–17, 19–32), but mightalso be important for a maximum positive modulationof signal transduction in addition to the well-knowneffect of tyrosine phosphorylation.

The participation of PKC-� in this early phosphory-lation event provides further evidence for a predomi-nantly positive function of atypical PKC isoforms in thephysiological process of insulin signal transduction.These kinases are activated by insulin (53, 54) and areimportant for insulin-stimulated glucose uptake (53,55, 56). Their positive role in insulin action is furthersupported by the impaired activation of atypical PKCisoforms in obese humans and type 2 diabetics (56–59). However, atypical PKCs appeared also to be in-volved in the self-attenuation of insulin signaling viaserine phosphorylation of IRS-1 (35, 60). Seven serineresidues proximal to the phosphotyrosine binding do-main including Ser-302, but not Ser-318 or Ser-307,could participate in this mechanism, because a seven-alanine mutant of IRS-1 confers protection from thepalmitic acid-induced inhibitory effects of PKC-�, (23,35). Because we have observed a dissociation ofPKC-� and IRS-1 after 10 min of insulin stimulation(42), an indirect negative feedback loop could be re-sponsible for the attenuation of insulin signaling, e.g.via the recently postulated PKC-�-dependent activa-tion of inhibitor of NF �B kinase (35).

Whereas Ser-302 was dephosphorylated by unde-fined phosphatases in the ongoing insulin action, thephosphorylation of Ser-318 was maintained, but notby PKC-�, as clearly demonstrated in this study. Thus,one residue could be temporally and sequentiallyphosphorylated by different kinases during the actionof the same biological stimulus. The phosphorylationof Ser-318 by insulin-stimulated kinase activity is ini-tially mediated by PKC-� followed within minutes by akinase of the mTOR/raptor-dependent pathway. Thefollowing insulin-induced JNK-mediated phosphoryla-tion of Ser-307 appeared not dependent on the phos-




phorylation of Ser-318, because the phosphorylationof Ser-307 was neither prevented by knockdown ofPKC-�, which abrogated the early phosphorylation ofSer-318, nor did the substitution with glutamate atposition 318 increase the phosphorylation of Ser-307.Moreover, mutation of Ser-302 and -318 to alaninealso had no effect on this phosphorylation. In a previ-ous report it has been suggested that the phosphor-ylation of Ser-307 could be required for efficient phos-phorylation of Ser-302 (11). Based on the findings ofthe present study, this interdependency appears un-likely, at least in skeletal muscle cells, during insulinstimulation, because phosphorylation of Ser-307 didnot precede the phosphorylation of Ser-302.

Whereas the simultaneous early phosphorylation atSer-302 and Ser-318 had a pronounced positive ef-fect, the influence of the late phosphorylation at Ser-307 together with Ser-318 on insulin signaling waslow, and no difference was found compared with wtIRS-1 in insulin-stimulated glucose uptake. This issomewhat surprising because both phosphorylation ofSer-307 and Ser-318 have been clearly implicated inthe attenuation of insulin signaling (12–20). In mostreports on the negative effects of single phospho-Ser-307 and phospho-Ser-318, data were obtained fromstudies using loss of function mutants (substitutionwith alanine), i.e. the simulation of an unphosphory-lated state at this site resulted in the detection of apositive effect on insulin signal transduction. When westudied the effect of the single-alanine mutants, wealso observed positive effects on insulin-stimulatedAkt phosphorylation using IRS-1 A307. Expression ofIRS-1 A318 resulted in reduced insulin action to anearly time point, whereas the attenuation of insulinaction after 60 min was abrogated as reported earlier(42). To rule out the possibility that the phosphoryla-tion of Ser-307 and Ser-318, which is simulated by theA302E307E318 mutant, does not occur in living cells andthis may cause the missing negative influence on sig-nal transduction, we investigated the phosphorylationof Ser-307 in the phospho-Ser-318-immunoprecipi-tated fraction of IRS-1. These data clearly indicatedthat Ser-307 and Ser-318 could be phosphorylated onthe same IRS-1 molecule in skeletal muscle cells. An-other possible explanation for the weak inhibitory ef-fect seen in this study is that in most studies reportingnegative effects of these sites the cells were stimu-lated only with insulin, but pretreated with other acti-vators of serine kinases such as TNF-� (12, 61), ani-somycin (51), phorbol ester (19), or fatty acids (33),which clearly induces other signaling pathways anddifferent phosphorylation patterns of signaling mole-cules including IRS-1 than stimulation with insulinalone. Therefore, we suggest that the phosphorylationof further serine sites of IRS-1 is required for the self-attenuation of insulin signaling.

Taken together, it is clearly the entire serine phos-phorylation pattern of IRS-1 that influences insulin sig-naling and not the individual action of one phosphor-ylated serine residue. However, it can only be

speculated whether the positive contribution to insulinaction is mediated by only a few serine residues andthe opposite negative function needs a complex pat-tern to down-regulate insulin signaling. Possibly, thesimultaneous phosphorylation of Ser-302 and -318leads to more significant changes in the structure ofIRS-1 than combined phosphorylation of Ser-307 and-318, but this can only be a hypothetical considerationso far because the reported crystal structure of anamino-terminal IRS-1 fragment encompasses only thepleckstrin homology and phosphotyrosine binding do-main (amino acid residues 4–271) (62), whereas theserine residues investigated in the present study arelocated distal to this region.

Taken together, our study indicates that, dependenton its duration, insulin stimulation generates differentphosphorylation patterns in defined regions of IRS-1,which differentially regulate signal transduction.

MATERIALS AND METHODS

Materials

C2C12 cells were from American Type Culture Collection(ATCC, Manassas, VA). Parental L6 cells and L6 GLUT4myccells were kindly provided from A. Klip (Toronto, Ontario,Canada). Cell culture media and supplements were fromCambrex (Verviers, Belgium). Oligonucleotides were synthe-sized by Life Technologies (Karlsruhe, Germany). Phospha-tase inhibitors and rapamycin were obtained from Sigma(Munich, Germany). The following antibodies were used:phospho-Akt Ser-473, phospho-IRS-1 Ser-302 (Cell Signal-ing Technology, Frankfurt, Germany); Akt, JNK (BD Bio-sciences, Inc., San Diego, CA); IRS-1 (06-248), phospho-IRS-1 Ser-307 (07-247), phosphotyrosine (4G10), phospho-c-Jun Ser-73 (06-659) (Upstate Biotechnology, Inc., LakePlacid, NY); PKC-�/� (sc-216), c-Jun (sc-1694) (Santa CruzBiotechnology, Inc., Santa Cruz, CA). Polyclonal anti-p-Ser-318 antiserum was raised against a synthetic peptide(SMVGGKPGpSFRVRASSD) flanking Ser-318 in IRS-1,which is conserved among mouse, rat, and human sequence.For immunoprecipitation, this antiserum was purified by atwo-step immunoaffinity chromatography. In the first step,antiserum was applied to an affinity column containing theunphosphorylated peptide. In the second step, the eluatewas purified using the phosphorylated peptide coupled to anaffinity column.

The cytomegalovirus promoter-based expression vectorsfor rat IRS-1 and human PKC-� are described in Ref. 63. 2-[3H(G)]-deoxy-D-glucose (SA 185–370 gBq/mmol) was fromPerkinElmer (Zaventem, Belgium).

Site-Directed Mutagenesis

Mutation of serine sites of rat IRS-1 to alanine or glutamate wasmade by oligonucleotide-mediated mutagenesis as describedelsewhere(42) or generated using the Stratagene QuikChangeXL Site-Directed Mutagenesis method (Stratagene, La Jolla,CA). Positive clones were verified by sequencing.

Cell Culture

For most transfection experiments, C2C12 myoblasts wereused because we observed higher expression levels, bettertransfection efficiencies, and no toxic effects from the trans-




fection reagents in these cells. L6 Glut4myc myoblasts wereused for determination of glucose uptake, because C2C12cells showed almost no insulin-dependent glucose uptake.C2C12 cells were cultured in DMEM containing 25 mM glu-cose, 10% fetal calf serum (FCS), 2 mM glutamine; 1 � 105

cells per well of a six-well plate were transfected using theCa3(PO4)2-DNA-coprecipitation-method (64). Forty hours af-ter transfection, the cells were starved in DMEM (5.5 mM

glucose) without FCS for 3 h and stimulated as indicated. L6cells were cultured in �-MEM containing 5.5 mM glucose,10% FCS, 100 U/ml penicillin, and 100 �g/ml streptomycinas described (65). For differentiation, 0.5 � 104 cells/cm2 L6cells were seeded in differentiation medium (�-MEM contain-ing 5.5 mM glucose, 2% FCS, 100 U/ml penicillin, and 100�g/ml streptomycin). Myotubes were used 7–8 d after cellseeding.

Cell Lysis, Immunoprecipitation, and WesternBlot Analysis

Cells were lysed with 200 �l of lysis buffer/well (50 mM

HEPES, pH 7.5; 150 mM NaCl; 1.5 mM MgCl2; 1 mM EDTA;10% glycerin; 1% Triton X-100; containing protease andphosphatase inhibitors). Total protein (400 �g) was used forimmunoprecipitation. Immunoprecipitated proteins or 40 �gprotein of the total extracts were separated by SDS-PAGE(7.5%), and Western blot analysis was performed as de-scribed elsewhere (43).

2-Deoxyglucose Uptake

L6 Glut4myc myoblasts seeded in 12-well plates were trans-fected with the IRS-1 mutants using lipofectamine 2000(Karlsruhe, Germany). Cells were serum-starved, 48 h aftertransfection, for 3 h in �-MEM before the experiments. Afterstimulation with 100 nM insulin, cells were washed withHEPES-buffered saline (containing 140 mM NaCl; 20 mM

HEPES-Na, pH 7.4; 5 mM KCl; 2.5 mM MgSO4; 1 mM CaCl2)and 300 �l/well of 2-deoxyglucose mix (HEPES-buffered sa-line containing 0.25 �Ci/ml [3H]2-deoxyglucose and 10 �M

2-deoxyglucose) was added. After incubation at 37 C, 5%CO2 for 7 min, cells were washed three times with 0.9% NaCland lysed with 250 �l of lysis buffer. Total amounts of celllysates were counted by liquid scintillation counting. Proteincontent of lysates generally varied less than 10%.

siRNA

siRNA oligonucleotides targeting mouse PKC-� (NM_008857),PKC-� (NM_008860), JNK1 (NM_016700), and JNK2 (NM_016961)were designed, synthesized, and annealed at Dharmacon Re-search (Lafayette, CO). siRNA oligonucleotides targeting mouseraptor (NM_028898) were from QIAGEN (Hilden, Germany). Anunrelated siRNA targeting firefly luciferase was used as controlin all experiments. Transfection was performed with CellPhect(Amersham Biosciences, Buckinghamshire, UK) with 50 nM

siRNA according to the instructions of the manufacturer.Briefly, 1 � 105 cells per well were seeded in six-well platesand transfected in DMEM containing 25 mM glucose, 10%FCS without antibiotics. Cells were stimulated as indicated24 h after the glycerol shock.

Statistical Analysis

Results presented are derived from at least four independentexperiments. Means � SEM values were calculated, andgroups of data were compared using Student�s t test. Sta-tistical significance was set at P � 0.05.

Acknowledgments

We thank Andreas Dittmar for excellent technical support.

Received March 28, 2008. Accepted October 10, 2008.Address all correspondence and requests for reprints to:

Rainer Lehmann, Division of Clinical Chemistry and Pathobio-chemistry, Department of Internal Medicine 4, University Hos-pital of Tubingen, Hoppe-Seyler-Strasse 3, D-72076 Tubingen,Germany. E-mail: [email protected].

This work was supported by Grant P-LS-Prot/29 fromLandesstiftung Baden-Wuerttemberg (to R.L.), Grant GRK1302/1 from the Deutsche Forschungsgemeinschaft (to E.S.),and a grant from the Deutsche Diabetes Gesellschaft (toR.L.).

Disclosure Statement: The authors have nothing todisclose.

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