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Department of Clinical Biochemistry (R.N., C.H.J., K.S., J.K.S.), University of Cambridge, Addenbrooke’s Hospital, Cambridge CB2 2QR, United Kingdom; and School of Life Sciences (A.G., C.P.D.), University of Dundee, Dundee DD1 5EH, United Kingdom
Address all correspondence and requests for reprints to: Jaswinder K. Sethi, Department of Clinical Biochemistry, University of Cambridge, Addenbrooke’s Hospital, Box 232, Hills Road, Cambridge CB2 2QR, United Kingdom. E-mail: jks30{at}cam.ac.uk.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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. By comparing the pharmacological characteristics of phorbol 12-myristate 13-acetate-stimulated Ser24 phosphorylation with phosphorylation at two other sites previously linked to PKC activity (Ser307 and Ser612), we show that PKC is likely to be directly involved in Ser24 phosphorylation, but indirectly involved in Ser307 and Ser612 phosphorylation. Using Ser24Asp IRS-1 mutants to mimic the phosphorylated residue, we demonstrate that the phosphorylation status of Ser24 does play an important role in regulating phosphoinositide binding to, and the intracellular localization of, the IRS1-PH domain, which can ultimately impinge on insulin-stimulated glucose uptake. Hence we provide evidence that IRS1-PH domain function is important for normal insulin signaling and is regulated by serine phosphorylation in a manner that could contribute to insulin resistance. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Uncoupling of insulin signaling can occur at numerous points along the pathway and by numerous mechanisms such as decreased expression and/or activity of key signaling components (1). In particular, insulin receptor substrates (IRS) have been implicated as major targets in insulin-resistant states (2). The IRS family members are defined by a characteristic tandem arrangement of N-terminal pleckstrin homology (PH) and phosphotyrosine-binding (PTB) domains. Both these domains are well conserved, particularly between IRS1 and IRS2, and are required for recruitment to, and association with, the insulin receptor (IR) (3, 4). The remainder of the proteins are poorly conserved across family members but do contain key receptor tyrosine kinase phosphorylation- and Src-homology (SH2) domain-binding sites. IRS family members play a pivotal role in transmitting insulin signals and are absolutely required for normal insulin-stimulated glucose uptake. Indeed, mice lacking either IRS1 or IRS2 exhibit peripheral insulin resistance and reduced neonatal growth (5). Despite the similarities between IRS1 and IRS2, some functional differences have also been reported. Whereas IRS2 is crucial for ß-cell growth and function, IRS1 is important in regulating metabolism in muscle and adipose tissue (5). Reductions in IRS1 protein levels and/or active tyrosine-phosphorylated IRS1 in these tissues are well documented in models of insulin resistance. Inducers of insulin resistance, such as FFA and TNF, can also cause posttranslational changes in IRS1, as can activators of serine kinases and/or inhibitors of serine phosphatases (1, 5, 6). Indeed, serine phosphorylation of IRS1 is currently the best-substantiated post-translational modification of IRS proteins, in addition to tyrosine phosphorylation.
Many serine kinases have now been implicated in mediating serine phosphorylation of IRS1 either directly or indirectly. These include casein kinase II, protein kinase C (PKC), PKCßI/II, PKC
, PKC
, PKC
, PKC
, mitogen-activated Erk kinase (MEK), p38MAPK, cJun NH2-terminal kinase (JNK), cRafK (Raf-1 kinase or v-raf-leukemia viral oncogene 1), PI3K (phosphatidylinositol 3-kinase), AKT/PKB (AKR mouse thymoma viral proto-oncogene/protein kinase B), glycogen synthase kinase (GSK)3ß, mammalian target of rapamycin (mTOR), inhibitor of 
B kinase ß (IKKß), salt-inducible kinase 2, AMP-activated protein kinase, and Rho-dependent kinase (reviewed in Refs. 1 , 6 , and 7). Most of these kinases appear to negatively regulate insulin signaling, although a few may play a role in positive feedback regulation (8). There are, however, some serine kinases, such as casein kinase II, whose role in insulin signaling remains to be defined.
The mechanistic basis by which serine phosphorylation may uncouple proximal insulin signaling is beginning to be revealed by mapping the specific residues that are targeted by these serine kinases (1). Recent estimates suggest that IRS1 may contain more than 70 potential serine/threonine phosphorylation sites. However, only 16 have been demonstrated to become phosphorylated in response to agonist stimulation in vivo (9). Most of these are located proximal to/or downstream of the PTB domain. Phosphorylation of sites that lie close to the PTB domain (e.g. Ser307) can disrupt IR-IRS interactions, thereby reducing tyrosine phosphorylation of IRS1 (7, 9, 10). However, serine phosphorylation in this region can also disrupt IRS-protein tyrosine phosphatase interactions, leading to sustained tyrosine phosphorylation of IRS1 (8). Further downstream, residues such as Ser612, Ser632, Ser662, and Ser570 occur near Src-homology 2-binding motifs where they can prevent tyrosine phosphorylation and/or p85 recruitment (11, 12). Some serine phosphorylation sites have been reported to impinge on intracellular trafficking of IR and/or signaling complex formation (13, 14). Finally, serine phosphorylation may also play a role in recruiting chaperones such as 14–3-3 that target IRS1 molecules for proteosomal degradation, thereby reducing IR-IRS signaling (15, 16, 17, 18).
Many of these mechanisms [especially those involving AKT, atypical PKC (aPKC), MAPK] are potentially targeted by insulin signaling itself and may represent physiologically relevant pathways involved in negative feedback. It is postulated that the perturbations induced during pathological states such as insulin resistance involve inappropriate activation of these inhibitory pathways. However, additional mechanisms may also exist that are not targeted by insulin but solely by agents that induce insulin resistance. Given the number of potential phosphoserine (pSer) sites it is also likely that multiple mechanisms are present by which serine phosphorylation of IRS1 can regulate insulin signaling. In this study, we report on the identification of a serine phosphorylation site located in the PH domain of IRS1. This site has recently been identified in an independent study and implicated as a target for IL receptor-associated kinase (19). Here we show that Ser24 is located in a substrate motif commonly targeted by a conventional PKC family of serine kinases and is indeed directly phosphorylated by PKC in vitro and in vivo. Unlike other serine phosphorylation sites that are linked to PKC activity, Ser24 is not phosphorylated after chronic insulin stimulation and exhibits a pharmacological profile that is distinct from that of Ser307 and Ser612. We also demonstrate that the phosphorylation status of Ser24 plays an important role in regulating lipid binding and intracellular localization of the IRS1-PH domain. Ultimately this can also impair insulin-stimulated glucose uptake. Hence, serine phosphorylation of the IRS1 PH domain may represent a novel regulatory mechanism that could be important in insulin resistance.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Despite the limitations of each bioinformatics tool, when used in combination and in conjunction with structural alignment analyses, they prove to be very powerful predictors of both potential phosphorylation sites as well as the kinases that may be involved. Indeed, motif scanning of all IRS1 proteins did highlight one serine residue, Ser24, as a putative PKC substrate site (Fig. 1A
). This residue also scored highly (>0.5) as a putative phosphorylation site in the NetPhos analysis. Sequence-structure alignment further revealed that Ser24 is highly conserved throughout all species of IRS1. However, it is present only in IRS1 and IRS3 and not in IRS2, IRS4, IRS5, or IRS6 (Fig. 1A). Chico, the Drosophila homolog for IRS1, also appeared to have a putative PKC phosphorylation site, albeit a threonine, in the same location. Serine 24 is located within the N-terminal PH domain of IRS1, and analysis of the crystal structure of IRS1 PH domain (db:1qqga) confirmed that it is located in the exposed variable loop 1 (VL1) region of this domain, a key area implicated in phosphoinositide binding and PH domain function (Fig. 1, A and B). Collectively, these data suggest that Ser24 is a candidate phosphorylation site for PKCs and may play a role in regulating PH domain function of IRS1.
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), a novel PKC (PKC), and an atypical PKC (PKC)]. The proteins were then separated by SDS-PAGE and immunoblotted with the anti-pSer24 antibody. A separate gel was run in parallel and stained for total protein. Figure 2A shows that incubation with all three active PKCs led to phosphorylation of IRS1PH at Ser24. Although equal amounts of kinase and substrate were used, PKC appeared consistently to produce the greatest amount of phosphorylation at Ser24, followed by PKC and then PKC. Similar reactions were performed to investigate whether casein kinase II would phosphorylate Ser24 in vitro. This enzyme has been reported to induce serine phosphorylation of IRS1 PH domain, albeit at Ser99 (21). Phosphorylation of Ser24 by casein kinase II was not observed (Fig. 2A), indicating that phosphorylation at this site is kinase selective. To confirm that the anti-pSer24 antibody was specific for this site, in vitro kinase assays were repeated in parallel with an IRS1PH peptide harboring a S24A mutation (Fig. 2B). The anti-pSer24 antibody detected phosphorylation of wild-type (wt) PH domain but not of the S24A mutant peptide.
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illustrates the resulting immunoblots wherein full-length IRS1 is also phosphorylated in an ATP-dependent manner on Ser24 by PKC, -, and - but not by casein kinase II. Again the magnitude of phosphorylation was greatest with PKC and least with PKC. The same membrane was sequentially stripped and reprobed with antibodies to pSer307 and pSer612. This allowed us to investigate the action of the kinases on two additional serine phosphorylation sites in IRS1. Figure 2C shows that casein kinase II was indeed active and that all four enzymes were able to directly phosphorylate Ser307 and Ser612 in vitro. Interestingly, each site exhibited a different profile with respect to the relative levels of phosphorylation achieved by the panel of kinases. For example, PKC most effectively mediated phosphorylation of Ser24, whereas casein kinase II was most effective at phosphorylating Ser612, and PKC was most effective for Ser307 phosphorylation. This suggests that site-specific differences may exist and can be reflected in vitro. Incubation with each of these serine kinases was also sufficient to induce a noticeable reduction in the electrophoretic mobility of rIRS1. This confirms that serine phosphorylation of IRS-1 is sufficient to account for the mobility shift reported for IRS-1 from insulin-resistant cells and tissues.
Phorbol 12-Myristate 13-Acetate (PMA), But Not C2 Ceramide or Chronic Insulin Treatment, Stimulates IRS1 Ser24 Phosphorylation in Vivo
To test whether insulin resistance-inducing agents could stimulate endogenous PKCs to phosphorylated IRS1 on Ser24 in vivo, NIH/hIR cells overexpressing either myc-rIRS1 wt or myc-rIRS1 S24A were stimulated with either PMA, C2 ceramide, or chronic insulin treatment. Anti-myc immunoprecipitates were then collected and analyzed for phospho-Ser24 immunoreactivity (Fig. 3). PMA induced detectable Ser24 phosphorylation of wt rIRS1 but not of the S24A mutant. PMA treatment also stimulated phosphorylation of IRS1 at Ser307 and Ser612 in both wt and S24A mutant. In contrast, neither ceramide nor insulin treatments stimulated detectable Ser24 phosphorylation in vivo but both induced phosphorylation at Ser307 and Ser612. Taken together, these data confirm that Ser24 can be phosphorylated in vivo. Furthermore, it is likely that PMA, C2 ceramide, and insulin activate different serine kinases and/or PKC isoforms, resulting in differential phosphorylation of Ser24 compared with Ser307 and Ser612.
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, ß, 
) and novel PKCs (nPKC: , , 
, ) are DAG dependent but can be distinguished by their requirement for calcium. In contrast, atypical PKCs (aPKC: , 
, µ) are calcium and DAG independent. We took advantage of these functional differences to characterize the PMA-stimulated phosphorylation of Ser24, Ser307, and Ser612. We reasoned that whereas 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM), a chelator of cytosolic free calcium, would inhibit only cPKCs, a DAG antagonist such as calphostin would inhibit both cPKC and nPKCs. PMA-induced phosphorylation of Ser24, Ser307, and Ser612 was sensitive to BAPTA-AM pretreatment (Fig. 4A). This suggests that calcium-dependent PKCs are activated by PMA and are required to induce phosphorylation of IRS-1 at these three serine residues. Consistent with this, we observed that serine phosphorylation induced by PMA was completely prevented by pretreatment with Gö6976, an inhibitor of cPKC, -ß, and -µ and Gö6983, an inhibitor of PKC and -ß but not -µ (Fig. 4A). In contrast, pretreatment with rottlerin, a selective inhibitor of nPKC and - was ineffective (Fig. 4, A and C). Unfortunately, calphostin treatment promoted significant degradation of IRS-1, making it difficult to analyze its impact on PMA-stimulated serine phosphorylation (Fig. 4A). Significant cytotoxicity was also observed using this concentration of calphostin (data not shown).
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shows that chronic treatment with PMA alone is sufficient to prevent the PMA-induced phosphorylation of Ser24, Ser307, and Ser612. The selective depletion of endogenous PKCs was confirmed in the same samples (Fig. 4B). PKC and - were significantly decreased whereas PKCµ and - levels were unaltered. Neither PKCß nor PKC was detected in these cells (data not shown). Taken together, these data suggest that PMA-stimulated phosphorylation at all three serine residues requires active conventional PKCs. Of these, PKC represents the most likely PKC isoform that is required for PMA-stimulated serine phosphorylation of IRS1 on Ser24, Ser307, and Ser612 in NIH3T3 cells.
PMA-Stimulated Phosphorylation of Ser24, Ser307, and Ser612 Exhibit Different Pharmacological Inhibitor Profiles
Having established that all three serine residues can be directly phosphorylated by PKCs in vitro (Fig. 3) and by PKC in vivo (Fig. 4), we next investigated whether PMA-stimulated PKC activation was acting directly on IRS1 or indirectly by activating other downstream serine kinase cascades. To address this, pharmacological inhibitor profiles were constructed for each of the three serine residues using a range of chemical inhibitors that selectively target kinases previously implicated in IRS1 serine phosphorylation. Figure 5A demonstrates that PMA-stimulated phosphorylation of Ser24 was insensitive to the entire panel of six inhibitors used. This argues against the involvement of MEK, JNK, p38, IKKß, mTOR, GSK3, and PI3K in Ser24 phosphorylation. In contrast, PMA-stimulated phosphorylation of Ser307 and Ser612 was sensitive to inhibition by all the kinase inhibitors (except SB203580) to varying degrees. Both Ser307 and Ser612 phosphorylation exhibited similar profiles but varied in the magnitude of response to individual kinase inhibition. These data suggest that although Ser307 and Ser612 are likely to be phosphorylated by similar kinases, they may vary in their relative affinities to individual kinases. In contrast, Ser24 is likely to be phosphorylated by a distinct kinase that is insensitive to the entire panel of inhibitors used in this experiment. Because PKC is insensitive to these inhibitors, it remains a good candidate for the direct phosphorylation of Ser24 and it is likely to play an indirect role in PMA-stimulated phosphorylation of Ser307 and Ser612 (Fig. 5B).
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Interacts with the IRS-1 PH Domain has been reported elsewhere (24). To test whether the IRS1-PH domain alone could mediate this interaction, glutathione-S-transferase (GST)-pull down assays were performed using recombinant PKC incubated with either GST alone or GST fused to the IRS1-PH domain. Figure 6 confirms that PKC can be identified only in GST immunoprecipitates that contain the IRS-1 PH domain. Subsequent in vitro kinase assays confirmed that this kinase-substrate complex contained sufficient levels of PKC to induce detectable phosphorylation on Ser24. These findings are in agreement with other studies suggesting that PKC can directly interact with IRS1 (24) and that the PH domain is sufficient for this interaction (25).
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). To determine whether phosphorylation of Ser24 can impact on PtdIns(4, 5)P2 binding to the IRS1-PH domain, we compared the binding properties of wt IRS1 PH domain with pseudophosphorylated (S24D) and nonphosphorylated (S24A) mutant peptides. As illustrated in Fig. 7B, binding by the S24D mutant is almost undetectable whereas S24A exhibits impaired PtdIns(4, 5)P2 binding affinity. To confirm that loss of PtdIns(4, 5)P2 binding affinity is not a result of misfolded mutants, but can be induced by phosphorylation-dependent events, the assay was conducted using wt peptide after in vitro kinase treatment in the presence and absence of PKC (Fig. 7C) or ATP (Fig. 7D). Both these treatments are sufficient to impair the lipid-binding capabilities of the IRS1 PH domain.
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, GFP alone is uniformly distributed throughout the cell cytoplasm and nucleus. However, the wt GFP-IRS-1PH domain is found in the nucleus but is also localized to the cell periphery. This is consistent with its ability to bind PtdIns(4, 5)P2, which is particularly abundant in the plasma membrane of these cells. In contrast, the S24D mutant does not exhibit any discernable peripheral localization whereas the S24A mutant can be found at the cell periphery (Fig. 8).
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). Western blot analysis demonstrates that similar levels of each S24 mutant were expressed in mature adipocytes and that the level of ectopic IRS-1 expression approximately matches that of endogenous IRS-1 because there appears to be a 2-fold increase in total IRS1 expression in the transfected cells relative to the empty vector controls (Fig. 9B). In dose-response assays for insulin-stimulated glucose uptake, those adipocytes expressing the pseudophosphorylated IRS1-Ser24D mutant consistently produced a significantly lower response to insulin than their wt counterparts (Fig. 9C), and the S24A mutant produced an intermediate response. This suggests that Ser24 phosphorylation may indeed impair insulin-stimulated glucose uptake in adipocytes.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Role of Serine 24 Phosphorylation in PH Domain Function
By analogy to the crystal structure of human IRS1 PH-PTB peptide, Ser24 of rIRS1 lies in a large exposed cationic patch that is present at the base of the PH domain. This region includes the putative phosphoinositide-interacting residues Lys 21, Lys 23 His 26 and Arg 28, Lys 61 and Arg 62 (highlighted in Fig. 1B) (26). Substitution of one of these, R28C, is sufficient to disrupt lipid binding to PH domain (30) as well as interactions with the pleckstrin homology domain interacting protein PHIP (31). Our findings suggest that the addition of a charged phosphate group on Ser24 can also induce significant changes to cationic patch and alter lipid interactions (Fig. 7, B and C). This modification is also sufficient to alter both intracellular localization (Fig. 8) and subsequent participation in insulin-stimulated glucose uptake (Fig. 9C). These observations are entirely consistent with a recent independent study demonstrating that the IRS1-S24D mutant shows impaired insulin-stimulated IR-IRS-1 interactions, tyrosine phosphorylation of IRS-1, recruitment/activation of PI 3-Kinase, and insulin-stimulated Glut4 translocation (19).
A surprising observation in our studies was that an alanine substitution of Serine 24 did not behave in a fashion akin to a nonphosphorylated peptide (Figs. 7B, 8, and 9C). Indeed, this mutation consistently produced actions that were intermediate between wt and the pseudophosphorylated Ser24. One possible explanation for these observations is that serine 24 is also involved in hydrogen bonding to a main-chain amide (Ref. 26 and Fig. 1), and substitution with an alanine is sufficient to disrupt this bond and hence alter the functionality of the PH domain. It is intriguing to speculate that, in addition to altering the electrostatic properties of the cationic patch, phosphorylation of Ser24 may also significantly alter PH domain structure by disrupting this hydrogen bond.
The PH domains of IRS1 and IRS2 are more similar to each other (58% identical and 78% similar) than to IRS3 (IRS1-PH is 37% identical and 63% similar to IRS3, and IRS2-PH is 33% identical and 57% similar to IRS3) (32). Hence, it is surprising that Ser24 is not conserved between IRS1 and IRS2, but rather that it is present in IRS3 and IRS_CHICO. It is interesting to note that functional differences between the PH domains of IRS1 and IRS2 have been reported (33, 34). Whether this is accomplished by differential regulation of PH domains by Ser24 phosphorylation remains a possibility.
Recently, two other proteins have been reported to be phosphorylated in their PH domains after PKC activation (30, 31). As with IRS1-Ser24, these phosphorylation events are also sufficient to disrupt phosphoinositide binding (35) and membrane translocation (36). Intriguingly, DGK1-PH is also phosphorylated by cPKCs, and the targeted serine site is also located in an analogous region. Both these serine sites are predicted as putative PKC phosphorylation sites in our bioinformatics analysis (data not shown). This further validates the use of bioinformatic software in hypothesis generation.
Role of PKC in Ser24 Phosphorylation
In this study, we show that in vitro, IRS1-Ser24 is a substrate for representatives of all three PKC subfamilies (, ß, and ). However, this relative lack of specificity is not recapitulated in vivo. In intact NIH/IR/IRS1 cells, agonist-induced phosphorylation of Ser24 was selective for specific PKC isoforms. Ser24 is phosphorylated after activation of phorbol ester-dependent kinases and not by C2-ceramide or chronic insulin treatment. This suggests an involvement of DAG-dependent PKCs. PMA-stimulated phosphorylation of Ser24 is likely to be mediated by conventional PKC, as it is sensitive to inhibition by the calcium chelator, BAPTA-AM, inhibitors of cPKC (i.e. Gö6976 and Gö6983), and chronic PMA treatment, and neither PKCß nor PKC is present in NIH3T3 cells (22). This conclusion is also consistent with the in vitro observations wherein PKC consistently produced the most robust phosphorylation at this site. It is therefore likely that PKC is a preferred kinase to phosphorylate Ser24.
It is interesting to note that Motif scan predicted Ser24 to be preferred by PKC as a substrate site. Also the amino acid sequence flanking Ser24 does not conform to two commonly cited consensus sequences for substrates of classic/generic PKCs, i.e. X-S/T-X-R/K (Prosite pattern and Ref. 37) or R-X-X-S/T (9). However, it is in absolute agreement with PKC isoform-specific sequence motifs reported by Cantley and colleagues (38). They report that all PKCs prefer substrates that have basic residues in the –3 position and a hydrophobic amino acid must occupy position +1. Additionally, cPKCs prefer substrates with basic residues particularly at positions +2 and +3, whereas nPKC and aPKC tend to prefer hydrophobic residues at these positions. In IRS1 Ser24 is flanked by such residues in the positions that confer this as a preferred phosphorylation site for cPKCs. Therefore our experimental evidence is entirely consistent with this particular motif-based prediction.1 This consensus motif, together with our in vivo and in vitro data, suggests that PKC is likely to directly phosphorylate Ser24. The finding that PMA-stimulated Ser24 phosphorylation is not affected by any of the non-PKC inhibitors (Fig. 5) further supports this notion, as does the findings that IRS1 and PKC are constitutively associated in vivo (39, 40) and that the PH domain itself is sufficient for this interaction (Fig. 6).
One possibility that cannot be excluded is that differences in endogenous levels, relative activities, and/or intracellular location of different PKC isoforms may also determine Ser24 phosphorylation in vivo. In this regard, PKC, - µ, and - are all expressed in NIH3T3 fibroblasts (Fig. 4B), but only PKC is involved in PMA-stimulated Ser24 phosphorylation. Whether this also occurs in other cell types that express cPKC ß- and/or -isoforms warrants further investigation.
Role of PKC in Insulin Resistance
Attempts to address the role of cPKCs in insulin signaling have produced conflicting data. Studies using PKC inhibitors and phorbol ester-induced PKC down-regulation have suggested that DAG-activated PKCs are not required for normal insulin-stimulated glucose uptake in adipocyte and muscle cells. In contrast, the targeted disruption of murine PKC does enhance insulin signaling through IRS1, leading to increased insulin-stimulated glucose transport in adipocytes and skeletal muscle (41). It is therefore possible that PKC may serve as a tonic endogenous inhibitor of IRS1-dependent pathways (23).
Although the role of cPKCs in physiological responses to insulin remains unresolved, it is clear that overexpression or activation of DAG-dependent PKCs can significantly impair insulin signaling (42, 43). Indeed, elevated PKC, -, and activities are implicated in insulin resistance in vivo (44, 45). Our observation that IRS1 Ser24 is not phosphorylated in response to insulin but only after PMA stimulation suggests that this site may represent a novel diagnostic marker of elevated cPKC activity that may occur in pathophysiological states such as insulin resistance and hyperglycemia (23).
pSer24 Exhibits Characteristics that Are Distinct from pSer307 and pSer612
As with Ser24, we found that PKCs could directly phosphorylate Ser307 and Ser612 in vitro, but PMA-stimulated phosphorylation required active PKC in vivo. This could suggest that these sites are also the direct targets of cPKC activity. However, neither site lies within a consensus motif for PKC substrates and, unlike PMA-stimulated pSer24, both pSer307 (312 in hIRS1) and pSer612 (616 in hIRS1) were sensitive to other serine kinase inhibitors. This implicates other downstream serine kinases in the direct interaction and phosphorylation of Ser307 and Ser612. Indeed, Ser612 was identified as a target for active PKCs and Erk (46), whereas Ser307 is targeted by numerous kinases including JNK, IKKß, AKT, and mTOR (47, 48, 49). Figure 5B summarizes the residue selective roles of PMA-responsive serine kinases in IRS1 phosphorylation. Future studies should help delineate the specific serine kinase and upstream kinase pathways that directly target these sites.
An additional novel observation made during our studies was that although casein kinase II has little effect on Ser24 phosphorylation, it does directly phosphorylate Ser307 and Ser612 in vitro. This activity on full-length IRS1 was also sufficient to cause the most significant mobility shift, suggesting that there may be a number of other sites. Indeed, IRS1 has 19 consensus sites for casein kinase II phosphorylation (S/T-X1-X2-E/D, where X1 is any amino acid except proline). This consensus motif is based on the requirement of acidic rather than basic residues (37). However, neither Ser307 nor Ser612 complies with this consensus and, as such, is unlikely to be directly phosphorylated by casein kinase II in vivo. This remains to be experimentally confirmed.
In summary, an increasing array of agonists, including insulin, IGF, TNF, FFA, C2 ceramide, and PMA, are being reported to stimulate serine phosphorylation of IRS1 at multiple sites including Ser307 and Ser612. It appears that many of these sites are promiscuous and open to regulation by multiple kinases (and phosphatases) and may be physiologically important in mediating feedback regulation of insulin signaling. In contrast, Ser24 is phosphorylated in vivo in response to a limited number of agonists and serine kinases. Once phosphorylated it can significantly alter PH domain function and impair insulin sensitivity. Future studies will address whether phosphorylated Ser24 has potential for use as a diagnostic marker for detection of pathological signaling events that occur during insulin resistance and hyperglycemia.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(Santa Cruz), anti-PKC, -ß, -, and -µ (BD Biosciences, Palo Alto, CA), anti-pSer643-PKC (New England Biolabs, Beverly, MA), anti-His6 (Santa Cruz), anti-GST (GST103, Siddle laboratory), and anti-p85 (Siddle laboratory). Unless otherwise stated, all other reagents were purchased from Sigma Chemical Co. (St. Louis, MO) or Calbiochem.
Bioinformatic Analysis
Protein sequences for IRS family members were systematically analyzed with two computational tools that predict putative phosphorylation sites. NetPhos 2.0 (www.cbs.dtu.dk/services/NetPhos/) is based on neural network predictions for serine, threonine, and tyrosine phosphorylation sites (50) whereas Motif Scan in Scansite (scansite.mit.edu/) identified experimentally determined protein binding or kinase substrate motifs in proteins (51). Structure-based homology alignment was performed using Fugue, (www-cryst.bioc.cam.ac.uk/
fugue/) (52) to compare IRS-PH domains with the crystal structure of hIRS1 PH domain (PDB:1qqga). Also used was Homstrad, a homologous structure alignment database (www-cryst.bioc.cam.ac.uk/homstrad/) (53), the output of which incorporates three-dimensional structural features that are annotated with JOY (www-cryst.bioc.cam.ac.uk/joy/) (54).
Generation of Anti-pSer24 IRS1 Antibody
The generation of anti-pSer24-IRS1 was commissioned from Cambridge Research Biochemicals Ltd. (Cambridge, UK). Briefly, rabbit antisera were generated against the following N-terminally, keyhole limpet hemocyanin-conjugated phosphopeptide: [C]YLRKPKS(p)MHKRFF. The phosphoreactive serum was then affinity purified on a Thiopropyl Sepharose 6B column derivatized with nonphosphorylated antigen. The unbound antiserum was then passed down a Thiopropyl Sepharose 6B column derivatized with the phosphorylated peptide. The resulting triethylamine eluate retained significant phosphospecific immunoreactivity on peptide-coated ELISA plates and was used in Western blot analysis as described below.
Plasmid Construction
C-terminally tagged full-length rIRS1-Myc-(His)6 in pcDNA3.1 was sequenced in full, and all nonconserved variations were corrected by using QuikChange XL (Stratagene, La Jolla, CA) such that rIRS1wt cDNA encoded the protein sequence NP_037101. This then was used as the template in further site-directed mutagenesis to construct the corresponding Ser24Ala and Ser24Asp mutants. Successful mutagenesis was confirmed by direct sequencing. The coding sequence for full-length myc-his tagged rIRS1 wt or S24 mutants was released from pcDNA3.1 with an NheI/AflII digestion and blunt ended with Klenow. They were then ligated into the SnaB1 site of the retroviral vector, pBabePuro. Diagnostic digests with BamHI confirmed both presence and correct orientation of the inserts.
All expression plasmids encoding IRS1-PH domains corresponded to amino acids 1–113 of rIRS1 (IRS1PH). The tagged constructs were generated by PCR amplification using appropriate mutant templates and primers also encoding compatible restriction sites to facilitate in-frame, directional ligation into either the N-terminal His6-tagging vector pMwHis6 or C-terminal GST-tagging vector pMwGST and C-terminal GFP tagging vector pEGFP-N1 (BD Biosciences; CLONTECH Laboratories, Inc., Palo Alto, CA). Correct orientation and in-frame positioning were verified by direct sequencing. Primer sequences are available on request.
Generation of Recombinant rIRS1PH Peptides
Competent cBL21DE3pLys cells were transformed with appropriate expression plasmids and peptide expressing, isopropyl-ß-D-thiogalactopyranoside (IPTG)-inducible clones were identified. For HIS6-tagged peptides, overnight starter cultures were used to inoculate small-scale (100 ml) expression cultures. These were grown at 37 C to 0.6 OD600 before being induced with 1 mM IPTG and left to express peptide during an overnight incubation at 22 C. The resulting bacterial cultures were pelleted (at 5000 x g for 10 min) and resuspended in ice-cold, sterile sonication buffer (10 mM HEPES, 200 mm NaCl, 25 mM MgCl2, DNAse, 4-(2-aminoethyl)benzenesulphonyl fluoride, 1% Triton X). After sonication these were clarified by centrifugation (at 10,000 x g for 10 min), and resulting supernatant was incubated with activated Ni2+ Sepharose beads (at 4 C for 1 h). Beads were washed twice with cold wash buffer (10 mM HEPES, 200 mm NaCl, 20 mM imidazole) and His-tagged peptides eluted in elution buffer (10 mm HEPES, 200 mM NaCl, 300 mm imidazole). For GST-tagged peptides, large-scale (1 liter) expression cultures were induced with IPTG and grown for a further 6 h at 37 C. Each was pelleted and lysed in Bug Buster (Novagen, Madison, WI). After centrifugation, clarified supernatants were incubated with reduced glutathione-sepharose beads (at room temperature for 1 h). Beads were washed three times with wash buffer (PBS and 1% Triton-X100) followed by a fourth wash with equilibration buffer (100 mM Tris-HCl, pH 8.8; 200 mm NaCl; 20% glycerol). GST-tagged peptides were then eluted in elution buffer (100 mM Tris-HCl, pH 8.8; 200 mm NaCl; 20% glycerol; and 15 mM glutathione). Peptide purification was confirmed by SDS-PAGE, and eluates were subsequently dialysed using Slide-A-Lyser cassettes (Pierce Chemical Co., Rockford, IL) with dialysis buffer (100 mm Tris-HCl, pH 8; 200 mM NaCl; and 20% glycerol) as per manufacturer’s instructions.
In Vitro Kinase Assays
Recombinant rIRS1 PH domains (5 µg) or immunoprecipitated full-length IRS1 was incubated with 0.2 µg of recombinant casein kinase II or PKC isoforms , , and in the appropriate kinase buffer. Casein kinase buffer comprised 20 mM Tris-HCl (pH 7.5), 50 mm KCl, 10 mM MgCl2, 100 µM Mg-ATP, whereas PKC required Ca2+-dependent kinase buffer (20 mM HEPES, 10 mm MgCl2, 0.5 mM CaCl2, 0.5 mM dithiothreitol, 100 µM Mg/ATP), and PKC and PKC required Ca2+-independent buffer (20 mM HEPES, 10 mm MgCl2, 0.5 mM EGTA, 0.5 mm dithiothreitol, 100 µM Mg/ATP). Additional lipid supplements included 10 µg/ml diolein and 100 µg/ml phosphatidylserine (for PKC and PKC) or 100 µg/ml phosphatidylserine (for PKC). Reactions were initiated with the addition of either kinase or ATP, incubated at 30 C for 2 h and terminated by the addition of reducing Laemmli loading buffer and heat inactivation.
In Vitro Lipid-Binding Assay
Lipid binding was determined using a time-resolved FRET assay as described elsewhere (27) in which increasing concentrations of a biotinylated phosphoinositide were bound to a FRET acceptor, streptavidin allophycocyanin conjugate, the FRET donor being a Europium chelate-labeled anti-GST antibody complexed with a GST fusion of the IRS1 PH domains. Binding of PH domain to lipid results in formation of a complex generating a FRET signal. The binding assays were carried out in 50 mM HEPES (pH 7.4), 150 mm NaCl, 2 mM dithiothreitol, and 0.02% Cholate with allophycocyanin conjugate-Streptavidin (Prozyme Ltd., San Leandro, CA) 32 nM, 0–20 pmol (as required) biotinylated, short-chain (diC8) phosphoinositides (Cell Signaling Technology, Danvers, MA) and 20–30 nM IRS1 PH domain with 21 nm Lance chelate-labeled anti-GST antibody in a final volume of 50 µl. For all assays, the samples were mixed in 96-well plates in Lumitrax 200 and the plates read in an LJL Analyst (Molecular Devices, Sunnyvale, CA) with the following settings: excitation filter, 360 –35 nm; emission filter, 665 nm; dichroic filter, 505 nm; digital sensitivity of PMT 1000 V, set to 2; flashes per well, 100; interval between flashes, 10 msec; read time after flash, 50 msec; and integration time, 1000 msec. Data are expressed as a FRET Ratio of the FRET signal divided by the total Europium fluorescence.
Cell Culture
BOSC23 cells and 3T3-L1 preadipocytes were routinely propagated in DMEM containing 4.5 g/liter glucose, 10% bovine calf serum (Hyclone Laboratories, Inc., Logan, UT), 50 U/ml penicillin, and 50 µg/ml streptomycin at 10% CO2. HEK293 cells were propagated in DMEM containing 4.5 g/liter glucose, 10% newborn calf serum, 50 U/ml penicillin, and 50 µg/ml streptomycin at 5% CO2. NIH/hIR cells, mouse fibroblasts overexpressing the human IR (and G418 resistant), were grown in the same medium as HEK293 cells but with the addition of 600 µg/ml G418.
Generation of Stable Cell Lines Expressing rIRS1 Mutants
3T3-L1 and NIH/hIR cells stably expressing myc-his-tagged IRS1 were generated by retroviral mediated gene transfer (55). Briefly, subconfluent BOSC23 packaging cells were transfected with pBabePuro-rIRS1mychis plasmids, and conditioned media was collected 48 h later. This was passed through a 0.45-µm filter, supplemented with 16 µg/ml of polybrene, and used to infect proliferating 3T3-L1 preadipocytes or NIH/hIR cells. Drug selection with puromycin (4 µg/ml for 3T3-L1 and 16 µg/ml for NIH/hIR) was initiated 2 d later. After a week in drug selection, stable cell lines were confirmed by Western blot analysis and expanded. Stocks were frozen in liquid nitrogen until required.
Adipocyte Differentiation and Glucose Uptake
3T3-L1 preadipocytes (2 d postconfluent) expressing either empty vector or full-length IRS1 mutants were maintained at 10% CO2 in adipocyte media AM [high-glucose DMEM containing antibiotics, 10% Cosmic Calf Serum (Hyclone), 4 µg/ml puromycin]. Adipocyte differentiation was induced by incubation for 2 d in AM supplemented with induction cocktail (5 µg/ml insulin, 0.5 mM isobutylmethlyxanthine, and 1 µM dexamethasone). This was followed by a further 2-d incubation in AM supplemented with insulin only. Thereafter, cells were fed unsupplemented AM every 2 d. Glucose-uptake assays were performed on fully differentiated adipocytes using 2-deoxy-D-[2,6 3H]-glucose uptake as described previously (56).
Confocal Fluorescence Microscopy
HEK293 cells were grown on poly-L-lysine-coated coverslips and transiently transfected with either pNEGFP, pNEGFP-IRS1-PH wt, pNEGFP-IRS1-PH S24A, or pNEGFP-IRS1-PH S24D. Monolayers were serum starved (overnight) 24–48 h after transfection, washed with PBS, fixed in 2% formalin, and mounted in 4',6-diamidino-2-phenylindole (DAPI) containing Vectorshield mount medium (Vector Laboratories, Inc., Burlingame, CA). Confocal fluorescence microscopy was performed using a LSM510 laser confocal microscope system (Carl Zeiss, Thornwood, NY) and a x60 oil emersion objective. Fluorescent images were captured using Argon 488-nm (GFP) and Krypton 413-nm (DAPI) lasers and a confocal slice thickness of less than 0.8µm.
Phosphoprotein Extraction and Analysis
Two-day postconfluent cells were washed and incubated with serum-free medium and starved for 4 h before stimulation with agonists. After stimulation, culture medium was removed and monolayers were transferred to 4 C. They were then washed with ice-cold PBS and lysed in modified RIPA buffer (50 mM Tris-HCl, pH 7.4; 1% NP-40; 0.25% sodium deoxycholate; 150 mm sodium chloride; and 1 mM EDTA) freshly supplemented with 50 mm ß-glycerol-2-phosphate, 5 µM 4-(2-aminoethyl)benzenesulphonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 1 mM sodium pervanadate, 1 mm sodium fluoride, and 50 nM okadaic acid. Cell lysates were scraped and clarified by centrifugation and the supernatant was collected. Protein quantification was determined using the Bio-Rad DC protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA).
For immunoprecipitation with anti-myc antibody (9E10), protein lysates were precleared with protein G beads and incubated overnight (with rotation, at 4 C) with prebound T9E10-protein G beads. Immunoprecipitates were subsequently washed three times with ice-cold PBS supplemented with 50 mM ß-glycerol-2-phosphate, 1 mM sodium pervanadate, 1 mm sodium fluoride, and 50 nM okadaic acid and resuspended in 1x Laemmli loading buffer and boiled for 5 min. Immunoprecipitated proteins were separated by SDS-PAGE, transferred onto polyvinylidine difluoride membranes (Amersham Pharmacia Biotech, Arlington Heights, IL), and used for immunodetection with the following primary antibody dilutions: anti-pSer24 (1:100), anti-pSer307 (1:500), anti-pSer616 (equivalent to pSer612 in rIRS1; 1:1000), and antirabbit (1:10,000). Sequential immunoblotting was performed only after blots had been stripped and complete removal of primary antibody had been confirmed.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Author disclosure summary: The authors, R.N., A.G., C.H.J., C.P.D., K.S., and J.K.S., have nothing to declare.
First Published Online March 30, 2006
Abbreviations: AKT/PKB, AKR mouse thymoma viral proto-oncogene/protein kinase B; aPKC, atypical PKC; BAPTA-AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester; cPKC, conventional PKC; DAG, diacylglycerol; DAPI, 4',6-diamidino2-phenylindole; FRET, fluorescence resonance energy transfer; GFP, green fluorescent protein; GSK, glycogen synthase kinase; GST, glutathione-S-transferase; HEK, human embryonic kidney; IKK, inhibitor of B kinase; IPTG, isopropyl-ß-D-thiogalactopyranoside; IR, insulin receptor; IRS, insulin receptor substrate; JNK, cJun NH2-terminal kinase; MEK, mitogen-activated Erk kinase; mTOR, mammalian target of rapamycin; nPKC, novel PKC; PH, pleckstrin homology; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; pSer, phosphoserine; PTB, phosphotyrosine binding; PtdIns(4 5 )P2, phosphatidylinositol 4,5-bisphosphate; wt, wild type.
1 A recent update of Motif scan appears to have recognized this inconsistency and now predict Ser24 as a putative phosphorylation site for basophilic kinases. 
Received for publication December 29, 2005. Accepted for publication March 20, 2006.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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in adipocyte metabolism. Semin Cell Dev Biol 10:19–29[CrossRef][Medline]: functional analysis and identification of novel phosphorylation sites. Biochemistry 43:5888–5901[CrossRef][Medline]
-torsion angle (italic). All numbering (except xIRS1) is according to Swissprot/Trembl. Sequence numbering for the xIRS1 sequence is taken from Ref. 
