Ser/Thr Phosphorylation of IRS Proteins: A Molecular Basis for Insulin Resistance

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Science's STKE  25 Jan 2005:
Vol. 2005, Issue 268, pp. pe4
DOI: 10.1126/stke.2682005pe4


S6K1, like other serine and threonine kinases activated by insulin (such as mTOR and PKCζ), has recently been shown to participate in negative feedback mechanisms aimed at terminating insulin signaling through IRS (insulin receptor substrate) phosphorylation. Such homeostatic mechanisms can also be activated by excess nutrients or inducers of insulin resistance (such as fatty acids and proinflammatory cytokines) to produce an insulin-resistant state that often leads to the development of diabetes. Identification of the specific kinases involved in such insulin resistance pathways can help lead to the rational design of novel therapeutic agents for treating insulin resistance and type 2 diabetes.

The duration and extent of signals induced by insulin, our major anabolic hormone, are tightly regulated to promote proper metabolism, energy balance, and maintenance of normal body weight. Impaired ability to generate these signals, termed insulin resistance, results in the development of diabetes, an emerging epidemic of the 21st century. Some of the mechanisms that control insulin signaling are set in motion immediately after the binding of insulin to its receptor and act to terminate insulin’s effects through the induction of serine and threonine (Ser/Thr) kinases that phosphorylate and uncouple various elements along the insulin signaling pathways. Negative feedback control through Ser/Thr phosphorylation also operates on a longer time scale, leading to a reduction in the cellular content of the insulin receptor, its substrates, and other signaling elements. Support for this concept is provided by a recent publication by Um et al. that illustrates the negative regulatory role on insulin action of S6 kinase 1 (S6K1), a Ser/Thr kinase activated along the insulin signaling pathway (1). Um et al. further show that obesity-induced insulin resistance results in elevated S6K1 activity, implicating S6K1 in the negative regulation of insulin action under both physiological and pathological conditions.

Ser Phosphorylation of IRS Proteins: A Negative Feedback Control Mechanism to Terminate Insulin Signaling

Control over insulin signaling can be achieved by autoregulation, whereby downstream enzymes inhibit upstream elements (homologous desensitization). Alternatively, signals from apparently unrelated pathways can inhibit insulin signaling through heterologous desensitization. The insulin receptor, which functions as a protein tyrosine (Tyr) kinase, and its major substrates, the IRS (insulin receptor substrate) proteins, are subject to a combination of homologous and heterologous desensitization. IRS proteins contain a conserved pleckstrin homology (PH) domain located at their N termini that anchors them to membrane phosphoinositides in close proximity to the insulin receptor (2). The PH domain is flanked by a phosphotyrosine-binding (PTB) domain, which enables binding to the insulin receptor (IR), the IGF-1 (insulin-like growth factor–1) receptor, and other receptors (3). The C-terminal region of IRS proteins contains multiple Tyr phosphorylation motifs that serve as docking sites for Src homology 2 domain–containing proteins, including the p85α regulatory subunit of phosphatidylinositol 3-kinase (PI3K). Association of PI3K with IRS proteins results in the production of phosphatidylinositol-3,4,5-trisphosphate (PIP3), which activates PDK1 (PIP3-dependent kinase 1) and its downstream effectors PKB (protein Ser/Thr kinase B, also named Akt), mTOR (mammalian target of rapamycin), S6K1, and atypical isoforms of protein kinase C (PKCζ and PKCλ), all leading to enhanced glucose transport and the synthesis of glycogen and protein (4, 5) (Fig. 1).

Fig. 1.

Mode of action of IRS kinases. Insulin-stimulated Ser/Thr kinases, including mTOR, S6K1, and PKCζ, mediate phosphorylation of IRS proteins. Such phosphorylation inhibits the function of the IRS proteins and is part of a negative feedback control mechanism induced by insulin to terminate its own signals. Excess nutrients as well as inducers of insulin resistance (such as fatty acids and proinflammatory cytokines) take advantage of this negative feedback control mechanism by activating either the same or additional IRS kinases, thus inducing an insulin-resistant state that often leads to the development of diabetes. aPKC denotes atypical isoforms of PKCζ and PKCλ (8).

IRS proteins contain more than 70 Ser/Thr residues that are potential targets for phosphorylation, with homologies to consensus phosphorylation sites of different kinases. Recent findings indicate that insulin-stimulated Ser/Thr phosphorylation of IRS proteins serves as a physiological negative feedback control mechanism that inhibits the activity of the IRS proteins (6). Ser/Thr phosphorylation can induce the dissociation of IRS proteins from the insulin receptor (7, 8), block Tyr phosphorylation sites of IRS proteins (9), release the IRS proteins from intracellular complexes that maintain them in close proximity to the receptor (10), induce degradation of IRS proteins (11), or turn IRS proteins into inhibitors of the insulin receptor kinase (IRK) (12). These multiple effects shine the spotlight on the Ser/Thr kinases that phosphorylate the IRS protein and on the Ser/Thr sites that are phosphorylated as key elements of this negative feedback control process.

The activity of insulin-stimulated IRS kinases is blocked by inhibitors of the PI3K pathway, implicating downstream effectors of PI3K as negative regulators of IRS protein function (8, 13). One potential candidate is mTOR, which enhances phosphorylation of Ser residues at the C terminus of IRS-1. This phosphorylation prevents insulin-stimulated Tyr phosphorylation of IRS-1 at this region and thus blocks its ability to bind PI3K (14). Another downstream effector of PI3K is PKCζ. PKCζ mediates the phosphorylation of IRS proteins (8, 15), which leads to the dissociation of IR-IRS complexes (8, 16). This inhibits the ability of IRS proteins to undergo further insulin-stimulated Tyr phosphorylation and, as a result, terminates insulin signaling. Ser318 is a potential target for PKCζ-mediated phosphorylation of IRS-1 (17). Because Ser318 is adjacent to the IRS-1 PTB domain, its phosphorylation presumably disrupts the interaction between the juxtamembrane domain of the IR and the PTB domain of IRS-1 in such a way as to inhibit insulin-stimulated Tyr phosphorylation of IRS-1.

Involvement of downstream effectors of PI3K in the negative regulation of insulin signaling was strongly substantiated by the recent publication by Um et al. (1). This study clearly demonstrated that S6K1, an effector of PI3K and of mTOR [which acts as an integrator of nutrient and insulin signals (18, 19)], negatively modulates insulin’s effects by phosphorylating IRS proteins. In doing so, S6K1 takes part in the physiological negative feedback control mechanism induced by insulin to shut off its own action.

This pivotal role played by S6K1 is further indicated by the fact that S6K1–/– mice placed on a high-fat diet (HFD) fail to fully autophosphorylate and activate their insulin receptors but still remain insulin sensitive, which suggests that the absence of S6K1 facilitates insulin signaling downstream of the insulin receptor (1). Indeed, whereas wild-type mice placed on HFD are insulin resistant, having increased levels of circulating insulin and a reduced capacity to activate the insulin receptor and its downstream effectors, S6K1–/– mice maintain their capacity to activate downstream effectors such as PKB. This suggests that S6K1 elicits a selective inhibitory effect on PKB activation at a point downstream of the insulin receptor. The target of S6K1 seems to be the IRS proteins. Indeed, S6K1 can directly phosphorylate IRS-1 in vitro (20), whereas lowering S6K1 expression with short interfering RNA (siRNA) reduces phosphorylation of IRS-1 on selected Ser residues (Ser307, Ser636, Ser639) and potentiates insulin-induced PKB phosphorylation (1). Expression of siRNA to S6K1 also restores the expression of IRS-1 in TSC–/– mice (TSC is the gene encoding tuberous sclerosis complex, an upstream inhibitor of S6K1), which is otherwise dampened by S6K1 in these animals (20, 21). Hence, S6K1 appears to act as both an IRS kinase and a central negative regulator of insulin action.

Ser Phosphorylation of IRS Proteins and Insulin Resistance

An interesting twist became apparent with the realization that inducers of insulin resistance [such as tumor necrosis factor–α (TNF-α), free fatty acids, and cellular stress] inhibit insulin action by activating IRS kinases, S6K1 included, that are also stimulated in response to prolonged insulin treatment. Indeed, phosphorylation of S6K1 substrates (the ribosomal protein S6 and IRS-1) is highly elevated in either nutritionally or genetically driven obesity (as is phosphorylation of S6K1 itself), but is absent in S6K1–/– mice (1). These observations suggest that nutrient-induced S6K1 activation suppresses insulin signaling by modulating the phosphorylation of IRS proteins. Other kinases, including c-Jun N-terminal kinase (JNK) and PKCζ, play a similar role. JNK is activated both by insulin (22) and by proinflammatory cytokines such as TNF-α, and its activity is abnormally elevated in obesity. Conversely, the absence of JNK1 results in decreased adiposity, significantly improved insulin sensitivity, and enhanced insulin receptor signaling capacity in two models of mouse obesity (23). The inhibitory effects of JNK on insulin signaling can be attributed, at least in part, to its ability to phosphorylate Ser307 of IRS-1, which uncouples IRS-1 from the insulin receptor (24). PKCζ, a downstream effector of insulin (25), is also stimulated in response to TNF-α. TNF-α activates a sphingomyelinase that leads to the production of ceramide, which stimulates PKCζ. Indeed, the inhibitory effects of TNF-α on insulin-stimulated Tyr phosphorylation of IRS proteins are mimicked by sphingomyelinase and ceramide analogs (7).

Concluding Remarks

Current findings implicate IRS proteins as key players in insulin-induced, phosphorylation-based, negative feedback control mechanisms that uncouple the insulin receptor from its downstream effectors and terminate insulin signaling under physiological conditions (26). The kinases involved are still under investigation, with current focus on PKCζ, inhibitor of nuclear factor κB kinase β (IKKβ), JNK, mTOR, and S6K1. These kinases are also activated by various inducers of insulin resistance, placing them at a point of convergence between physiological and pathological stimuli. Still, the list of IRS kinases is incomplete and additional candidates are likely to emerge. Furthermore, the signaling cascades that result in Ser phosphorylation of IRS proteins need to be established. Although all of the kinases mentioned above promote Ser phosphorylation of IRS proteins, some are likely to be direct IRS kinases, whereas others are presumably their upstream activators. mTOR is such an example: It might be a direct IRS kinase, an upstream activator of S6K1, or both. The fact that several IRS kinases with different substrate specificities promote phosphorylation of the same site (for instance, S6K1, JNK, and IKKβ all stimulating the phosphorylation of IRS-1 Ser307) supports such an idea.

The spatial and temporal activation of IRS proteins is likely to involve a challenging and intricate signaling cascade. This assumption is based on the fact that some IRS kinases are mediators of insulin action. S6K1 promotes insulin-induced protein synthesis through the phosphorylation of the ribosomal protein S6 (27), whereas PKCζ mediates insulin-stimulated glucose transport by an as yet unknown mechanism (25). Hence, their negative regulatory role as IRS kinases is expected to take place only after completion of their positive effects on insulin action.

Because each IRS kinase has a unique substrate specificity, the question remains as to which Ser/Thr sites are modified by each kinase and what are the consequences of such phosphorylation. Present studies indicate that negative regulatory sites are found in close proximity to the PTB domain (for instance, Ser307 and Ser408) and at the C-terminal end of IRS-1 (for instance, Ser612) (1, 24, 28). It remains to be determined whether phosphorylation of Ser residues, either scattered along the IRS molecule or clustered within a confined region, yields a given functional change (such as enhanced dissociation of IR-IRS complexes). Given the number of stimuli, pathways, kinases, and potential sites involved, it appears that Ser/Thr phosphorylation of IRS proteins represents a combinatorial consequence of the activity of several kinases, activated by different pathways, acting in concert to phosphorylate multiple sites. Still, despite the complexity and dynamics of the system, unraveling IRS kinases and their biological role is an exciting opportunity to provide a novel approach to uncovering the molecular basis for insulin resistance. The work of Um et al. is a substantial step in that direction: It enables the use of rational drug design to selectively inhibit the activity of the relevant kinases and thereby generate a novel class of therapeutic agents for treatment of insulin resistance and type 2 diabetes.


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