Regulation of insulin sensitivity by serine/threonine phosphorylation of insulin receptor substrate proteins IRS1 and IRS2

Abstract

The insulin receptor substrate proteins IRS1 and IRS2 are key targets of the insulin receptor tyrosine kinase and are required for hormonal control of metabolism. Tissues from insulin-resistant and diabetic humans exhibit defects in IRS-dependent signalling, implicating their dysregulation in the initiation and progression of metabolic disease. However, IRS1 and IRS2 are regulated through a complex mechanism involving phosphorylation of >50 serine/threonine residues (S/T) within their long, unstructured tail regions. In cultured cells, insulin-stimulated kinases (including atypical PKC, AKT, SIK2, mTOR, S6K1, ERK1/2 and ROCK1) mediate feedback (autologous) S/T phosphorylation of IRS, with both positive and negative effects on insulin sensitivity. Additionally, insulin-independent (heterologous) kinases can phosphorylate IRS1/2 under basal conditions (AMPK, GSK3) or in response to sympathetic activation and lipid/inflammatory mediators, which are present at elevated levels in metabolic disease (GRK2, novel and conventional PKCs, JNK, IKKβ, mPLK). An emerging view is that the positive/negative regulation of IRS by autologous pathways is subverted/co-opted in disease by increased basal and other temporally inappropriate S/T phosphorylation. Compensatory hyperinsulinaemia may contribute strongly to this dysregulation. Here, we examine the links between altered patterns of IRS S/T phosphorylation and the emergence of insulin resistance and diabetes.

Fig. 1

Insulin signalling and feedback pathways initiated by IRS (a) Tyrosine phosphorylation of IRS by IR kinase enables binding of SH2 domain proteins p85, GRB2, SHP2 and others (not shown). Metabolic signalling is accomplished largely by the recruitment of dimeric (p85•p110) PI3K to IRS, which stimulates (via PDK1) the activation of aPKCs (λ/ι, ζ) and AKT/PKB. In the absence of inhibitory phosphorylation by AKT, key regulatory proteins: (1) limit GLUT4-dependent glucose uptake (Akt substrate of 160 kDa; AS160); (2) inhibit the storage of glucose as glycogen (GSK3); (3) promote hepatic gluconeogenic gene transcription (forkhead box O1; FOXO1); and (4) block the stimulation of protein synthesis via mTORC1→S6K1 signalling (TSC2) [173]. IRS-mediated activation of the RAS→ERK kinase cascade is less important for metabolic control. Tyrosine phosphatase SHP2 negatively regulates insulin sensitivity, potentially in part by direct action on IRS phosphotyrosines. Multiple insulin-regulated S/T kinases (black ovals) can directly phosphorylate IRS, mediating positive and/or negative feedback regulation of insulin signalling (for more details, see Fig. 3). Arrows indicate stimulation/ activation, blocked lines indicate inhibition; green ‘P’, activating S/T phosphorylation; red ‘P’, inhibitory S/T phosphorylation. (b) Positive and negative regulation of AKT by tyrosine and feedback S/T phosphorylation of IRS. AKT is activated via PI3K/PDK1-dependent phosphorylation on T308 (lost in tissues lacking IRS), primed by phosphorylation on S473 by mTORC2 [21]. Excessive activation of mTORC1→S6K1 signalling by deletion of TSC1/2—or overexpression of their target, RHEB (not shown)—downregulates AKT activation, in part via mTOR- and S6K1-mediated S/T phosphorylation of IRS. Equivalent S/Ts on mouse (M:) and human (H:) IRS1 that are directly phosphorylated by mTOR and S6K1 in vitro are in bold type; those in parentheses are likely targets of mTOR. Related pathways that contribute to the regulation of AKT include: (1) mTORC1 phosphor-ylation/ stabilisation of GRB10 [174], which inhibits productive interaction of IRS with a key phosphotyrosine in IR [175]; (2) inhibition of mTORC2 by S6K1-mediated phosphorylation of rictor [176]; and (3) transcriptional upregulation, via hypoxia inducible factor 1α (HIF1α), of the phosphatidylinositol phosphatase known as phosphatase and tensin homologue (PTEN) [177]. The mTORC1, but not mTORC2, kinase complex is acutely sensitive to the inhibitor rapamycin

Fig. 2

Structure/function of IRS and relationship to S/T phosphorylation. (a) The IRS1 and IRS2 proteins consist of similar amino-terminal PH and PTB domains (~100 amino acids each), followed by long C-terminal tail regions that are apparently unstructured. Shown are phosphorylated tyrosines (p-Y) within IRS1 and IRS2 (conserved tyrosines lacking evidence of phosphorylation are in parentheses). The tyrosines on IRS1 demonstrated by mutation or MS to recruit dimeric PI3K, GRB2 or SHP2 signalling proteins are indicated by the red bars [18, 178]. IRS2 uniquely contains the kinase regulatory loop binding (KRLB) ‘domain’, which modulates interaction IR [28]. (b) S/T phosphorylation of IRS1. Shown above the sequence are 20 phospho-S/T residues (p-S/T) for which the function has been investigated using site-specific methods; these include S→A (alanine) or S→E (glutamic acid) mutation to ablate or mimic phosphorylation, as well as descriptive investigation with phospho-specific antibodies (commercially available antibodies in green). Shown below the sequence are 34 additional conserved phospho-S/T sites identified only by MS of mouse/rat IRS1; different/additional phospho-S/T are known in human IRS1 (hS629 and not shown). The size of the circles is proportional to the number of PubMed references and/or MS datasets per site at the time of writing within the curated database at www.phosphosite.org (accessed 31 December 2011). Four phospho-S/T were added from [58]. The mouse amino acid numbering is used here except as noted. ^aFor the corresponding human phospho-S/T, please see Fig. 3

Fig. 3

Functional effects of IRS1 S/T phosphorylation by insulin-regulated and heterologous kinase pathways. Shown are S/T sites and their known/implicated kinase(s) uniquely investigated by mutation in cells or mice, in vitro kinase assay, or discriminatory kinase-activating agents. Positive effects on insulin signalling (green-filled circles)— including temporally separable positive/negative effects (green centre)—are unique to S/T phosphorylation via insulin-regulated kinases. In contrast, only negative effects on insulin signalling (red-filled circles) are observed by phosphorylation of IRS1 by heterologous (non-insulin) signalling pathways. In some studies, negative effects of multiple S/T kinases are implied or cannot be ruled out (light red-filled circles): The letters next to a circle indicate that the function is inferred from a rodent (r) or human (h) study. S332/6 and S632/5 phosphorylations are assayed by commercial antibodies to paired phosphorylation sites