In addition to type 2 diabetes, insulin resistance is associated with a variety of physiological states, including trauma and infection, hypertension, glucose intolerance, and obesity; however, the molecular mechanisms that modulate insulin signaling under these conditions are difficult to resolve. Many studies suggest that serine phosphorylation of the IRS proteins might inhibit insulin signaling, but the identification of physiologically relevant sites and the regulation of their phosphorylation and dephosphorylation has progressed slowly (26
). Recently, we discovered that JNK-1 associates with IRS-1 and promotes phosphorylation of Ser307
. Although our current results show that JNK is not the only kinase that phosphorylates Ser307
, several results including a Ser307
→Ala substitution reveals that the phosphorylation of Ser307
inhibits insulin-stimulated tyrosine phosphorylation of IRS-1 in cultured cells (26
The phosphorylation of specific residues in IRS-1 is an attractive regulatory mechanism because it provides specificity at multiple levels, including the interaction between specific serine kinases and IRS-1, regulation of the associated kinase activity, and selection of specific phosphorylation sites. However, it is difficult to demonstrate that serine phosphorylation of IRS-1 changes under specific physiological conditions, because IRS-1 contains nearly 100 potential serine phosphorylation sites that complicate ordinary biochemical analysis. Immunodetection of specific serine or threonine phosphorylation sites is successful in many cases. Applying this strategy to IRS-1, we prepared αpS307, a polyclonal antiserum generated in rabbits against the peptide sequence around phosphorylated Ser307 in IRS-1. Several experiments demonstrate that αpS307 recognizes specifically the phosphorylated Ser307 residue: αpS307 fails to immunoblot IRS-1 immunoprecipitated from quiescent cells; αpS307 strongly immunoblots IRS-1 immunoprecipitated from cells stimulated with anisomycin, TNF-α, IGF-1, or insulin; under stimulated conditions, dephosphorylation of IRS-1 immunoprecipitates with alkaline phosphatase inhibits immunoreactivity of αpS307; and phosphorylated peptides used to generate αpS307 inhibit immunoblotting, whereas unphosphorylated peptides have no inhibitory effect. Finally, αpS307 fails to recognize the mutant IRS-1 molecule containing the Ser307→Ala substitution. Together these results demonstrate that αpS307 reacts specifically with phosphorylated Ser307 in IRS-1 from mouse, rat, and human tissues; it is unreactive with other IRS proteins because the peptide sequence around Ser307 is unique to IRS-1.
The use of αpS307
provides clear evidence that Ser307
phosphorylation is regulated by several mechanisms. Although previous work suggested indirectly that anisomycin and TNF-α stimulate Ser307
phosphorylation, immunoblots with αpS307
confirm this conclusion for the first time. Moreover, these results support the hypothesis that the inhibitory effect of anisomycin and TNF-α on insulin-stimulated tyrosine phosphorylation is mediated by the phosphorylation of Ser307
). Moreover, immunoblotting with αpS307
can help establish biologic relevance, by assessing Ser307
phosphorylation in IRS-1 isolated from tissues. For example, Ser307
is phosphorylated in liver, fat, and muscle isolated from rats subjected to thermal injury, suggesting that TNF-α or other inflammatory cytokines might also have effects in animals (data not shown).
Surprisingly, insulin and IGF-1 stimulate phosphorylation of Ser307 in 3T3-L1 fibroblasts and adipocytes. This unexpected result appears to be physiologically important, as injection of insulin into mice or rats promotes Ser307 phosphorylation in muscle. In humans, pharmacological insulin levels achieved during a hyperinsulinemic euglycemic clamp stimulate Ser307 phosphorylation on IRS-1 immunopurified from muscle biopsies. Thus, the regulated phosphorylation of Ser307 might be a common pathway to counterregulate the insulin signal during traumatic stress or chronic hyperinsulinemia.
The phosphorylation of Ser307
in IRS-1 was first observed during in vitro phosphorylation experiments with recombinant IRS-1 and JNK-1. JNK-1 associates specifically with IRS-1 and, during this association, mediates phosphorylation of Ser307
). Because JNK-1 is activated by TNF-α or anisomycin, these results reveal a pathway to regulate IRS-1 serine phosphorylation. However, the finding that insulin stimulates Ser307
phosphorylation of IRS-1 suggests that other kinases might also be involved. Interestingly, inhibition of PI 3-kinases by LY294002 and wortmannin abrogates phosphorylation of Ser307
induced by insulin and IGF-1, but not by TNF-α. In contrast, inhibition of MEK1 by PD98059 abolishes phosphorylation of Ser307
induced by TNF-α, but not by insulin or IGF-1; this finding is unexpected because PD98059 is not reported to inhibit JNK or kinases upstream of JNK activation and suggests that another kinase might mediate the effect of TNF-α on IRS-1. Thus, at least three kinases mediate phosphorylation of Ser307
, including JNK, serine kinases in the PI 3-kinase cascade that are activated by insulin or IGF-1, and MEK1-sensitive kinase cascades during TNF-α stimulation. Thus, Ser307
phosphorylation might integrate signals mediated through multiple serine kinases, including those promoted by heterologous ligands as well as insulin itself.
Interestingly, although PD98059 blocks the MEK/ERK pathway activated by both insulin/IGF-1 and TNF-α, it only inhibits TNF-α-induced Ser307 phosphorylation. These observations suggest that the MEK/ERK cascade might be required, but is not sufficient for Ser307 phosphorylation. Perhaps other signals that are specific to TNF-α but not insulin/IGF-1 act together with the MEK/ERK pathway to induce Ser307 phosphorylation. In this scenario, the MEK/ERK pathway is not involved in phosphorylation of Ser307 in response to insulin and IGF-1. Alternatively, an unidentified, PD98059-sensitive kinase(s) that is activated by TNF-α but not insulin/IGF-1 mediate TNF-α-stimulated Ser307 phosphorylation.
Our results suggest that phosphorylation of Ser307 in IRS-1 might be a common site integrating heterologous inhibition of the insulin signal by certain cytokines with feedback inhibition during chronic hyperinsulinemia (Figure ). Consistent with this conclusion, insulin and IGF-1 act synergistically with TNF-α to promote phosphorylation of Ser307 to a much higher extent. A potential mechanism for peripheral insulin resistance emerges from these results. During the early stages of glucose intolerance, moderate hyperinsulinemia compensates for peripheral insulin resistance. Although this strategy promotes adequate metabolic regulation in moderate cases, chronic hyperinsulinemia might increase the steady-state level of Ser307 phosphorylation, eventually reducing the available pool of active IRS-1 molecules. Compensation might continue to adjust to this dynamic interaction until heterologous mechanisms, such as cytokine-stimulated phosphorylation of Ser307, exacerbate the situation and diabetes ensues because β cells fail to produce sufficient insulin (Figure ).
Figure 9 Models for the function of Ser307 phosphorylation. Upon insulin stimulation, IRS-1 is tyrosyl-phosphorylated by the IR, resulting in the activation of PI 3-kinase that mediates Ser307 phosphorylation. Ser307 phosphorylation subsequently inhibits the ability (more ...)
At the molecular level, the mechanism of inhibition of IRS-1 by Ser307
phosphorylation is an important question, but the answer is unclear. Previous reports suggest that serine or threonine phosphorylation of IRS-1 impairs its association with the insulin receptor (23
). This inhibition might result from dysregulation of PTB domain. Previous reports indicate that TNF-α might inhibit the ability of the PTB domain in IRS-1 to interact with the phosphorylated NPEY-motif in the activated insulin receptor (23
). Given that Ser307
is adjacent to the PTB domain in IRS-1, this mechanism must be directly tested. Alternatively, phosphorylation of Ser307
might create a binding site for inhibitory molecules, including the binding of protein tyrosine phosphatases.
In summary, TNF-α, insulin, and IGF-1 stimulate phosphorylation of Ser307 in IRS-1 in cultured cells. Insulin also stimulates phosphorylation of Ser307 in skeletal muscle of mice, rats and humans. Insulin– and IGF-1–induced phosphorylation of Ser307 depends on PI 3-kinase, whereas phosphorylation of Ser307 induced by TNF-α requires the activation of MEK1 or an unknown PD98059-sensitive kinase. Phosphorylation of Ser307 inhibits subsequent tyrosyl phosphorylation of IRS-1 and activation of IRS-1–mediated signaling pathways in response to insulin, which might contribute to insulin resistance.