Although insulin is generally considered an anabolic hormone that supports protein synthesis and inhibits general protein degradation (21
), evidence has accumulated over the past 5 years suggesting that prolonged exposure of cells to insulin promotes IRS-1 degradation (23
). We previously identified the proteasome degradation pathway as a major mechanism that specifically regulates the cellular level of IRS-1 but not IRS-2 during prolonged insulin exposure in a cell culture system (68
). In the current study, we have demonstrated, for the first time, that ubiquitin conjugation of IRS-1 is a prerequisite for insulin-induced IRS-1 proteasome degradation. The N-terminal region of IRS-1 contains the structural element that specifically targets IRS-1 to the ubiquitin-proteasome degradation pathway. Both tyrosyl phosphorylation of IRS-1 and PI 3-kinase activation are needed to activate the IRS-1 ubiquitin-proteasome degradation pathway. We have provided evidence against mTOR's playing a significant role in the process of IRS-1 proteasome degradation in CHO cells. Together, these results provide insight into the mechanism of activation of the IRS-1 ubiquitin-proteasome degradation pathway during prolonged insulin exposure, which may underlie the molecular mechanism of insulin resistance.
Proteasome degradation is an important cellular system that not only functions to eliminate malfunctioning or misfolded proteins but also acts as a regulatory mechanism to precisely control the levels of cellular proteins and thus their biological functions in a timely manner (9
). Proteasome degradation is a highly controlled and specific process and depends largely on ubiquitination of the targeted protein (10
). In addition to the three-step ubiquitination reaction involving E1, E2, and E3 ligases, recent studies have shown that ubiquitination of targeted proteins is regulated by a signaling pathway which activates a specific E3 ligase (10
Although we and others have previously shown by immunoblotting with antiubiquitin antibodies, or a similar technique, that IRS-1 is indeed ubiquitinated (35
), the requirement for ubiquitination of IRS-1 in terms of proteasome degradation had not been established prior to this study. ts20 cells were derived from chemically mutagenized E36 cells and shown to contain a temperature-sensitive mutation in the ubiquitin-activating enzyme E1 (22
). ts20 cells which overexpress the human insulin receptor (ts20/IR) were used to test the requirement for ubiquitination in the insulin-induced degradation of IRS-1. Inactivating E1 in ts20/IR cells by raising the temperature to 40°C completely blocked the insulin-induced degradation of IRS-1, clearly demonstrating that insulin-induced degradation of IRS-1 is a ubiquitin-dependent process. This result further suggests that prolonged insulin exposure activates a signaling pathway that mediates the ubiquitination and proteasome degradation of IRS-1 (IRS-1 ubiquitin-proteasome degradation pathway).
The insulin receptor is absolutely required for the insulin-induced degradation of IRS-1, suggesting that the activation of the IRS-1ubiquitin degradation pathway is mediated by insulin receptor signaling. Binding of insulin to its receptor activates the intrinsic protein tyrosyl kinase, which subsequently phosphorylates its intracellular substrates on tyrosine residues, including IRS proteins (77
). The NPXY960
motif at the juxtamembrane region of the insulin receptor has been shown to interact with the PTB domain of IRS-1 when it is phosphorylated (20
), and mutation at tyrosine960
significantly decreases its ability to phosphorylate IRS-1 (27
). Tyrosyl phosphorylation of IRS proteins is an important step to activate insulin intracellular signaling (77
Here we showed that tyrosyl phosphorylation of IRS-1 is also a critical step for the insulin-induced degradation of IRS-1. First, cells (CHO, E36, and ts20 cells) with low levels of insulin receptors displayed only a modest tyrosyl phosphorylation of IRS-1 in response to acute insulin stimulation and a modest degradation of endogenous IRS-1 during prolonged insulin exposure. However, overexpression of insulin receptor in these cells dramatically increased insulin-induced tyrosyl phosphorylation of endogenous IRS-1 and concomitantly increased insulin-induced ubiquitin-proteasome-dependent degradation of IRS-1. Second, a good correlation between decreased tyrosyl phosphorylation of IRS-1 and decreased insulin-induced degradation of IRS-1 in cells overexpressing IRA960 or IRF960 was observed at all insulin concentrations tested (from 1 to 100 nM) compared with the cells overexpressing wild-type insulin receptors. Finally, fetal bovine serum failed to induce tyrosyl phosphorylation of IRS-1 and degradation of IRS-1, although it activated PI 3-kinase and Akt. Taken together, these data strongly suggest that tyrosyl phosphorylation of IRS-1 is an initial step in the activation of the IRS-1 ubiquitin-proteasome degradation pathway.
An IRS-1 mutant in which 18 potential tyrosyl phosphorylation sites were replaced by phenylalanines (IRS-1F18
) is unable to undergo tyrosyl phosphorylation or mediate activation of PI 3-kinase and p70S6K
during insulin stimulation (41
). To determine if it can be degraded by the ubiquitin-proteasome system, IRS-1F18
was overexpressed in CHO/IR cells, in which the IRS-1 ubiquitin-proteasome degradation pathway is activated by endogenous IRS-1 during prolonged insulin exposure. The insulin-stimulated tyrosyl phosphorylation of the endogenous IRS proteins and PI 3-kinase activity in CHO/IR/IRS-1F18
cells was comparable to that in CHO/IR cells suggesting that IRS-1F18
does not function as a dominant negative molecule to block endogenous IRS-1 signaling. Therefore, it is reasonable to assume that in CHO/IR/IRS-1F18
cells, the IRS-1 ubiquitin-proteasome degradation pathway is activated normally through the endogenous IRS-1 during prolonged insulin exposure. Interestingly, IRS-1F18
was degraded at the same rate as wild-type IRS-1 in CHO/IR cells during prolonged insulin exposure, suggesting that unphosphorylated IRS-1 can be targeted for ubiquitin-proteasome degradation.
Tyrosyl phosphorylation of IRS-1 promotes the interaction of IRS-1 and SH2 domain-containing proteins, including PI 3-kinase, Grb2, Nck, Fyn, and SHP-2, leading to the activation of signaling pathways mediated by these molecules (77
). To identify the downstream molecules that mediate the activation of the IRS-1 ubiquitin-proteasome degradation pathway, we used a wide range of inhibitors for serine kinases, including PKCs, MAP kinases, mTOR, and PI 3-kinase. Among the inhibitors tested, LY294002 and wortmannin effectively blocked the insulin-induced degradation of IRS-1, suggesting that PI 3-kinase activity is essential for the IRS-1 ubiquitin-proteasome degradation pathway.
The insulin-induced activation of PI 3-kinase is mediated by tyrosyl-phosphorylated IRS-1 (4
), consistent with the requirement of tyrosyl phosphorylation of IRS-1 for activation of the IRS-1 ubiquitin-proteasome degradation pathway. More interestingly, LY294002 was able to completely block IRS-1 degradation even after the IRS-1 ubiquitin-proteasome degradation pathway had been activated for up to 5 h. This suggests that the activity of PI 3-kinase is essential not only to activate but also to maintain the IRS-1 ubiquitin-proteasome degradation pathway.
The essential role of PI 3-kinase in IRS-1 degradation raises the important question of whether the activation of PI 3-kinase is sufficient for IRS-1 degradation. Fetal bovine serum contains various growth factors which can effectively activate PI 3-kinase and downstream signaling molecules, including Akt and mTOR. Our data clearly demonstrate that fetal bovine serum failed to induce IRS-1 degradation, suggesting that PI 3-kinase activation alone is not sufficient for IRS-1 degradation. The fact that fetal bovine serum failed to induced tyrosyl phosphorylation of IRS-1 further supports the notion that activation of PI 3-kinase via interaction with tyrosyl-phosphorylated IRS-1 is essential for the IRS-1 ubiquitin-proteasome degradation pathway.
The location of the activated PI 3-kinase may be important, since platelet-derived growth factor (PDGF)-activated-PI 3-kinase associates mainly with the PDGF receptor at the plasma membrane, whereas the majority of insulin-stimulated IRS-1-associated PI 3-kinase activity is in the low-density microsomes (42
). Overexpressing a constitutively active form of PI 3-kinase, p110CAAX
, in the adenovirus system has been shown to induce IRS-1 degradation in the absence of insulin in 3T3-L1 adipocytes (16
), suggesting that PI 3-kinase activation is sufficient for IRS-1 degradation. The reason for this contradiction is not clear. It is possible that overexpression of a large quantity of a constitutively active form of PI 3-kinase may overcome the need for tyrosyl phosphorylation of IRS-1. Another possibility is that p110CAAX
is a membrane-targeted construct and may target PI 3-kinase activity to a compartment to which wild-type PI 3-kinase is never translocated without binding tyrosyl-phosphorylated IRS-1.
Rapamycin, a specific inhibitor of the mammalian target of rapamycin (mTOR) and a downstream molecule of PI 3-kinase (58
), has been shown to block the insulin-induced degradation of IRS-1 (23
) and tumor necrosis factor alpha-induced serine636
phosphorylation in IRS-1 in 3T3-L1 adipocytes (44
). Our data agree with these studies in that rapamycin did prevent serine phosphorylation of IRS-1 manifested by prevention of the IRS-1 mobility shift on SDS-PAGE, and increased insulin-induced tyrosyl phosphorylation of IRS-1 to some degree, and the activation of p70S6K
in CHO/IR cells. However, rapamycin failed to block the insulin-induced degradation of IRS-1, suggesting that a rapamycin-sensitive molecule (most likely mTOR) is not a major player in the activation of the IRS-1 ubiquitin-proteasome degradation pathway in CHO/IR cells. The reason for these different findings remains unknown. Insulin-induced degradation of IRS-1 in 3T3-L1 adipocytes can be blocked by a calcium chelator or specific inhibitors for calpain, indicating a Ca2+
-dependent and calpain-mediated process (62
). Furthermore, IRS-1 can be degraded by calpain in vitro (61
). One possible explanation is that in 3T3-L1 adipocytes, in addition to the proteasome degradation pathway (a Ca2+
-independent process), IRS-1 degradation may also be controlled by a Ca2+
-dependent calpain degradation pathway which may be sensitive to rapamycin.
Serine phosphorylation has been shown to be an important regulatory mechanism in the ubiquitin-proteasome degradation pathway. A growing number of E3 ligases have been identified as SCF (Skp/Cullin/F-box protein) complexes which couple the protein kinase signaling pathway to the control of protein abundance (18
). F-box proteins in SCF complexes serve as receptors that recruit serine-phosphorylated substrates to the SCF ubiquitin-ligase complexes, a mechanism that controls not only the substrate specificity but also the timing (60
). For example, the IκB-ubiquitin ligase complex only ubiquitinates IκBα when it is phosphorylated (64
), whereas β-catenin ubiquitination is preceded by phosphorylation via the glycogen synthase kinase 3β/Axin kinase complex, which coexists in the ubiquitin ligase complex (31
Studies using in vivo 32
P metabolic labeling of CHO/IR/IRS-1 have shown that insulin induces serine/threonine phosphorylation of IRS-1 (69
), which is prevented by preincubation of cells with wortmannin (unpublished data). Inactivation of PI 3-kinase by LY294002 before or after insulin stimulation not only blocked the degradation of IRS-1, but also prevented the mobility shift of IRS-1 on SDS-PAGE (an indication of serine phosphorylation of IRS-1) and increased the insulin-induced tyrosyl phosphorylation of IRS-1, suggesting that PI 3-kinase-dependent serine phosphorylation of IRS-1 might be involved in the regulation of IRS-1 ubiquitin-proteasome degradation. Although direct evidence is lacking, it is tempting to speculate that the IRS-1 ubiquitin ligase complex may associate with an LY294002-sensitive serine kinase which phosphorylates IRS-1, triggering ubiquitination. Since PI 3-kinase has been reported to phosphorylate IRS-1 in αIRS-1 immune complexes in a wortmannin-sensitive manner (74
), it is possible that PI 3-kinase is the serine kinase. Further work needs to be done to test this idea.
The IRS-1 ubiquitin-proteasome degradation pathway is specific for IRS-1 and is completely ineffective for IRS-2 in both CHO/IR cells and 3T3-L1 adipocytes (48
). This suggests that a unique structural element present in IRS-1 but absent in IRS-2 is important for IRS-1 degradation. The results from IRS chimeras confirmed that this structural element does exist. IRS-1 with its N-terminal region replaced by that of IRS-2 lost the ability to be degraded, whereas IRS-2 with its N-terminal region replaced by that of IRS-1 gained the ability to be degraded, suggesting that the structural element for IRS-1 degradation resides within the N-terminal region of IRS-1, where the PH, PTB, and IH3 domains (71
) are located. Interestingly, this region of IRS-1 shows the highest homology with IRS-2 (71
Exactly how the N-terminal region of IRS-1 plays a role in controlling the ubiquitin-proteasome degradation of IRS-1 is not known. It may contain ubiquitination sites, phosphorylation sites for recognition by the IRS-1 ubiquitin ligase complex, or a domain that interacts with the ubiquitin ligase. Further investigation should answer these key questions.