Shuichi Okada

Molecular Basis of Insulin-stimulated GLUT4 Vesicle Trafficking LOCATION! LOCATION! LOCATION!

Among all the diverse actions of insulin, one of the most critical and intensively studied is its regulation of glucose homeostasis. In the postabsorptive state, insulin action in muscle and adipose tissue results in increased glucose uptake from the circulation, thereby maintaining plasma euglycemia and preventing hyperglycemia (1–3). Defects in this pathway result in insulin resistance, a condition in which excessive concentrations of insulin are required to reduce blood glucose levels and often precede the development of frank Type II diabetes (3, 4). Although it has been appreciated for almost two decades that this major action of insulin results from a redistribution of glucose transporter proteins from intracellular storage sites to the plasma membrane (5, 6), the cellular mechanisms responsible for these trafficking events and the defects associated with insulin resistance have remained largely enigmatic.

Synip: A Novel Insulin-Regulated Syntaxin 4–Binding Protein Mediating GLUT4 Translocation in Adipocytes

Insulin-stimulated glucose transport and GLUT4 translocation require regulated interactions between the v-SNARE, VAMP2, and the t-SNARE, syntaxin 4. We have isolated a novel syntaxin 4-binding protein, Synip, which specifically interacts with syntaxin 4. Insulin induces a dissociation of the Synip:syntaxin 4 complex due to an apparent decrease in the binding affinity of Synip for syntaxin 4. In contrast, the carboxyterminal domain of Synip does not dissociate from syntaxin 4 in response to insulin stimulation but inhibits glucose transport and GLUT4 translocation. These data implicate Synip as an insulin-regulated syntaxin 4-binding protein directly involved in the control of glucose transport and GLUT4 vesicle translocation.

The 66-kDa Shc isoform is a negative regulator of the epidermal growth factor-stimulated mitogen-activated protein kinase pathway.

In addition to tyrosine phosphorylation of the 66-, 52-, and 46-kDa Shc isoforms, epidermal growth factor (EGF) treatment of Chinese hamster ovary cells expressing the human EGF receptor also resulted in the serine/threonine phosphorylation of approximately 50% of the 66-kDa Shc proteins. The serine/threonine phosphorylation occurred subsequent to tyrosine phosphorylation and was prevented by pretreatment of the cells with the MEK-specific inhibitor PD98059. Surprisingly, only the gel-shifted 66-kDa Shc isoform (serine/threonine phosphorylated) was tyrosine phosphorylated and associated with Grb2. In contrast, only the non-serine/threonine-phosphorylated fraction of 66-kDa Shc was associated with the EGF receptor. To assess the relationship between the three Shc isoforms in EGF-stimulated signaling, the cDNA encoding the 66-kDa Shc species was cloned from a 16-day-old mouse embryo library. Sequence alignment confirmed that the 66-kDa Shc cDNA resulted from alternative splicing of the primary Shc transcript generating a 110-amino acid extension at the amino terminus. Co-immunoprecipitation of Shc and Grb2 from cells overexpressing the 52/46-kDa Shc isoforms versus the 66-kDa Shc species directly demonstrated a competition of binding for a limited pool of Grb2 proteins. Furthermore, expression of the 66-kDa Shc isoform markedly accelerated the inactivation of ERK following EGF stimulation. Together, these data indicate that the serine/threonine phosphorylation of 66-kDa Shc impairs its ability to associate with the tyrosine-phosphorylated EGF receptor and can function in a dominant-interfering manner by inhibiting EGF receptor downstream signaling pathways.

Insulin regulates the dynamic balance between Ras and Rap1 signaling by coordinating the assembly states of the Grb2–SOS and CrkII–C3G complexes

Insulin stimulation of Chinese hamster ovary cells expressing the human insulin receptor resulted in a time‐dependent decrease in the amount of GTP bound to Rap1. The inactivation of Rap1 was associated with an insulin‐stimulated decrease in the amount of Rap1 that was bound to Raf1. In parallel with the dissociation of Raf1 from Rap1, there was an increased association of Raf1 with Ras. Concomitant with the inactivation of Rap1 and decrease in Rap1–Raf1 binding, we observed a rapid insulin‐stimulated dissociation of the CrkII–C3G complex which occurred in a Ras‐independent manner. The dissociation of the CrkII–C3G was recapitulated in vitro using a GST–C3G fusion protein to precipitate CrkII from whole cell detergent extracts. The association of GST–C3G with CrkII was also dose dependent and demonstrated that insulin reduced the affinity of CrkII for C3G without any effect on CrkII protein levels. Furthermore, the reduction in CrkII binding affinity was reversible by tyrosine dephosphorylation with PTP1B and by mutation of Tyr221 to phenylalanine. Together, these data demonstrate that insulin treatment results in the de‐repression of Rap1 inhibitory function on the Raf1 kinase concomitant with Ras activation and stimulation of the downstream Raf1/MEK/ERK cascade.

Insulin and epidermal growth factor receptors regulate distinct pools of Grb2-SOS in the control of Ras activation.

Insulin and epidermal growth factor (EGF) stimulate a rapid but transient increase in the amount of GTP bound to Ras that returns to the basal GDP-bound state within 10-30 min. Although insulin stimulation resulted in a dissociation of the Grb2·SOS complex, EGF did not affect the Grb2·SOS complex but instead induced dissociation of Grb2-SOS from tyrosine-phosphorylated Shc. The dissociation of Grb2-SOS from Shc was not due to dephosphorylation as Shc remained persistently tyrosine-phosphorylated during this time. Furthermore, there was no decrease in the extent of insulin receptor substrate 1, insulin receptor, or EGF receptor tyrosine phosphorylation. Surprisingly, however, despite the EGF-induced decrease in the amount of Grb2-SOS bound to Shc, the extent of Grb2 associated with Shc remained constant, and there was a concomitant increase in the amount of SOS associated with Grb2. In addition, after the insulin-stimulated dissociation of Grb2 from SOS, EGF treatment induced the reassociation of the Grb2·SOS complex. Quantitative immunoprecipitation demonstrated that only a small fraction of the total cellular pool of Grb2 was associated with SOS. Similarly, only a small fraction of SOS and Grb2 were co-immunoprecipitated with Shc. Together, these data suggest the presence of distinct Grb2-SOS pools that are independently utilized by insulin and EGF in their recruitment to tyrosine-phosphorylated Shc.

Adenovirus-mediated transfer of a modified human proinsulin gene reverses hyperglycemia in diabetic mice

The human proinsulin cDNA was introduced into a replication-defective adenovirus and was found to confer proinsulin expression to a hepatocyte (H4-II-E) cell line upon infection. A second virus was constructed in which the dibasic prohormone convertase recognition sequence was mutated to a tetrabasic furin cleavage site. Cells infected with this virus synthesized both proinsulin and mature insulin. Gel filtration chromatography, competition of insulin binding, and activation of the insulin receptor kinase activity demonstrated that this mature insulin was functionally identical to that of authentic processed insulin. Injection of these viral constructs into the external jugular vein of mice resulted in insulin gene expression in the liver. Expression from the mutated proinsulin virus dramatically improved the glycemic state of diabetic mice. However, the effects of the viral infection were transient, being maximal at ∼5-7 days and returning to steady-state levels by 14-21 days. These data demonstrate that somatic cell insulin gene delivery by the use of recombinant adenovirus can be used to transiently reverse the diabetic state in mice.

Interactions between Src Homology (SH) 2/SH3 Adapter Proteins and the Guanylnucleotide Exchange Factor SOS Are Differentially Regulated by Insulin and Epidermal Growth Factor

Co-immunoprecipitation of whole cell extracts demonstrated that the guanylnucleotide exchange factor SOS was associated with the small adapter proteins Grb2, CrkII, and Nck. In vitro binding indicated a similar binding affinity of SOS for all three adapter proteins but with a slightly lower Kd for Grb2 (∼2.5-fold) compared with Nck and CrkII. Insulin stimulation resulted in co-immunoprecipitation of tyrosine-phosphorylated IRS1 with Grb2 and to a lesser extent CrkII. Although Grb2 also associated with tyrosine-phosphorylated Shc, there was no detectable interaction of CrkII with Shc. In contrast, EGF stimulation resulted in the predominant co-immunoprecipitation of Grb2 with the EGF receptor, whereas CrkII primarily associated with an unidentified 120-130-kDa protein. Similar to the ability of insulin to induce the dissociation of the Grb2-SOS complex, there was a concomitant time-dependent dissociation of the CrkII-SOS and Nck-SOS complexes. However, EGF stimulation had no effect on the association state of the Grb2-SOS or the Nck-SOS complexes but did result in a time-dependent dissociation of the CrkII from SOS. Together, these data demonstrate that different cellular pools of SOS associate with different adapter proteins forming various signaling complexes, each undergoing distinct patterns of assembly/disassembly following growth factor stimulation.

Insulin-induced Desensitization of Extracellular Signal-regulated Kinase Activation Results from an Inhibition of Raf Activity Independent of Ras Activation and Dissociation of the Grb2-SOS Complex

Previous studies have suggested that the interaction between the small adaptor protein Grb2 with the Ras guanyl nucleotide exchange factor SOS is functionally important in the regulation of the Ras activation/inactivation cycle. To examine the relationship between the Grb2-SOS complex and Ras activation, we observed that insulin stimulation results in a rapid but transient activation of Ras and the extracellular-signal regulated kinase (ERK) followed by dissociation of the Grb2-SOS complex. Although treatment with the phorbol myristate acetate resulted in ERK activation and complete dissociation of the Grb2-SOS complex, there was no effect on subsequent insulin-stimulated Ras activation. Similarly, insulin stimulation followed by insulin removal resulted in a time-dependent restoration of the Grb2-SOS complex but which was significantly slower than the recovery of insulin-stimulated Ras activation. In addition, although insulin was able to activate Ras under these conditions, there was a complete desensitization of Raf and ERK activation. This apparent homologous desensitization of insulin action was specific for Raf and ERK as the insulin re-stimulation of insulin receptor autophosphorylation and protein kinase B activation were unaffected. Together, these data demonstrate the presence of a pathway independent of the Grb2-SOS complex that can lead to Ras activation but that the desensitization of Raf accounts for the homologous desensitization of ERK.

Insulin and Epidermal Growth Factor Stimulate a Conformational Change in Rap1 and Dissociation of the CrkII-C3G Complex

Insulin and epidermal growth factor (EGF) stimulation of Chinese hamster ovary cells expressing the human insulin and EGF receptors resulted in a time-dependent decrease in the ability of a Rap1 antibody (amino acid epitope 121–136) to immunoprecipitate Rap1 from whole cell detergent extracts. This was due to an apparent masking of Rap1 as heat denaturation of the whole cell detergent extracts (5 min at 100u2009°C) resulted in equal immunoprecipitation of Rap1 with this epitope-specific antibody. The time-dependent change in Rap1 immunoreactivity was paralleled with an insulin-stimulated dissociation of the CrkII-C3G complex. Similarly, EGF treatment also resulted in a time-dependent dissociation of the CrkII-C3G complex that occurred concomitant with the masking of the 121–136 Rap1 epitope. Furthermore, pretreatment of the cells with the tyrosine kinase inhibitor, genistein, decreased both the basal and insulin-stimulated tyrosine phosphorylation of CrkII that directly correlated with the amount of CrkII that was immunoprecipitated with C3G. Together, these data suggest that insulin and EGF stimulation result in the dissociation of the CrkII-C3G complex, thereby inducing an apparent conformation change in Rap1.

Insulin Receptor-mediated Dissociation of Grb2 from Sos Involves Phosphorylation of Sos by Kinase(s) Other than Extracellular Signal-regulated Kinase

The Ras signaling pathway is rapidly activated and then down-regulated following stimulation of multiple cell-surface receptors including the insulin receptor (IR). Much recent attention has focused on elucidating the mechanism of Ras inactivation following IR engagement. Previous data suggest that IR-mediated serine/threonine phosphorylation of the Ras guanine nucleotide exchange factor Sos correlates with its decreased affinity for the adapter protein Grb2. This phosphorylation-induced disassembly of the Grb2·Sos complex is thought to be responsible, at least in part, for diminishing Ras activity in Chinese hamster ovary cells. In this report, we confirm the causal relationship between Sos phosphorylation and Grb2/Sos dissociation. We then examine several putative phosphorylation sites of Sos that could potentially regulate this event. Since a number of reports suggest that extracellular signal-regulated kinase (ERK) phosphorylates Sos, we generated a Sos mutant lacking all seven canonical phosphorylation sites for ERK. This mutant is a poor substrate of activated ERK in vitro and fails to undergo a change in its electrophoretic mobility following IR stimulation. It is, however, phosphorylated after IR stimulation when expressed in Chinese hamster ovary cells. Interestingly, the mutant protein still dissociates from Grb2 following insulin stimulation, suggesting that ERK is not the kinase responsible for regulating the stability of the Grb2·Sos complex.