The pseudophosphatase phogrin enables glucose-stimulated insulin signaling in pancreatic β cells

Seiji Torii,Chisato Kubota,Naoya Saito,Ayumi Kawano,Ni Hou,Masaki Kobayashi,Ryoko Torii,Masahiro Hosaka,Tadahiro Kitamura,Toshiyuki Takeuchi,Hiroshi Gomi

doi:10.1074/jbc.ra117.000301

Abstract

Autocrine insulin signaling is critical for pancreatic β-cell growth and activity and is at least partially controlled by protein-tyrosine phosphatases (PTPs) that act on insulin receptors (IRs). The receptor-type PTP phogrin primarily localizes on insulin secretory granules in pancreatic β cells. We recently reported that phogrin knockdown decreases the protein levels of insulin receptor substrate 2 (IRS2), whereas high-glucose stimulation promotes formation of a phogrin-IR complex that stabilizes IRS2. However, the underlying molecular mechanisms by which phogrin affects IRS2 levels are unclear. Here, we found that relative to wildtype mice, IRS2 levels in phogrin-knockout mice islets decreased by 44%. When phogrin was silenced by shRNA in pancreatic β-cell lines, glucose-induced insulin signaling led to proteasomal degradation of IRS2 via a negative feedback mechanism. Phogrin overexpression in a murine hepatocyte cell line consistently prevented chronic insulin treatment-induced IRS2 degradation. In vitro, phogrin directly bound the IR without the assistance of other proteins and protected recombinant PTP1B from oxidation to potentiate its activity toward the IR. Furthermore, phogrin expression suppressed insulin-induced local generation of hydrogen peroxide and subsequent PTP1B oxidation, which allowed progression of IR dephosphorylation. Together, these results suggest that a transient interaction of phogrin with the IR enables glucose-stimulated autocrine insulin signaling through the regulation of PTP1B activity, which is essential for suppressing feedback-mediated IRS2 degradation in pancreatic β cells.

Highlights

Autocrine insulin signaling is critical for pancreatic ␤-cell growth and activity and is at least partially controlled by protein-tyrosine phosphatases (PTPs) that act on insulin receptors (IRs)
Phogrin expression suppressed insulin-induced local generation of hydrogen peroxide and subsequent PTP1B oxidation, which allowed progression of IR dephosphorylation. These results suggest that a transient interaction of phogrin with the IR enables glucosestimulated autocrine insulin signaling through the regulation of PTP1B activity, which is essential for suppressing feedback-mediated insulin receptor substrate 2 (IRS2) degradation in pancreatic ␤ cells
A slight reduction in IA-2 protein expression was observed in phogrin-deficient islets, but there were no significant changes in other insulin granule proteins, such as carboxypeptidase E (CPE), secretogranin III (SgIII), Rab27, and VAMP2 (Fig. 1F)

Summary

Results

To assess the effect of phogrin deletion on pancreatic ␤ cells, phogrinflox/flox mice (Fig. 1A) were crossed with rat insulin promoter (RIP)-cre mice to produce conditional knockout mice that have phogrin expression depletion in ␤ cells. IRS2 levels in KO mouse islets were consistently lower than those of control mice at different ages (Fig. 1F and Fig. S2) This result suggests that the proliferative activity of pancreatic ␤ cells is decreased by phogrin knockout via down-regulation of IRS2 protein levels. The Y2F mutant did not overexpress well (Fig. 3A, left), the protein content of Y2F and Y3F but not Y1F in the phogrin immunoprecipitates was significantly decreased compared with that of IR-EGFP WT (Fig. 3A, right) This result suggests that phosphorylated tyrosines in Y2 and Y3 sites are involved in phogrin binding. The precipitated IR-EGFP was incubated with purified glutathione S-transferase (GST)-fused PTP proteins, and bound proteins were analyzed by immunoblotting with anti-GST antibody Both phogrin and IA-2 bound to tyrosine-phosphorylated IR-EGFP, and not to EGFP (data not shown), but this binding was less efficient than TCPTP (Fig. 3B). Expression levels of IRS2, IA-2, CPE, SgIII, IR␤, cyclin D2, Rab, or VAMP2 protein quantified by densitometric imaging are shown as -fold increases Ϯ S.E. (n ϭ 4) compared with control

25 IRS2 mRNA

Discussion

Experimental procedures