Characterisation of IRTKS, a novel IRSp53/MIM family actin regulator with distinct filament bundling properties

Abstract

IRSp53 is a scaffold protein that contains an IRSp53/MIM homology domain (IMD) that bundles actin filaments and interacts with the small GTPase Rac. IRSp53 also binds to the small GTPase Cdc42 and to Scar/WAVE and Mena/VASP proteins to regulate the actin cytoskeleton. We have characterised a novel IMD-containing protein, insulin receptor tyrosine kinase substrate (IRTKS), which has widespread tissue distribution, is a substrate for the insulin receptor and binds Rac. Unlike IRSp53, IRTKS does not interact with Cdc42. Expression of IRTKS induces clusters of short actin bundles rather than filopodia-like protrusions. This difference may be attributable to a short carboxyl-terminal (Ct) extension present on IRTKS, which resembles a WASP-homology 2 (WH2) motif. Addition of the Ct extension to IRSp53 causes an apparent shortening of bundles induced by the IMD in vitro, and in cultured cells, suggesting that the Ct extension of IRTKS modulates the organising activity of the IMD. Lastly, we could not detect actin monomer sequestration by the Ct extension of IRTKS as would be expected with a conventional WH2 motif, but it did interact with actin filaments.

Deficiency of IRTKS as an adaptor of insulin receptor leads to insulin resistance

“…To test this hypothesis, we evaluated the tyrosine phosphorylation state of endogenous IRTKS in response to insulin stimulation by an immunoprecipitation assay. The data indicate that IRTKS is tyrosine-phosphorylated in the liver cancer cell line Huh-7 (Supplementary information, Figure S4A), which is consistent with previous studies in COS-7 cells co-transfected with ectopic IRTKS and IRβ [10]. These suggest that IRTKS could be a component of insulin signaling.…”

“…MIM has been shown to be involved in cancer invasion and metastasis [18]. Similar to IRSp53, IRTKS plays an important role in the formation of membrane protrusions [10,19]. It should be noted that although IRSp53 and IRTKS were known to be tyrosinephosphorylated in response to insulin stimulation [6,10], very little is known about the role of these proteins in insulin signaling and resistance and the development of diabetes.…”

“…However, the molecular mechanism by which IRTKS contributes to insulin signaling is unknown. We noted that unlike IRSp53 expression, which is relatively enriched in the brain, IRTKS expression is widespread and is particularly found in the liver [10], raising the possibility that IRTKS might play an important role in liver. To explore the contribution of IRTKS to insulin signaling, insulin resistance and diabetes development, we generated IRTKS-deficient mice by targeting the gene via homologous recombination.…”

“…www.cell-research.com | Cell Research Figure S2G) [10]. We also evaluated the expression of certain genes involved in gluconeogenesis in livers from IRTKS-deficient mice by real-time RT-PCR.…”

“…However, whether IRSp53 deficiency disrupts insulin signaling and leads to insulin resistance was not evaluated. Similarly to IRSp53, IRTKS can induce the clustering of short actin bundles, cause membrane protrusions [10] and trigger pathogen-driven actin assembly [11][12][13]. Tyrosine phosphorylation of IRTKS occurred in response to insulin stimulation when ectopic IRTKS and insulin receptor (IR) β-subunit (IRβ) were co-transfected into COS-7 cells [10], which implies that IRTKS could be a component of insulin signaling.…”