Abstract

Insulin receptor substrate 1 (IRS1) and IRS2 are well-characterized adapter proteins that relay signals from receptor tyrosine kinases to downstream components of signalling pathways. In contrast, the function of IRS4 is not well understood. IRS4 overexpression has been associated with acute lymphoblastic leukaemia and subungual exostosis, while point mutations of IRS4 have been found in melanomas. Here, we show that while IRS4 expression is low in most cancer cell lines, IRS4 mRNA and protein levels are markedly elevated in certain cells including the NCI-H720, DMS114, HEK293T and HEK293AAV lines. Surprisingly, IRS4 expression was also strongly induced when HEK293 cells were infected with retroviral particles and selected under puromycin, making IRS4 expression a potential off-target effect of retroviral expression vectors. Cells with high expression of IRS4 displayed high phosphatidylinositol (3,4,5)-trisphosphate (PIP3) levels, as well as elevated Akt and p70 S6 kinase activities, even in the absence of growth factors. PI 3-kinase (PI3K) signalling in these cells depends on IRS4, even though these cells also express IRS1/2. Knockdown of IRS4 also inhibited cell proliferation in cells with high levels of IRS4. Together, these findings suggest IRS4 as a potential therapeutic target for cancers with high expression of this protein.

Highlights

  • The insulin receptor substrate (IRS) proteins are a family of cytoplasmic adaptors that couple activation of the insulin receptor and other receptor tyrosine kinases to downstream PI 3-kinase (PI3K)–Akt and Ras signalling pathways [1,2,3,4,5]

  • In this study we show that while expression of IRS4 is generally low in the studied panel of cancer cell lines, it is high in NCIH720, DMS114, HEK293T and HEK293AAV cells and that PI3K signalling in these cell lines relies on IRS4, but not Insulin receptor substrate 1 (IRS1)

  • Elevated IRS4 expression was not associated with any particular cancer type, since high expression of IRS4 mRNA was found only in a small set of cell lines that had been derived from different cancer types

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Summary

Introduction

The insulin receptor substrate (IRS) proteins are a family of cytoplasmic adaptors that couple activation of the insulin receptor and other receptor tyrosine kinases to downstream PI3K–Akt and Ras signalling pathways [1,2,3,4,5]. Based on the phenotypes of knockout mice, IRS1 and IRS2 have complementary roles in insulin and growth factor signalling, while IRS1 and IRS3 have complementary roles in adipogenesis [6,7,8]. IRS4 is generally reported to be expressed at low levels, being picked up originally using sensitive phosphotyrosine antibodies in human embryonic kidney (HEK) cells and by PCR in rodent hypothalamus, where it functions in signalling from the insulin and leptin receptors [3,9,10,11,12]. IRS4 knockout mice exhibit mild defects in growth, reproduction and glucose homeostasis [13]. Overexpression of IRS4 rescues the effects of IRS1 and/or IRS2 knockout in rodent cells, and IRS4 levels were found to be increased during regeneration of resected rodent liver [14,15,16]. Compared with rodents, the relative roles of the IRS proteins may be different in humans, which lack IRS3

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