Effect of IRS4 levels on PI 3-kinase signalling - PubMed (original) (raw)

Effect of IRS4 levels on PI 3-kinase signalling

Gerta Hoxhaj et al. PLoS One. 2013.

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.

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Conflict of interest statement

Competing Interests: The authors thank the UK Medical Research Council and pharmaceutical companies that support the Division of Signal Transduction Therapy (DSTT) at University of Dundee (AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline, Merck-Serono and Pfizer) for financial support. The Oncomine data (Supporting Table S1) was searched as part of a collaboration with AstraZeneca. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials. AstraZeneca had no further role in the study design, data collection and analysis, nor in the decision to publish, nor preparation of the manuscript.

Figures

Figure 1

Figure 1. Expression of IRS4, IRS1 and IRS2 in various cell lines.

(A) The graph represents the IRS4 mRNA expression in 298 cancer cell lines. Data in Oncomine (

www.oncomine.com

) was searched for mRNA expression in cell lines, normalised with multiple probe sets . The higher the value, the higher the expression of IRS4 mRNA. (B) Cell lysates from the indicated cell lines were subjected to immunoblotting with the IRS4, IRS1, IRS2 and GAPDH antibodies

Figure 2

Figure 2. Effects of IRS1 and IRS4 knockdown on the PI3K pathway in cells with low and high IRS4 protein levels.

(A) Cell lines exhibiting high levels of IRS4 such as DMS114, NCI-H720, HEK293AAV, HEK293T were transfected with 100 nM final concentration of control (Scrambled), IRS4 or IRS1 siRNAs using Dharmafect 1 transfection reagent following manufacturer's instructions. After 48 h, cells were harvested and lysates (20 µg) were immunoblotted using the indicated antibodies to test the activation status of the PI3K pathway. (B) As in (A) except that the experiment was performed in cells with low expression of IRS4 such as HeLa, A431, HEK293, MDA-MB-231 and A549.

Figure 3

Figure 3. Effect of IRS4 knockdown on growth of DMS114, HEK293T and 293AAV cells.

(A) DMS114, (B) HEK293T and (C) 293AAV cells were transfected with control (Scrambled) and IRS4 siRNA oligos. At 24 h post transfection, cells were seeded in 24-well plates (20 000 cells per well). For each time point, cells were washed with phosphate-buffered saline (PBS), fixed in 4% (v/v) paraformaldehyde in PBS for 15 min, washed in water once, stained with 0.1% Crystal Violet in 10% ethanol for 20 min and washed thrice with water. Crystal Violet was extracted from cells with 0.5 ml of 10% (v/v) acetic acid for 20 min while shaking at room temperature and the absorbance measured at 590 nm.

Figure 4

Figure 4. HEK293 clones with increased IRS4 expression have elevated PI3K signalling.

(A) Clones of HEK293 cells expressing different levels of IRS4 (#1-low, #2- and #3-high) obtained after retroviral infection and puromycin selection as described in materials and methods, were serum starved and then stimulated with IGF1 or 10% FBS. Cell lysates (30 µg) were subject to immunoblotting with the indicated antibodies. (B) As in (A), except that cells were starved of amino acids and then stimulated with amino acids or 10% FBS for 20 min. (C and D) The cells expressing different levels of IRS4 were serum deprived overnight and assayed for the catalytic activity of immunoprecipitated endogenous Akt and S6K1 using Crosstide peptide as substrate, as described in materials and methods. (E) Clones #1 and #3 of HEK293 cells were serum starved overnight and assayed for PIP3 as in . Cells from clone #1 were serum starved and treated with IGF1+/− PI-103 (1 µM, 30 min) and used as positive and negative controls, respectively. (F) Cells expressing low (#1) or high (#2 and #3) levels of IRS4 were transfected with control, IRS4 or IRS1 siRNA oligos and the activity of PI3K pathway components was analysed as in (A) under serum starved conditions.

Figure 5

Figure 5. The effects of IRS4 overexpression on the PI3K pathway.

(A) U2OS-Flpin™ cells expressing empty vector or IRS4-FLAG protein were analysed for the activation of PI3K pathway components under serum starved (SS), amino acid-starved (-AA) and 10% FBS conditions. Cells were deprived of serum for 12 h (SS) and starved of amino in EBSS for 2 h (-AA). (B) U2OS-Flpin™ cells expressing empty vector, full length IRS4-FLAG (WT), and the indicated fragments containing residues from 200–end-IRS4-FLAG or 336–end-IRS4-FLAG proteins were analysed for the activation of PI3K pathway components, in the presence or absence of serum. (C) As in B, except that the cells were treated with IGF1 (50 ng/ml, 20 min). The FLAG tagged proteins expressed were analysed for the Tyrosine phosphorylation as well as the binding to the p85 regulatory subunit of PI3 kinase.

Figure 6

Figure 6. Deletion of PH domain S4 targets IRS4 to the nucleus.

U2OS-Flpin™ cells expressing full length IRS4-FLAG and the indicated fragments containing residues from 200–end-IRS4-FLAG or 336–end-IRS4-FLAG proteins were analysed for their subcellular localization, under serum starved (SS) or addition of IGF1(50 ng/ml, 20 min). Cells were stained for FLAG antibody and with DAPI as described in Materials and Methods.

Figure 7

Figure 7. Gene synteny analyses of human IRS1, IRS2, IRS4 and the IRS3P pseudogene.

A circle plot links the human gene synteny clusters containing human IRS1, IRS2, IRS4 genes and the IRS3P pseudogene. Plots were generated using the Synteny Database (teleost.cs.uoregon.edu/synteny_db) with the Branchiostoma genome as outgroup. Arcs around the circumference represent chromosomes, while those within the circle connect pairs of related genes. The human IRS1 (chromosome 2q36), IRS2 (13q34) and IRS4 (Xq22.3) genes and also genes within 500 kb of these three genes, were used as queries. Different chromosomes were tested in the fourth position until a strong synteny trace was observed for chromosome 7 as shown, and these synteny lines were then confirmed to converge on the human IRS3P pseudogene at 7q22.1.

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