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. 2017 Dec 6;474(24):4105-4118.
doi: 10.1042/BCJ20170620.

Regulation of insulin-like growth factor receptors by Ubiquilin1

Zimple Kurlawala  1   2   3 , Rain Dunaway  1 , Parag P Shah  1   2   3 , Julie A Gosney  1   2   3 , Leah J Siskind  1   2   3 , Brian P Ceresa  1   2   3 , Levi J Beverly  4   2   3
Affiliations

Regulation of insulin-like growth factor receptors by Ubiquilin1

Zimple Kurlawala et al. Biochem J. .

Abstract

Insulin-like growth factor-1 receptor (IGF1R) is a receptor tyrosine kinase that mediates growth, proliferation and survival. Dysregulation of IGF pathway contributes to the initiation, progression and metastasis of cancer and is also involved in diseases of glucose metabolism, such as diabetes. We have identified Ubiquilin1 (UBQLN1) as a novel interaction partner of IGF1R, IGF2R and insulin receptor (INSR). UBQLN family of proteins have been studied primarily in the context of protein quality control and in the field of neurodegenerative disorders. Our laboratory discovered a link between UBQLN1 function and tumorigenesis, such that UBQLN1 is lost and underexpressed in 50% of human lung adenocarcinoma cases. We demonstrate here that UBQLN1 regulates the expression and activity of IGF1R. Following loss of UBQLN1 in lung adenocarcinoma cells, there is accelerated loss of IGF1R. Despite decreased levels of total receptors, the ratio of active : total receptors is higher in cells that lack UBQLN1. UBQLN1 also regulates INSR and IGF2R post-stimulation with ligand. We conclude that UBQLN1 is essential for normal regulation of IGF receptors. UBQLN-1-deficient cells demonstrate increased cell viability compared with control when serum-starved and stimulation of IGF pathway in these cells increased their migratory potential by 3-fold. As the IGF pathway is involved in processes of normal growth, development, metabolism and cancer progression, understanding its regulation by Ubiquilin1 can be of tremendous value to many disciplines.

Keywords: Ubiquilin; insulin-like growth factor; receptor tyrosine kinases; trafficking; turnover.

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Conflict of interest statement

Conflict of Interest: The authors declare that they have no conflicts of interest with the contents of this article.

Figures

FIGURE 1
FIGURE 1. UBQLN1 interacts with IGF1R, IGF2R and INSR
(A) Schematic of Ubiquilin1 protein. UBQLN1 is a 590 amino acid protein with an N-terminal UBL (ubiquitin-like) domain, four STI chaperone like domains in the middle and a C-terminal UBA (ubiquitin-associated) domain. (B) HEK293T cells were transfected with FLAG-tagged UBQLN1 followed by co-immunoprecipitation (IP) by anti-FLAG antibody and mass spectrometry (MS) analysis. IGF1R, IGF2R and INSR were identified as some of the top interacting partners of FLAG-UBQLN1. (C) HEK293T cells were transiently transfected with siRNA for UBQLN1 knock down (siUBQLN1) followed by co-immunoprecipitation by anti-UBQLN1 antibody and Western Blot analysis. (D) Confocal microscopy images of indirect immunofluorescence staining for FLAG-UBQLN1 (red) and IGF1R (green) in HeLa cells. Co-localization was determined for using JACop plugin of ImageJ software. Automatic threshold for images were determined by the Costes method and overlap coefficients (Mander’s Correlation Coefficients) were calculated. For the 2 chosen fields, 15.8–33.5% of FLAG-UBQLN1 overlaps with IGF1R.
FIGURE 2
FIGURE 2. First two STI-1 domains of UBQLN1 are responsible for interaction with IGF1R
(A) Schematic of UBQLN1WT and engineered FLAG-tagged domain deletion constructs missing individual domains. (B) HEK293T cells were transfected with FLAG-UBQLN1ΔUBL, FLAG-UBQLN1ΔSTI-1 and FLAG-UBQLN1ΔUBA constructs followed by indirect immunofluorescence staining for FLAG-UBQLN1. (C) HEK293T cells were transfected with FLAG-tagged constructs of UBQLN1 (in A) followed by co-immunoprecipitation by anti-FLAG antibody and Western Blot analysis. (D) HEK293T cells were transfected with empty vector or FLAG-UBQLN1 and cells were cultured in complete media (COMPLETE) or serum-free media supplemented with IGF1 (SF, IGF1) or Linsitinib (SF, LINSITINIB), a small molecule inhibitor of IGF1R activity, followed by co-immunoprecipitation by anti-FLAG antibody and Western Blot analysis (IGF1:50ng/ml, Linsitinib:1uM). (E) HEK293T cells were transfected with empty vector or FLAG-UBQLN1 showed that FLAG-UBQLN1 interacts with both immature pro-form of IGF1R at 135kD and mature, processed IGF1R at 100kD.
FIGURE 3
FIGURE 3. UBQLN1 regulates expression and activity of IGF1R
(A) Expression and activity of IGF1R were tested in A549 lung cancer cells following downregulation of UBQLN1 with two different siRNA (U1 KD#1 and U1 KD#2). Cells were serum starved (SF) overnight (12 hours), incubated with protein synthesis inhibitor Cycloheximide one hour prior to supplementing serum-free media with IGF1. 6 hours later, cells were harvested analyzed by Western Blot. (B) Data are normalized to Actin in non-targeting siRNA control in unstimulated cells and represented as mean+/−SEM from 2 experiments. *p ≤ 0.05. Expression of IGF2R (C) and INSR (D) were tested in A549 lung cancer cells following downregulation of UBQLN1 with two different siRNA (U1 KD#1 and U1 KD#2). Cells were cultured as in (A). IGF2R and INSR expression decreases post IGF1 stimulation in UBQLN1 deficient cells. Expression of P-AKT (D) is increased in UBQLN1 deficient cells under serum-free and stimulated conditions while T-AKT expression remains unchanged compared to control. Data are normalized to Actin in non-targeting siRNA control in unstimulated cells. (E) There is a 2-fold decrease in IGF1R mRNA expression in A549 cells that have siRNA mediated loss of UBQLN1 (p=0.0015, SEM=0.04 for U1 KD#1, p=0.0094, SEM=0.06 for U1 KD#2). Data are represented as represented as mean+/−SEM from 3 independent quantitative real-time PCR experiments done in triplicates.
FIGURE 4
FIGURE 4. Loss of UBQLN1 accelerates loss of IGF1R
A549 cells expressing shRNA against UBQLN1 and non-targeting control were serum starved for 12 hours, followed by incubation with Cycloheximide (20uM), an inhibitor of de novo protein synthesis to study loss of IGF1R expression in UBQLN1 deficient cells, post-stimulation with IGF1. Cells were harvested at the indicated time points and Western Blot analysis for phosphorylated (A) and total receptor (B) levels were performed and graphed. Data are normalized to Actin in non-targeting shRNA control in unstimulated cells and represented as mean+/−SEM from 2 experiments.
FIGURE 5
FIGURE 5. UBQLN1 stabilizes IGF1R during its trafficking
A549 cells expressing shRNA against UBQLN1 and non-targeting control were serum starved for 12 hours followed by incubation with Monensin (10uM) (an early endosome inhibitor that traps internalized receptors and slows down recycling and receptors’ lysosomal turnover) an hour prior to stimulation with IGF1 and harvested post-stimulation at the indicated time points. Western Blot analysis for phosphorylated (A) and total receptor (B) levels were performed and graphed in both control and UBQLN1 deficient cells. Data are normalized to Actin in non-targeting shRNA control in unstimulated cells and represented as mean+/−SEM from 2 experiments.
FIGURE 6
FIGURE 6. UBQLN1 deficient A549 cells show increased cell viability and migration potential when serum starved and upon stimulation of the IGF pathway
(A,B) A549 cells expressing stable shRNA against UBQLN1 (U1 KD#1, U1 KD#2, and control) were cultured in conditions of serum-free media and serum-free media supplemented with IGF1 for 4 days. Alamar Blue readings were recorded every 24 hours and relative cell viability of UBQLN1 deficient cells were compared to control cells on each day. Day 2 onwards, UBQLN1 deficient cells gradually outsurvived control cells when cultured in serum-free media (A) and supplementing serum-free media with IGF1 (B) enhanced survival of these cells. Data are represented as mean±SEM from 2 independent experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, all relative to control in serum-free conditions. (C) A549 cells expressing stable shRNA against UBQLN1 (U1 KD#1, U1 KD#2, and control) were seeded in a transwell setup to assess cell migration in response to IGF1 stimulation. Cells were cultured in the top chamber in one of 3 conditions – serum-free media, serum-free media supplemented with IGF1 and serum-free media supplemented with IGF1 and Linsitinib, a small molecule inhibitor of IGF1R activity. Media supplemented with 10% FBS was used as chemo-attractant in the bottom chamber. At the end of 24 hours, cells were fixed and probed with HEMA 3 stain. Number of migrated cells were quantified by ImageJ software and analyzed by two-way ANOVA (D). Data are represented as mean±SEM from 3 independent experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, all relative to control.
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