The SH3 domain distinguishes the role of I-BAR proteins IRTKS and MIM in chemotactic response to serum

Abstract

The family of inverse BAR (I-BAR) domain proteins participates in a range of cellular processes associated with membrane dynamics and consists of five distinct members. Three of the I-BAR proteins, including insulin receptor tyrosine kinase substrate (IRTKS), contain an SH3 domain near their C-termini. Yet, the function of the SH3 domain of IRTKS remains uncharacterized. Here we report that in contrast to MIM, which is a prototype of I-BAR proteins and does not contain an SH3 domain, IRTKS promoted serum-induced cell migration along with enhanced phosphorylation of mitogen activated kinases Erk1/2 and p38, and activation of small GTPases Rac1 and Cdc42. In addition, cells overexpressing IRTKS exhibited an increased polarity characterized by elongated cytoplasm and extensive lamellipodia at leading edges. However, a mutant with deletion of the SH3 domain attenuated both cellular motility and p38 phosphorylation but had little effect on Erk1/2 phosphorylation. Also, a chimeric mutant in which the N-terminal portion of MIM is fused with the C-terminal IRTKS, including the SH3 domain, was able to promote chemotactic response to serum and cellular polarity. In contrast, a chimeric mutant in which the N-terminal IRTKS is fused with the C-terminal MIM failed to do so. Furthermore, treatment of cells with SB203580, a selective inhibitor of p38, also neutralized the effect of IRTKS on cell migration. These data indicate that the SH3 domain distinguishes the function of IRTKS in promoting cell migration and inducing signal transduction from those of SH3-less I-BAR proteins.

Keywords: Cell migration; Cell morphology; IRTKS; MIM; SH3; Small GTPases; p38.

Fig. 1. IRTKS promotes cell motility

(A, B) HeLa cells expressing IRTKS-GFP or GFP were migrated through Transwell plates towards FBS at various concentrations for 16 hr (A) or towards 15% FBS for various times (B). (C, D) HeLa cells expressing MIM-GFP or GFP cells were also analyzed by Transwell assay for chemotactic response to FBS at varying concentrations (C) or to 15% FBS for different times (D). (E) Schematic presentation of IRTKS, MIM, and IRTKS mutants. CT, C-terminal; NT, N-terminal; I-BAR, inverse BAR domain; PRD, proline rich domain; and W, WH2 domain. (F) Comparison of the motility response of cells expressing MIM and different IRTKS mutants to 15% FBS. ***, P<0.001, n=3.

Fig. 2. IRTKS regulates MAPKs and small GTPases upon serum treatment

HeLa Cells expressing IRTKS-GFP, MIM-GFP or GFP were stimulated by 10% FBS for the indicated times, and then subjected to examination for p38 phosphorylation (A), Erk1/2 phosphorylation (B) and Rac1 activation. Cdc42 activation was also examined in cells expressing IRTKS-GFP or GFP (D), MIM-GFP or GFP (E) after treated with 10% FBS for the indicated times. Quantification of these activated proteins was based on normalization to the total amount of individual these proteins. **, P<0.0.1, and ***, P<0.001, n=3.

Fig. 3. The SH3 domain is indispensable for IRTKS-mediated p38 phosphorylation

(A) Hela cells expressing GFP, IRTKS-GFP, and IRTKSΔSH3-GFP were treated with 10% FBS for 5 min and subjected to Western blot analysis for the level of phosphorylated p38. As a negative control, IRTKS-GFP cells were pre-treated with 10 μM SB203580 1 hr prior to adding serum. (B) Phosphorylation of Erk1/2 in cells expressing IRTKS-GFP or IRTKSΔSH3-GFP was evaluated after treated with 10% FBS for the indicated times. (C) Cells expressing IRTKS mutants were analyzed for the chemotactic response to serum in the presence and absence of 10 μM SB203580. ***, P<0.001, n=3.

Fig. 4. The SH3 domain is required for IRTKS-mediated cell shape changes

HeLa cells expressing GFP, MIM-GFP, and IRTKS-GFP (A) or HeLa cells expressing MIM-I-BAR-IRTKS-CT-GFP, IRTKS-I-BAR-MIM-CT-GFP and IRTKSΔSH3-GFP (B) were cultured in serum-containing medium and co-stained with GFP antibody and Alexa flour 594 tagged phalloidin. The stained cells were inspected by fluorescent microscopy. Bars: 80 μm. (C) Quantification of the degree of cell elongation as described in the Materials and Methods. The data shown is mean ± SEM of three independent experiments. In each experiment, 50 cells were analyzed. ***, P<0.001.