Molecular basis for autoinhibition of RIAM regulated by FAK in integrin activation

Abstract

RAP1-interacting adapter molecule (RIAM) mediates RAP1-induced integrin activation. The RAS-association (RA) segment of the RA-PH module of RIAM interacts with GTP-bound RAP1 and phosphoinositol 4,5 bisphosphate but this interaction is inhibited by the N-terminal segment of RIAM. Here we report the structural basis for the autoinhibition of RIAM by an intramolecular interaction between the IN region (aa 27-93) and the RA-PH module. We solved the crystal structure of IN-RA-PH to a resolution of 2.4-Å. The structure reveals that the IN segment associates with the RA segment and thereby suppresses RIAM:RAP1 association. This autoinhibitory configuration of RIAM can be released by phosphorylation at Tyr45 in the IN segment. Specific inhibitors of focal adhesion kinase (FAK) blocked phosphorylation of Tyr45, inhibited stimulated translocation of RIAM to the plasma membrane, and inhibited integrin-mediated cell adhesion in a Tyr45-dependent fashion. Our results reveal an unusual regulatory mechanism in small GTPase signaling by which the effector molecule is autoinhibited for GTPase interaction, and a modality of integrin activation at the level of RIAM through a FAK-mediated feedforward mechanism that involves reversal of autoinhibition by a tyrosine kinase associated with integrin signaling.

Keywords: RIAM; autoinhibition; focal adhesion kinase; integrin signaling; small GTPase.

Fig. 1.

Identification of an autoinhibitory region in RIAM. (A) Schematic representation of the domain organization of RIAM. Full-length RIAM (FL) possesses residues 1–670. Talin-binding site (TBS) is colored in green; IN segment is in orange; CC segment is in red; poly-proline (PP) segment is in black; the Ras-association domain (RA) is in yellow; and the Pleckstrin-homology domain (PH) is in cyan. TBS includes residues 1–30; IN includes residues 27–93; NT includes residues 1–93; ΔTBS: RIAM lacking residues 1–30; ΔIN: RIAM lacking residues 50–85; ΔNT: RIAM lacking residues 1–93; IN-RA-PH represents the protein construct used in the crystallographic study, including residues 27–437 with residues 94–149 deleted. (B) In vitro pull-down of His-tagged RIAM RA-PH by various GST-tagged RIAM fragments. (C) Coexpression of the GFP-tagged RIAM constructs with mCherry-RAP1-G12V in Jurkat T cells. (D) Quantification of the PM colocalization of the indicated GFP-RIAM with mCherry-RAP1-G12V.

Fig. 2.

Crystal structure of RIAM in an autoinhibitory configuration. (A) Ribbon diagram of RIAM in an autoinhibited configuration represented by the crystal structure of RIAM IN-RA-PH, with the IN segment in orange, the cc segment in red, the RA domain in yellow, the PH domain in cyan, and the linker between RA and PH in gray. The salt-bridge interaction supporting the cc helix is illustrated in the Right. (B, Left) Superposition of the autoinhibited RIAM structure with the RAP1:RIAM complex structure. The RAP1:RIAM complex structure is showed in a surface representation. RAP1 is colored light gray. The RA-PH module in the RAP1:RIAM complex is omitted. (B, Upper Right) Side-chain interactions formed by Lys213 from the RA domain and Glu60/Asp63 from the IN segment. (B, Lower Right) The canonical Ras:effector interaction mediated by Lys213 of RIAM and Asp33 of RAP1. (C) Surface representation of the hydrophobic cluster formed by the residues from the IN segment (orange) and the RA domain (yellow). (D) 2Fo-Fc electron density map of the IN and CC segments. Side chain of Tyr45 is shown in stick representation. (E) Residues in the proximity of Tyr45 in the autoinhibitory state of RIAM are shown in stick representation.

Fig. 3.

The IN of RIAM inhibits translocation to the PM and blocks T cell adhesion. (A) In vitro pull-down of His-tagged RA-PH by GST-tagged TBS, IN, and various mutations of IN. Input samples were examined by Coomassie staining. (B) Coimmunoprecipitation of HA-RAP1 and full-length RIAM, or various RIAM mutants. (C, Upper) Coexpression of the GFP-tagged point mutations of RIAM with mCherry-RAP1-G12V in Jurkat T cells. (C, Lower) Quantification of the PM colocalization of the indicated GFP-RIAM with mCherry-RAP1-G12V. Data shown are mean ± SD, n = 3. (D) Jurkat T cells were transfected with indicated constructs. Twenty-four hours later, adhesion assay was performed as described and adherence was determined by analyzing GFP-positive cells using flow cytometry. All results were normalized to untransfected Jurkat cells. Data shown are mean ± SD, n = 4 (**P < 0.001).

Fig. 4.

Phosphorylation of Tyr45 is regulated by FAK upon TCR activation. (A) GST-tagged recombinant IN, IN45E (Y45E), and IN45F (Y45F), were incubated with Jurkat cell lysates with or without anti-CD3 antibodies treatment. Tyrosine phosphorylation was visualized by Western blot. The recombinant protein inputs are shown as the Lower, and Western blot of Jurkat lysate inputs were shown to the Right. (B) Tyrosine phosphorylation of IN was detected as in A. Jurkat cells were treated with DMSO, or FAK inhibitors (PM431396 and PM572271) for 1 h before anti-CD3 antibodies stimulation. Then cell lysates were incubated with GST-IN, and the phosphorylation was detected by p-tyrosine antibody. The blots were quantified by Licor Odyssey and normalized to the control. Data shown are mean ± SD, n = 4. (C) Coexpression of the GFP-tagged RIAM and mCherry-RAP1-G12V in the control or anti-CD3 antibodies stimulated Jurkat T cells with or without FAK inhibitors. Quantification of the cells translocated to the PM is shown in the bar graph. Data shown are mean ± SD, n = 3. (D) The PM translocation of the GFP-tagged RIAM bearing the Y45E or E60A/D63A mutations was quantified in Jurkat cells when coexpressing with mCherry-RAP1-G12V. Data shown are mean ± SD, n = 3.