Oligomerization and Phosphorylation Dependent Regulation of ArgBP2 Adaptive Capabilities and Associated Functions

<div><p>ArgBP2 (Arg-Binding Protein 2/SORBS2) is an adaptor protein involved in cytoskeleton associated signal transduction, thereby regulating cell migration and adhesion. These features are associated with its antitumoral role in pancreatic cancer cells. Tyrosine phosphorylation of ArgBP2, mediated by c-Abl kinase and counterbalanced by PTP-PEST phosphatase, regulates many of its interactions. However, the exact mechanisms of action and of regulation of ArgBP2 remain largely unknown. We found that ArgBP2 has the capacity to form oligomers which are destabilized by tyrosine phosphorylation. We could show that ArgBP2 oligomerization involves the binding of one of its SH3 domains to a specific proline rich cluster. ArgBP2 self-association increases its binding to some of its molecular partners and decreased its affinity for others. Hence, the phosphorylation/oligomerization state of ArgBP2 directly regulates its functions by modulating its adaptive capabilities. Importantly, using a human pancreatic cancer cell model (MiaPaCa-2 cells), we could validate that this property of ArgBP2 is critical for its cytoskeleton associated functions. In conclusions, we describe a new mechanism of regulation of ArgBP2 where tyrosine phosphorylation of the protein interfere with a SH3 mediated self-interaction, thereby controlling its panel of interacting partners and related functions.</p></div

Mutation of P1 cluster of proline motifs affects ArgBP2's oligomerization.

<p><b>A</b>) Lysates from HEK293T cells, expressing myc and Flag tagged version of WT or mutants ArgBP2, were subjected to immunoprecipitation (IP) with an anti-Myc antibody and membranes were immunoblotted (IB) with an anti-Flag antibody. After stripping, membranes were blotted with an anti-Myc antibody to control the amount of precipitated material. The expression of the different constructs was controlled by blotting total cell lysates (TCL) with the corresponding antibodies. <b>B</b>) BRET assay using ArgBP2 WT or P1 mutant. A constant amount of Luciferase fused to ArgBP2 WT or P1 was co-transfected in HEK293T cells with increasing amount of EYFP fused ArgBP2 WT or P1 respectively. BRET signal has been calculated as described in experimental procedures and one representative experiment is shown. <b>C</b>) PLA assay for ArgBP2 oligomerization using NIH-3T3 cells transfected with Myc and Flag tagged ArgBP2 WT, or P1 mutant. <b>D</b>) Lysates from HEK293T cells transfected with Myc tagged ArgBP2 WT or ArgBP2 TM were treated (+ DMP) or not (- DMP) with a cross-linking reagent (Dimethyl pimelimidate, DMP). Then, lysates were subjected to immunoprecipitation with an anti-Myc antibody and membranes were immunoblotted with an anti-Myc antibody. Expression of both constructs was controlled by blotting total cell lysates (TCL) with an anti-Myc antibody. <b>E</b>) Gel filtration assay. Macromolecular complexes from lysates of HEK293T cells expressing either ArgBP2 WT or ArgBP2 P1 mutant, were separated by FPLC as described in experimental procedures. Fractions were subjected to immunoprecipitation using an anti-Myc antibody and membranes were immunoblotted using anti-ArgBP2 antibody.</p

ArgBP2 oligomerization can modulate its binding to some of its partners.

<p><b>A</b>) Co-immunoprecipitation of ArgBP2 WT or ArgBP2 P1 with WAVE1. Lysates from HEK293T cells, transfected as indicated, were subjected to immunoprecipitation (IP) using an anti-ArgBP2 antibody. Membranes were first immunoblotted (IB) with an anti-WAVE antibody then, after stripping, membranes were blotted with an anti-ArgBP2 antibody to control the amount of precipitated material. Expression of all proteins was controlled by blotting total cell lysates (TCL) with the corresponding antibodies. <b>B</b>) Effect of the P1 peptide upon the ArgBP2/WAVE1 co-immunoprecipitation. Lysates from cells expressing WAVE1 and ArgBP2 WT were treated or not with the P1 peptide prior to IP using an anti-ArgBP2 antibody. Membranes were first immunoblotted with an anti-WAVE antibody then, after stripping, with an anti-ArgBP2 antibody to control the amount of precipitated material. Proteins expressions were controlled by blotting TCL with the corresponding antibodies. <b>C</b>) As in <b>A</b>), co-immunoprecipitation of ArgBP2 WT or P1 mutant with PTP-PEST. <b>D</b>) As in <b>B</b>), effect of P1 peptide upon the ArgBP2/PTP-PEST co-immunoprecipitation. <b>E</b>) As in <b>A</b>), co-immunoprecipitation of ArgBP2 WT or P1 mutant with c-Abl, and resulting phosphorylation of ArgBP2. <b>F</b>) As in <b>B</b>), effect of P1 peptide upon the ArgBP2/c-Abl co-immunoprecipitation and ArgBP2 phosphorylation.</p

Oligomerization of ArgBP2.

<p><b>A</b>) HEK293T cells were transfected with myc-ArgBP2 and flag-ArgBP2 expressing vectors as indicated. Lysates were subjected to immunoprecipitation (IP) with an anti-Myc antibody and membranes were first immunoblotted (IB) with an anti-Flag antibody. After stripping, membranes were blotted with an anti-Myc antibody to control the amount of precipitated material. The expression of the different constructs was controlled by blotting total cell lysates (TCL) with the corresponding antibodies. <b>B</b>) PLA study of endogenous ArgBP2 self-interaction in BxPC3 cells. Cells were transfected with ArgBP2 specific siRNA or control siRNA (48 h before) and anti-ArgBP2 monoclonal antibody (C1) conjugated to positive PLA probe was mixed with the same antibody conjugated to negative PLA probe to perform PLA experiments. <b>C</b>) Schematic representation of ArgBP2 indicating the different deletion mutants used. <b>D</b>) Lysates from HEK293T cells expressing Flag-tagged ArgBP2 N-terminal (ArgBP2 N-ter) or C-terminal (ArgBP2 C-ter) part (see Fig. B) were subjected to GST pull-down with N-terminal and C-terminal parts of ArgBP2 as indicated. The upper parts of membranes were immunoblotted with anti-Flag (ArgBP2 N-terminal or C-terminal), and the lower part with anti-GST antibody. <b>E</b>) Scheme of ArgBP2 parts involved in its interaction.</p

The interaction takes place between the SH3 B domain of one ArgBP2 protein and a proline motif of another one.

<p><b>A</b>) Lysates from cells expressing myc-ArgBP2 were subjected to GST pull-down with different SH3 domains of ArgBP2. <b>B</b>) Schematic representation of ArgBP2 showing the three clusters of proline motifs contained in the C-terminal part of ArgBP2. Three mutants of ArgBP2 were generated in which the important prolines residues of each clusters were changed to alanines (ArgBP2 P1, ArgBP2 P2 and ArgBP2 P3). A mutant owning the triple mutation was generated (ArgBP2 TM). Mutated prolines are indicated in bold. <b>C</b>) Lysates from HEK293T cells transfected with proline clusters mutants of ArgBP2, were subjected to GST pull-down with different SH3 domains of ArgBP2 as indicated. The upper parts were immunoblotted (IB) with an anti-myc antibody (ArgBP2) and the lower parts with an anti-GST antibody. <b>D</b>) Far western blot of ArgBP2 WT and P1. Lysates from cells expressing Flag tagged ArgBP2 WT or P1 were probed with GST or GST-ArgBP2 fusion protein. Membranes were then revealed using anti-GST immunoblot. <b>E</b>) GST pull-down of myc-ArgBP2 WT using GST fused to SH3 domains or C-terminal part of ArgBP2 (as in A) in presence or not of 50 µM of P1 peptide.</p

Role of ArgBP2 oligomerization regarding cell spreading and migration.

<p><b>A</b>) Miapaca-2 cells stably expressing ArgBP2 WT, ArgBP2 P1 mutant, or GFP as control, were seeded onto fibronectin coated culture plates. After two hours, cells were fixed and stained using Coomassie blue. Ten pictures of each were taken and the size of each cell has been evaluated using ImageJ software (NIH). Three independent experiments were used to generate the data which are represented as mean +/- SD and Student T test has been performed to compare GFP with ArgBP2 WT and ArgBP2 WT with ArgBP2 P1 expressing cells. <b>B</b>) Miapaca-2 cells stably expressing ArgBP2 WT, ArgBP2 P1 mutant, or GFP as control, were subjected to cell migration assays in Boyden chambers during 7 hours. Migrating cells were then fixed and stained using Coomassie blue. Each experiment was done in triplicate and five random pictures were taken from each well. Three independent experiments were pulled together and cell migration is expressed as a percentage of control cells (GFP) migration +/- SD and student T test has been done to compare GFP with ArgBP2 WT and GFP with ArgBP2 P1 expressing cells.</p

Phosphorylation of ArgBP2 by c-Abl regulates its oligomerization.

<p><b>A</b>) HEK293T cells were transfected with Myc and Flag tagged ArgBP2, wild type c-Abl (WT) and kinase-inactive c-Abl (KD) expressing vectors, as indicated. Lysates were subjected to immunoprecipitation (IP) using an anti-Myc antibody and membranes were immunoblotted (IB) using an anti-Flag (ArgBP2) antibody at ArgBP2 level, and with an anti-c-Abl antibody at c-Abl level. After stripping, membranes were blotted with an anti-phosphotyrosine (PY) antibody to estimate the phosphorylation of ArgBP2 and, in a third time, with an anti-Myc antibody (to control the amount of precipitated material). Expression of all proteins was controlled by blotting total cell lysates (TCL) with the corresponding antibodies. <b>B</b>) BRET analysis of the effect of c-Abl upon ArgBP2 self-association. HEK293T cells were transfected with a constant amount of Luciferase fused to ArgBP2 WT and an increasing amount of EYFP fused ArgBP2 WT. BRET signal has been calculated as described in experimental procedures. <b>C</b>) PLA study of ArgBP2 WT oligomerization in presence or not of c-Abl, using NIH-3T3 cells transfected with Myc and Flag tagged ArgBP2 WT with or without c-Abl expressing vectors. <b>D</b>) HEK293T cells were transfected with myc and flag tagged ArgBP2, wild type (WT) or five tyrosines mutant (5Y). Lysates were subjected to IP with an anti-Flag antibody and membranes were blotted using anti-Myc antibody. After stripping, membranes were blotted using anti-Flag antibody to control the amount of precipitated material. The expression of the different constructs was controlled by blotting TCL with the corresponding antibodies. <b>E</b>) Lysates from 293T cells expressing Flag tagged ArgBP2 WT together with c-Abl WT, the kinase deficient (KD), the SH2 domain mutant (SH2), or the SH3 domain mutant (SH3), were subjected to IP using an anti-Flag antibody to precipitate ArgBP2. The amount of co-precipitated c-Abl was revealed by blotting the upper part of the membrane with an anti-c-Abl antibody, and the amount of precipitated ArgBP2 was controlled by blotting the lower part with an anti-Flag antibody. The expression of all constructs was verified in TCLs. <b>E</b>) (right) Graphical representation of pooled data from three independent experiments.</p

Mdm2 interacts with NUB1.

<p>(A) Lysates from HEK-293T cells expressing Myc-NUB1 alone or together with Mdm2 were subjected to immunoprecipitation with an anti-Mdm2 antibody. Proteins were separated through SDS-PAGE and NUB1 was detected by western blotting using an anti-Myc antibody (9E10). Amount of precipitated Mdm2 and expression levels of both proteins in cell extracts were controlled. (B) Lysates from HEK-293T cells expressing Mdm2 alone or together with Myc-NUB1 were subjected to immunoprecipitation with the anti-Myc 9E10 antibody. Immunoprecipitates and cell extracts were analyzed by western blotting using an anti-Mdm2 and anti-Myc antibodies. * Shifted NUB1.</p

Mdm2 triggers the ubiquitination of NUB1.

<p>(A) HEK-293T cells were transfected with Myc-NUB1 alone or together with Mdm2, and twenty-four hours later lysates were prepared using an hypotonic / Triton X-100 buffer supplemented or not with N-Ethylmaleimide (NEM). Proteins were separated through SDS-PAGE and NUB1 was detected by western blotting using the anti-Myc antibody. (B) HEK-293T cells were transfected with Myc-NUB1 alone or together with wild type (WT) or catalytically inactive (C462A) Mdm2. Lysates were prepared and proteins were separated through SDS-PAGE and analyzed by western blotting with the appropriate antibodies.</p

NUB1 is ubiquitinated by Mdm2 on lysine 159.

<p>(A) Workflow of the protocol used to identify NUB1 ubiquitinated lysine. (B) METTRE SPECTRES (C) HEK-293T cells were transfected with wild type (WT), K134R, K159R or K134,159R (2KR) mutants Myc-NUB1 together with 6HF-Ubiquitin and Mdm2 constructs. 6HF-Ubiquitinated proteins were isolated by Ni2+ pull down and NUB1 was revealed by western blotting using an anti-NUB1 antibody. (D) Lysates from HEK-293T cells expressing Mdm2 alone or together with wild type (WT), K134R or K159R mutants Myc-NUB1 were subjected to immunoprecipitation with the anti-Myc 9E10 antibody. Immunoprecipitates and input samples were analyzed by western blotting using an anti-Mdm2 antibody.</p