Fig 2: Activation of CXCR4 promotes phosphorylation of eIF4B, 4E-BP1, and PDCD4. 231S cells were stimulated with 10–20 nM CXCL12 for the indicated period. Phosphorylation status of (A) p-eIF4B S422 (B) p-PDCD4 S67 and (C) p-4E-BP1 Thr70 was determined by Western blotting (n = 3). Fold change indicates the densitometry ratio of (phospho-protein/total protein)/ß-tubulin signal with 0 min. set to 1. (D) Status of p-eIF4B, p-PDCD4 S67, and p-4E-BP1 Thr70 in MCF7 vector, wild-type CXCR4, and CXCR4 ?CTD cells (n = 3). Fold change indicates the densitometry ratio of (phospho-protein/total protein)/ß-tubulin signal with MCF7 vector cells set to 1. (E) Proposed model of CXCR4 signaling and its effects on the eIF4F complex.

Fig 3: LASP1 interacts with the eIF4F complex in a CXCL12-dependent manner. (A) Co-immunoprecipitation assay of eIF4A and LASP1 in 231S cells following stimulation with 20 nM CXCL12 (n = 2). (B) Co-immunoprecipitation assay of eIF4B and LASP1 in 231S cells following stimulation with 10 nM CXCL12 (n = 3). (C) Co-immunoprecipitation assay of LASP1 and eIF4A/B following stimulation with 20 nM CXCL12 in 231S cells (n = 2). Fold change was calculated based off the densitometry ratio of co-immunoprecipitated/immunoprecipitated protein signal with 0 min. set to 1. (D–E) m7GTP pulldown assay in 231S cells following stimulation with 20 nM CXCL12 and 100 nM AMD3465 examining the interaction between: (D) LASP1-eIF4E (n = 3) and (E) eIF4G-eIF4E (n = 3). Fold change was calculated based off the densitometry ratio of co-precipitate (LASP1 or eIF4G)/precipitate (eIF4E) protein signal with 0 min. set to 1.

Fig 4: LASP1 directly interacts with both eIF4A and eIF4B. (A–C) 1 mg of 231S lysate was incubated with 1.5 nmoles of GST or GST-LASP1. (A) Presence of eIF4A was detected by Western blotting (n = 3). (B) 2 mM ATP and 3mM MgCl2 were exogenously added to the 231S lysate. Presence of eIF4A was then detected by Western blotting (n = 3). (C) Presence of eIF4B was detected by Western blotting (n = 3). (D–E) Purified eIF4A or eIF4B was incubated with 1.5 nmoles GST or GST-LASP1. Amounts of purified proteins are indicated in parenthesis. (D) Presence of eIF4B was detected by Western blotting (n = 3). (E) Presence of eIF4A was detected for by Western blotting (n = 3). Ponceau S stains of each blot are shown below to confirm loading of GST or GST-LASP1 following the elution from glutathione agarose beads. (F) Purified eIF4A and eIF4B were mixed with purified GST or GST-LASP1 in an equimolar ratio and in solution. Proteins complexes were then captured with glutathione beads and detected for by Western blotting (n = 1). (G) Imperial Protein Stain of purified eIF4A, eIF4B, GST, and GST-LASP1 (n = 1).

Fig 5: Proposed Model of the CXCR4-LASP1-eIF4F Axis. An illustration of CXCR4 and its relation to the eIF4F complex upon stimulation with CXCL12. This relationship is occurring through two distinctive mechanisms. First, LASP1 dissociates from CXCR4 and directly interacts with eIF4A and eIF4B. Second, phosphorylation of PDCD4, 4E-BP1, and eIF4B is promoted through G protein-coupled receptor signaling. As a result, both complex formation increases along with the function of eIF4A. Consequently, the translation of oncogenic proteins is promoted.