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. 2015 Jul 3;6(28):26308–26321. doi: 10.18632/oncotarget.4763

Figure 1. CD44 phosphorylation status affects P-glycoprotein mediated drug resistance.

Figure 1

A. A split ubiquitin yeast two-hybrid assay was used to detect protein-protein interactions between P-gp and CD44. Budding yeast DSY-1 cells expressing MDR1 (in pTMBV4) and CD44s (in pDL2-X-Cub) as indicated, were grown on medium lacking leucine, tryptophan and histidine (HIS3) or containing X-Gal (LacZ), showing activation of the HIS3 and lacZ reporters respectively. When both MDR1 and CD44 genes were expressed yeast colonies grew in minimal media. B. Subcellular distribution of the expressed P-gp and CD44s. Membrane expression of both full length proteins is shown. C. DSY-1 cells expressing CD44, P-gp or both CD44 and P-gp along with untransformed wild type were grown overnight and serially diluted in liquid culture to an OD600 reading of 0.2, 0.1, 0.05 and 0.025 and spotted on a minimal synthetic plate with different concentrations of valinomycin as indicated. Colony spots were photographed as shown. D. Phosphorylation of CD44 at Ser 325 using an antibody specific for the CD44 Ser325 phosphorylated residue on Western blot analysis. Ovarian cancer cell line, SKA (Lanes 1 and 2) known to be P-gp(−)/CD44(+) and chemosensitive was compared with the MCF7/Adr cell line (Lanes 3-5) that is P-gp(+)/CD44(+). Cells were treated with PMA to induce PKC and hence phosphorylation of Ser291 of CD44 (Lane 2 and 4). Cells were treated with the PKC inhibitor bisindolylmaleimide to inhibit phosphorylation at Ser291 (Lane 5). Total amounts of CD44 and P-gp were determined by probing with the respective monoclonal antibodies. Anti-tubulin antibody was used as loading control. E. CD44 Ser to Ala mutations at either residue 291 or 325 were created and co-transformed with P-gp in the yeast two hybrid system. Serially 10-fold dilution cultures grown overnight were spotted on a minimal synthetic plate with or without 50μM valinomycin. Plates were incubated at 30°C for 3 days. Colony spots were photographed. F. CD44 Ser to Asp mutations at either residue 291 or 325 were created and co-transformed with P-gp in the yeast two hybrid system. Serially 10-fold dilution cultures grown overnight were spotted on a minimal synthetic plate with or without 50μM valinomycin. Plates were incubated at 30°C for 3 days. Colony spots were photographed.