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Figure 8


Fig. 8. The N-terminal ENTH domain of epsin is necessary and sufficient for phosphorylation of Hip1r and membrane localization. (A,B) In wild-type (A, WT) and Hip1r-null cells (B, Hip1r-), epsin-GFP (epsin:GFP) localizes within puncta on the plasma membrane. (C,D) In contrast to wild-type cells (C), Hip1r puncta (anti-Hip1r) are not restricted to the cell periphery in epsin-null cells (D, epsin-). (E) Expression of the ENTH domain in epsin-null cells [epsin-(ENTH:GFP)] restores the peripheral localization of Hip1r. Bar, 10 µm. (F) Epsin influences the phosphorylation state of Hip1r. Immunoblots of cell lysates of WT cells, epsin-null cells expressing epsin-GFP (epsin-, epsin:GFP) and the ENTH domain of epsin (epsin-, ENTH:GFP) probed with antibody against Hip1r exhibit double bands, indicative of phosphorylation of Hip1r. In epsin-null cells (epsin-) and cells expressing only the epsin C-terminus (epsin-, C-term), Hip1r is not phosphorylated.





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