Podocyte Injury Associated with Mutant α-Actinin-4

Figure 3

α-actinin-4 K256E “chokes” the proteasome and exacerbates ER stress. (a) GFP-α-actinin-4 K256E undergoes ubiquitination. COS-1 cells were transiently transfected with GFP-α-actinin-4 wild type (WT) or K256E mutant (Mut), and the cells were incubated with the proteasome inhibitor, MG132. Lysates were immunoprecipitated with anti-GFP antibody (+), or nonimmune IgG (−) in controls. Then, the immunoprecipitates were immunoblotted with antibodies to ubiquitin (upper panel) or GFP (lower panel). (b, c) COS cells were transiently cotransfected with the GFP^U proteasome reporter (or GFP for comparison), plus GFP-α-actinin-4 K256E or wild type. Lysates were immunoblotted with anti-GFP antibody. In COS cells transfected with α-actinin-4 wild type, expression of the GFP^U reporter is substantially lower than GFP, since GFP^U is readily degraded by the ubiquitin-proteasome pathway, but GFP is stable. Mutant α-actinin-4 did not affect GFP expression. GFP^U expression was enhanced by cotransfection of α-actinin-4 K256E, indicating that this mutant impaired degradation of GFP^U by the ubiquitin-proteasome system. (c) ^∗𝑃<.035 GFP^U mutant versus wild type. (d, e) COS cells were transiently transfected with GFP-α-actinin-4 wild type or mutant. Lysates were immunoblotted with antibodies to GFP, grp94 or CHOP. NS, nonspecific band-loading control. ^∗𝑃<.03, ^∗∗𝑃<.045 mutant versus wild type. The figure is adapted from [35] with permission of the American Physiological Society.