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  current news   Press   selected story    
     
  10th July 2009  
 

Vimentin Regulates Scribble Activity by Protecting It from Proteasomal Degradation

 
 




Authors
Dominic C. Y. Phua, Patrick O. Humbert, and Walter Hunziker

Abstract
Cell polarization—the asymmetric distribution of cellular components into functionally separate regions—is fundamental to processes such as proliferation and movement. Its deregulation plays a central role in human diseases, in particular cancer. The multidomain protein Scribble (Scrib) is a key polarity regulator and neoplastic tumor suppressor in Drosophila. Mammalian Scrib is implicated in epithelial cell–cell adhesion and polarization during directed cell migration. In this article, we characterize a novel interaction between Scrib and the intermediate filament protein vimentin, which has a stabilizing effect on Scrib levels. Vimentin depletion results in the proteasome-dependent degradation of Scrib, which consequently leads to defective epithelial cell–cell adhesion and deregulated cell migration, closely phenocopying Scrib depletion. Double knockdown of Scrib and vimentin causes a phenotype similar to single silencing and suggests that the two proteins function in a single linear pathway. This stabilization of Scrib expression and function by vimentin is consistent with previously reported observations that vimentin is upregulated during epithelial wound healing. The findings imply a possible regulatory function for vimentin in Scrib homeostasis during epithelial migration.

 
 


 
 

 

Figure legend: Proteasome-dependent degradation of Scrib is inhibited by its interaction with vimentin.

(A) Vimentin expression in MDCK cells was silenced using siRNA over 3 days. Cells were subsequently re-seeded to sparse and confluent cultures and Scrib protein levels were monitored by Western blot analysis on Day 4. K18 was detected to check for equal cell lysate loading.

(B) Quantitative representation of Scrib downregulation relative to levels of vimentin silencing in MDCK cells.

(C) MDCK cells expressing EGFP-hScrib WT (~250 kDa), LRR (~130 kDa), PDZ (~150 kDa) or, as a negative control, EGFP alone, were treated with vimentin (+) or non-targeting (-) siRNA. hScrib expression was analyzed by Western blot using antibodies to GFP. GAPDH served as a control for equal lysate input.

(D) MDCK cells expressing EGFP-hScrib WT (panels a-d), LRR (panels e-h) or PDZ (panels i-l) were treated with non-targeting (panels a, b, e, f, i and j) or vimentin (panels c, d, g, h, k and l) siRNA and EGFP-hScrib (panels b, f, j, d, h and l, green color) and vimentin (panels a, e, i, c, g and k, red color) expression was visualized by fluorescence microscopy.

(E) MDCK cells exogenously expressing EGFP vimentin, ECFP-K8, EYFP-K18 or EGFP alone were analyzed by Western blot for expression of Scrib. GAPDH served as a control for equal lysate input. (F, G and H) Effect of a proteasome inhibitor on Scrib turnover.

(F) Western blot. MDCK cells expressing EGFP-hScrib WT, were treated with vimentin (+) or non-targeting (-) siRNA for 3 days and subsequently in the presence of a proteasome inhibitor for 0, 3, 6 or 9 hrs. Scrib levels and vimentin expression levels were then analyzed by Western blot. Note how in vimentin depleted cells, EGFP-hScrib (250 kDa) as well as endogenous Scrib (220 kDa) degradation is blocked by the proteasome inhibitor (see also H, below). Actin served as a control for equal lysate input.

(G) Immunofluorescence microscopy. MDCK cells expressing EGFP-hScrib WT were treated with vimentin siRNA and subsequently, a proteasome inhibitor for 0 hrs (panels a and b) or 9 hrs (panels c and d) and EGFP-hScrib (panels a and c, green color) and vimentin (panels b and d, red color) expression was visualized by fluorescence microscopy.

(H) Western blot endogenous Scrib. MDCK cells were treated with vimentin (+) or non-targeting (-) siRNA and subsequently in the presence of a proteasome inhibitor for 0, 3, 6 or 9 hrs. Endogenous levels of canine Scrib and vimentin were then analyzed by Western blot. GAPDH served as a control for equal lysate input.

(I) hScrib-EGFP of non-targeting or vimentin siRNA treated MDCK cells in the 9 hr presence (+) or absence (-) of proteasome inhibitor was immunoprecipitated and ubiquitinated hScrib detected by Western blot. Normal IgG served as a negative control.

Mol Biol Cell. 2009 Jun;20(12):2841-55

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