E-cadherin JMD Level is Regulated by Proteasomal Degradation.

<p>(A) Top: schematic representation of the Juxtamembrane domain (JMD) expression construct (left) and ActA control construct (right). Bottom: mouse E-cadherin JMD sequences aligned to demonstrate differences in mutations utilized in this study. Lysine to arginine mutations are denoted by rectangular boxes (K#R and Red “R”). Red AAA denotes mutations to abolish E-cadherin JMD/p120-catenin binding. E-cadherin octapeptide sequence required for binding p120-catenin is underlined. Numbers above the sequence represent the amino acid position number, and the corresponding amino acid position in murine E-cadherin sequence in parenthesis. (B) MDCK cells stably expressing ActA or WT-JMD were incubated for 6 hours with cycloheximide to determine protein turnover. β-catenin used as a degradation control and GAPDH as a loading control. Number(s) on the side of gels represent apparent molecular weights of protein standards (× 10^∧3). (C) Quantification of immunoblots of MDCK cells stably expressing ActA or WT-JMD in cycloheximide time course experiment (see B). Band intensities normalized to GAPDH to account for the reduction in overall protein levels. Results were averaged (+/− standard error of the mean (s.e.m.)) from 3 different experiments (N = 3). (D) Fluorescence images of MDCK cells transiently expressing RFP (ActA) or E-cadherin JMD RFP fusion protein (WT-JMD) targeted to the mitochondria; boxed regions in “RFP” are shown below at a higher magnification in “RFP-inset.” Scale bar is 25 µm in 100X images, and 5 µm in insets. (E) WT-JMD levels increase 4-fold upon proteasome inhibition. WT-JMD migrated with an apparent molecular weight that was ∼15kDa greater than ActA. Number(s) on the side of the gels are the apparent molecular weights of protein standards (× 10^∧3). (F) Quantification of immunoblot RFP protein levels averaged (+/− s.e.m.) from 3 different experiments; *p≤0.034.