Loss of Par-3 leads to elevated levels of phospho-cofilin. (A) MDCK cells were transfected with control or pSUPER–Par-3 (Par-3 KD) to suppress Par-3 expression followed by calcium switch. HCM, high calcium medium. Total cell lysates were analyzed by immunoblotting. (B) Ectopic Par-3c reduces phospho-cofilin levels in Par-3–depleted cells. A construct encoding human Par-3c was cotransfected with pSUPER–Par-3 into MDCK cells followed by calcium switch and Western blot analysis of total cell lysates. Numbers indicate relative levels of phospho-cofilin normalized against the total cofilin level. LCM, low calcium medium. (C) Depletion of endogenous Par-3 in HeLa cells increases phospho-cofilin levels. HeLa cells were transfected with human Par-3–specific (Par-3 KD) or control siRNAs. Cell lysates were prepared 48 and 72 h later and blotted with indicated antibodies. (D) NRG treatment abolishes the increase in phospho-cofilin in Par-3–depleted MCF-7 cells. 1 d after transfection with Par-3–specific or control siRNAs, MCF-7 cells were serum starved overnight followed by 45 min of treatment with 50 ng/ml NRG in serum-free medium before cells were lysed.

Par-3 negatively regulates LIMK2 activity. (A) Par-3 inhibits LIMK2 kinase activity in vitro. HA-tagged LIMK2 or Par-3b was expressed in COS cells and immunoprecipitated. The immunocomplexes were subjected to in vitro kinase assays using GST-cofilin as the substrate or GST-Rac as a control. Coomassie brilliant blue (CBB) staining showed equal amounts of LIMK2 and GST-cofilin in the assays. (B) aPKC is not required for Par-3 to inhibit LIMK2 activity in vitro. HA-tagged Par-3c variant, which does not bind to aPKC, was used in the kinase assay together with Par-3b. (C) Ectopic Par-3 inhibits cofilin phosphorylation after LPA treatment. Control COS cells or cells expressing Par-3c were serum starved for 4 h and briefly treated with 5 μM LPA for indicated times. Total cell lysates were probed with specified antibodies. Numbers on the bottom of the blot indicate relative levels of phospho-cofilin normalized against the total cofilin level. Graph is representative of two independent experiments.

Expression of nonphosphorylatable cofilin S3A mutant promotes tight junction assembly in Par-3 KD MDCK cells. (A) MDCK cells were transfected with control, pSUPER–Par-3 (Par-3 KD), or pSUPER–Par-3 together with a construct expressing cofilin S3A (+cofilin S3A). Cells were subjected to calcium switch and stained for occludin at various times after calcium readdition. (B) Quantification of occludin cortical localization after calcium readdition. Asterisks denote a significant difference from Par-3 KD cells (P < 0.05) by t test. 6-h time point: control, n = 6; Par-3 KD, n = 7; cofilin S3A, n = 6. 7-h time point: control, n = 6; Par-3 KD, n = 7; cofilin S3A, n = 7. (C) Western blot analysis of total cell lysates from A. White lines indicate that intervening lanes have been spliced out. (D) Cofilin S3A expression partially rescues the development of transepithelial electrical resistance (TER) in Par-3 KD MDCK cells during calcium switch. MDCK cells were transfected with indicated constructs, and the kinetics of TER establishment was monitored for 13 h after the readdition of calcium. Each value is the mean of triplicate measurements (n = 3). Error bars represent SD.

Suppressing LIMK2 expression promotes tight junction assembly in Par-3 KD MDCK cells. (A) ROCK activity is required for the elevated phospho-cofilin in Par-3–depleted cells. Control or Par-3 KD MDCK cells were subjected to calcium switch and treated with 10 μM H-1152 for indicated times after the readdition of calcium. Cells without calcium switch (HCM) were treated with H-1152 for 1 h. Total cell lysates were analyzed by immunoblotting. (B) Suppression of LIMK2 level in MDCK cells. MDCK cells were transfected with control vector or two different pSUPER-LIMK2 (LIMK2 KD) constructs targeting distinct regions of canine LIMK2 mRNA. Total cell lysates were probed with the indicated antibodies. pSUPER–LIMK2 #2 reduced LIMK2 levels and was used for subsequent studies. (C) Double KD of LIMK2 and Par-3 blocks the increase in phospho-cofilin levels. MDCK cells were transfected with control vector, pSUPER–Par-3 (Par-3 KD) alone, or pSUPER–Par-3 together with pSUPER-LIMK2 (double KD). Total cell lysates were probed with the indicated antibodies. Numbers indicate relative levels of phospho-cofilin normalized against the total cofilin level. (D) Double KD of LIMK2 and Par-3 partially rescues occludin localization during calcium switch. MDCK cells were transfected as in C followed by calcium switch and staining for occludin at various times after calcium readdition. (E) Quantification of occludin cortical localization after the readdition of calcium. Asterisks denote significant differences from Par-3 KD cells (P < 0.05) by t test. Control, n = 5; Par-3 KD, n = 6; Par-3/LIMK2 KD, n = 6. Error bars represent SD.

Par-3 interacts with LIMK2 in vivo and in vitro. (A) Schematic diagrams of Par-3, LIMK2, and their various deletion fragments. Amino acid residue numbers are shown. Par-3c is a splice variant that lacks the aPKC-binding site. (B) Association of LIMK2 but not LIMK1 with recombinant Par-3 fragments. S-tagged Par-3 fragments immobilized on beads were incubated with COS cell lysates expressing Flag-tagged LIMK1 or LIMK2. Bound proteins were analyzed by Western blotting. (C) The COOH terminus of Par-3 is sufficient to associate with LIMK2. COS cells transfected with the indicated constructs were lysed, and the myc-tagged Par-3 fragments and Pals1 were immunoprecipitated by an anti-myc antibody. The associated LIMK2 was detected with an anti-HA antibody. Arrowhead points to LIMK2-HA. IP, immunoprecipitation. (D) Association of endogenous Par-3 and LIMK2 in MDCK cells. Endogenous LIMK2 was immunoprecipitated with an anti-LIMK2 antibody. Control immunoprecipitations were performed with anti-GFP and anti–β-catenin antibodies. (E) The NH₂-terminal region of LIMK2 is required for association with the Par-3 COOH terminus. COS cells were transiently transfected with constructs expressing the COOH terminus of Par-3 (Par-3c-E) together with various LIMK2 deletion fragments. LIMK2 fragments were immunoprecipitated with an anti-HA antibody, and the associated Par-3c-E fragment was detected with an anti-myc antibody. Asterisks indicate LIMK2 fragments. (F) Binding of LIMK2 NH₂ terminus to Par-3 fragments in vitro. GST–LIMK2 ΔC or GST-Rac was incubated with S-tagged Par-3 fragments immobilized on beads, and their interactions were analyzed by Western blotting. Asterisks indicate breakdown products. Arrowhead points to nonspecifically associated GST-Rac. White lines indicate that intervening lanes have been spliced out.