Given the close connections between actin reorganization and tight junction assembly, we examined the effects of reduced Par-3 expression on the phosphorylation of cofilin. The specificity of Par-3 suppression by RNAi has been confirmed previously (Chen and Macara, 2005
). Depletion of Par-3 caused a substantial increase in cofilin Ser3 phosphorylation ( and Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200510061/DC1
). In contrast, depletion of the tight junction protein occludin had only a small effect on phospho-cofilin levels (Fig. S1 B). Epithelial (E) cadherin knockdown (KD) also resulted in elevated levels of phospho-cofilin (Fig. S1 B), but depletion of Par-3 did not affect E-cadherin levels or disrupt adherens junctions under normal calcium conditions and only had minor effects on adherens junction assembly during calcium switch (Chen and Macara, 2005
). Moreover, when MDCK cells were subjected to calcium depletion overnight to disrupt all cell–cell junctions, the phospho-cofilin levels in Par-3–depleted cells were still higher than in control cells (, LCM), indicating that Par-3 can regulate phospho-cofilin levels independently of cell junction status and extracellular calcium levels. In addition, a double KD of Par-3 and E-cadherin led to an additive increase in phospho-cofilin compared with that achieved by a single KD of Par-3 or E-cadherin (Fig. S1 C), further suggesting that Par-3 and E-cadherin regulates cofilin phosphorylation through distinct mechanisms. Transient expression of human Par-3c, which is not recognized by the short hairpin RNA (shRNA) targeting canine Par-3, partially reversed the increase in phospho-cofilin (), supporting the argument that the increased phospho-cofilin is caused by Par-3 depletion rather than by off-target effects. Par-3c is a splice variant that lacks the aPKC-binding site (Gao et al., 2002
), suggesting that the ability of Par-3 to regulate cofilin phosphorylation does not involve aPKC.
Figure 1. 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 (more ...)
To determine whether regulation of phospho-cofilin by Par-3 is conserved across different cell types, we suppressed the expression of endogenous Par-3 in HeLa cells and found that cofilin phosphorylation was also enhanced after Par-3 depletion (). Similarly, depletion of endogenous Par-3 in MCF-7 breast carcinoma cells also markedly elevated the pool of phospho-cofilin (). Neuregulin (NRG)-1β treatment of MCF-7 cells activated the cofilin-specific phosphatase Slingshot (Nagata-Ohashi et al., 2004
) and completely blocked the increase in phospho-cofilin in Par-3–depleted cells (), indicating that the loss of Par-3 does not interfere with the activation of Slingshot. These data suggest that Par-3 is generally involved in down-regulating cofilin phosphorylation.
Because the depletion of Par-3 results in phosphorylation of cofilin and its inactivation, we investigated whether reduced cofilin activity might contribute to the defects in tight junction assembly caused by Par-3 depletion. Using the transmembrane protein occludin as a marker, we observed, as reported previously, that MDCK cells lacking Par-3 had disrupted tight junctions for many hours after a calcium switch (; Chen and Macara, 2005
). However, the normal cortical distribution of occludin was partially rescued at later stages of junction assembly by transient expression of a phosphorylation-defective, constitutively active cofilin mutant (S3A; ). Quantification of the mean length per cell of occludin at cell–cell contacts confirmed the significance of the enhanced cortical localization of occludin (). Importantly, the expression of cofilin S3A did not affect the efficiency of Par-3 KD (). As shown previously, the loss of Par-3 substantially delayed the development of transepithelial electrical resistance (TER) after calcium readdition, and this was partially reversed by the expression of cofilin S3A (; Chen and Macara, 2005
). Silencing of cofilin expression alone did not disrupt tight junctions, however, suggesting that cofilin activity is not a limiting regulator of junction assembly (Fig. S1, E–G). Altogether, these results suggest that cofilin activity contributes to tight junction formation during epithelial cell polarization and that one of the functions of Par-3 is to prevent inappropriate cofilin phosphorylation.
Figure 2. 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 (more ...)
We have shown previously that Par-3 binds to Tiam1 to down-regulate Rac activity (Chen and Macara, 2005
). However, a double KD of Par-3 and Tiam1 had no effect on the augmented phospho-cofilin levels induced by Par-3 suppression (Fig. S1 D). Furthermore, the expression of a constitutively active fragment of Tiam1 (Tiam1 C1199; Chen and Macara, 2005
) did not elevate phospho-cofilin levels (Fig. S1 D), supporting the idea that the elevated phospho-cofilin in Par-3 KD cells is independent of misregulated Tiam1 activity. Interestingly, cofilin S3A expression did not further improve the TER establishment in Par-3 and Tiam1 double KD cells (Fig. S1 H), suggesting that cofilin activity and the Tiam1–Rac pathway may regulate similar aspects or stages of tight junction assembly.
We next asked whether Par-3 might directly or indirectly regulate the activities of LIMKs or phosphatases to influence cofilin phosphorylation. LIMKs are activated by Rho small GTPases through their downstream effectors Rho-associated kinase (ROCK) and p21-activated kinase (Edwards et al., 1999
; Sumi et al., 2001
). No specific binding of Par-3 to the Slingshot phosphatase was detected (unpublished data), which is consistent with our observation that Slingshot activation is not altered by the loss of Par-3 (). However, recombinant Par-3 fragments (Par-3c-B and Par-3c-D) specifically pulled down LIMK2 but not LIMK1 from COS cell lysates (). Furthermore, the COOH terminus of Par-3 (Par-3c-E) alone coimmunoprecipitated LIMK2 just as efficiently as a longer fragment of Par-3 (Par-3c-D; ), suggesting that the COOH terminus of Par-3 is sufficient to mediate the interaction with LIMK2. In contrast, another PDZ domain–containing polarity protein, Pals1, did not coimmunoprecipitate LIMK2. When endogenous LIMK2 from MDCK cells was immunoprecipitated, an association of endogenous Par-3 with LIMK2 was detected (). These data demonstrate that Par-3 and LIMK2 interact in vivo.
Figure 3. 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 (more ...)
To further dissect the region of LIMK2 that is involved in its interaction with Par-3, deletion fragments of LIMK2 were tested for their abilities to coimmunoprecipitate the COOH terminus of Par-3 (). Deletion of the entire NH2
-terminal half of LIMK2 (LIMK2 ΔN) or of the two LIM domains (LIMK2 ΔLIM) severely abrogated association with the Par-3 COOH terminus, indicating that the LIM domains are required for the interaction. In contrast, LIMK2 ΔC or a kinase-dead mutant of LIMK2 was able to associate with Par-3 ( and Fig. S2 A, available at http://www.jcb.org/cgi/content/full/jcb.200510061/DC1
). To examine whether Par-3 fragments can bind to the NH2
terminus of LIMK2 in vitro, recombinant Par-3 fragments immobilized on beads were incubated with GST–LIMK2 ΔC or with GST-Rac as a negative control ().GST–LIMK2 ΔC interacted very weakly with the Par-3c-A fragment, but it showed more robust interaction with the Par-3c-B and Par-3c-D fragments, which is in agreement with the pull-down experiments.
LIMK2 can be activated by ROCK downstream of RhoA (Sumi et al., 2001
). A specific ROCK inhibitor H-1152 greatly reduced the phospho-cofilin level in Par-3 KD MDCK cells (), supporting the involvement of LIMK2 in cofilin phosphorylation. To confirm that endogenous LIMK2 mediates cofilin phosphorylation in Par-3–depleted cells, we partially suppressed the expression of LIMK2 in MDCK cells by RNAi without affecting the total levels of Par-3 and cofilin (). Double KD of both Par-3 and LIMK2 inhibited the increase in phospho-cofilin caused by Par-3 depletion (), suggesting that LIMK2 is important for increasing phospho-cofilin levels after Par-3 depletion. Consistent with the reduction in phospho-cofilin level, the suppression of both Par-3 and LIMK2 also promoted the assembly of tight junctions after a calcium switch, as monitored by the improved localization of occludin to cell–cell borders () and by the small but reproducible enhancement in TER establishment (Fig. S2 C).
Figure 4. 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 (more ...)
RhoA activity is not altered in Par-3 KD cells (Chen and Macara, 2005
), and we could not detect any interaction between Par-3 and ROCK (Fig. S2 B). The ability of Par-3 to interact with LIMK2 suggests that Par-3 is acting directly on LIMK2. Interestingly, cells lacking Par-3 showed a small increase in the level of phosphorylation on Thr505 of LIMK2 (Fig. S3 A, available at http://www.jcb.org/cgi/content/full/jcb.200510061/DC1
). Phosphorylation at this site is mediated by ROCK and leads to LIMK2 activation (Sumi et al., 2001
). Therefore, Par-3 might be involved in negatively regulating LIMK2 activation by ROCK in vivo.
We next examined whether Par-3 can mediate the inhibition of LIMK2 kinase activity in vitro. Immunoprecipitated LIMK2 specifically phosphorylated GST-cofilin, and the presence of Par-3b inhibited this activity (). Par-3b was also phosphorylated during the kinase assay, which was likely caused by endogenous aPKC associated with Par-3b (Gao et al., 2002
). However, the Par-3c splice variant also inhibited the kinase activity of LIMK2 (). This variant does not bind to aPKC (Gao et al., 2002
), and it was minimally phosphorylated compared with Par-3b. To test the specificity of the inhibition, LIMK1 was used, and its activity was not inhibited by Par-3 (Fig. S3 B). Moreover, when we replaced Par-3 with another PDZ domain protein, Pals1, no inhibition of LIMK2 activity was observed (Fig. S3 C).
Figure 5. 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 (more ...)
To determine whether Par-3 can inhibit cofilin phosphorylation in vivo, Par-3c was transiently expressed in COS cells followed by treatment with lysophosphatidic acid (LPA), which activates the Rho–ROCK–LIMK2 pathway (). Overexpression of Par-3c decreased the basal phospho-cofilin level (0 min) and inhibited the increase in cofilin phosphorylation induced by LPA. Together, our data suggest that Par-3 binding to LIMK2 mediates the inhibition of LIMK2 activity and the down-regulation of cofilin phosphorylation.
LIMK1 and 2 share similar domain structures and have 50% overall amino acid identity (Okano et al., 1995
), but they are regulated by distinct mechanisms. LIMK1 is activated specifically by p21-activated kinase and is inhibited by association with bone morphogenetic protein type II receptor, which binds to the NH2
-terminal region of LIMK1 (Foletta et al., 2003
). LIMK2 is activated specifically by ROCK and, as we show here, is inhibited by association through its NH2
-terminal region with Par-3. The two kinases also exhibit different tissue distributions, with LIMK1 highly expressed in the brain and LIMK2 broadly expressed in the epithelia of nonneuronal tissues (Mori et al., 1997
), which is consistent with a role for LIMK2 in epithelial polarization.
Although Par-3 is known to be a key regulator of epithelial polarization, its molecular actions have remained elusive. We have now identified a novel function for Par-3 in the regulation of LIMK2, which phosphorylates and inactivates cofilin. Depletion of Par-3 leads to elevated cofilin phosphorylation, and the restoration of cofilin activity by either active cofilin mutant or double KD of Par-3 and LIMK2 partially rescues tight junction assembly, indicating that the suppression of cofilin phosphorylation by Par-3 plays a positive role in junction assembly. Although it is possible that other indirect mechanisms contribute to the increase in phospho-cofilin after Par-3 KD, our data strongly suggest that Par-3 is directly involved in regulating cofilin phosphorylation through its interaction with LIMK2. Together with our previous finding that Par-3 regulates Rac activity through its interaction with Tiam1 (Chen and Macara, 2005
), our current study suggests a possible role for Par-3 in modulating actin dynamics to facilitate junction assembly and polarization. On the other hand, actin organization is required for the correct localization of Bazooka (the Drosophila melanogaster
homologue of Par-3) in Drosophila
embryonic epithelia (Harris and Peifer, 2005
). We propose that one of the functions of Par-3 is to coordinate and fine-tune the actin remodeling processes associated with junction assembly through its abilities to interact with and regulate multiple signaling pathways that control actin reorganization. It will be interesting to examine whether this mechanism of Par-3–directed cofilin activation is used in other cellular processes that require polarization, such as cell migration.