Extracellular Signal-regulated Kinase (ERK) Regulates Cortactin Ubiquitination and Degradation in Lung Epithelial Cells*

Background: Cortactin is essential for barrier integrity. Results: ERK inhibition attenuates cortactin phosphorylation, ubiquitination, and degradation via the β-Trcp-mediated ubiquitin-proteasome system. Conclusion: Cortactin stability is coordinately regulated by stress kinases and the ubiquitin-proteasomal network. Significance: Understanding cortactin stability provides new targets to regulate epithelial barrier integrity. Cortactin, an actin-binding protein, is essential for cell growth and motility. We have shown that cortactin is regulated by reversible phosphorylation, but little is known regarding cortactin protein stability. Here, we show that lipopolysaccharide (LPS)-induced cortactin degradation is mediated by extracellular regulated signal kinase (ERK). LPS induces cortactin serine phosphorylation, ubiquitination, and degradation in mouse lung epithelia, an effect abrogated by ERK inhibition. Serine phosphorylation sites mutant, cortactinS405A/S418A, enhances its protein stability. Cortactin is polyubiquitinated and degraded within the proteasome, whereas a cortactinK79R mutant exhibited proteolytic stability during cyclohexamide (CHX) or LPS treatment. The E3 ligase subunit β-Trcp interacts with cortactin, and its overexpression reduced cortactin protein levels, an effect attenuated by ERK inhibition. Overexpression of β-Trcp was sufficient to reduce the protective effects of exogenous cortactin on epithelial cell barrier integrity, an effect not observed after expression of a cortactinK79R mutant. These results provide evidence that LPS modulation of cortactin stability is coordinately regulated by stress kinases and the ubiquitin-proteasomal network.

Cortactin was originally identified as a substrate of Src kinase, which plays a critical role in actin polymerization via interaction with the Arp2/3 complex (1,2). Cortactin regulates cell migration (3)(4)(5) and cell-cell junctions (6 -8). It accumulates in lamellipodia (5,9), an area that represents the leading edge in migratory cells. Down-regulation of cortactin decreases cell migration and invasion (5,10,11), whereas overexpression or activation of cortactin has the opposite effect (10,11). Cortactin also associates with cadherin (7,12) and ␤-catenin (13), which plays a critical role in maintaining cell-cell adherens junction. Cortactin depletion increases pulmonary endothelial permeability in vitro (14,15) and in vivo (16). Cortactin expression promotes barrier function via interacting with myosin light chain kinase in pulmonary endothelial cells (17,18). However, the role of cortactin in epithelial barrier function is still unclear. Another major function of cortactin is to regulate receptor-mediated endocytosis. Zhu et al. showed that suppression of cortactin expression by siRNA reduced transferrin uptake (19). Cortactin regulates clathrin-coated vesicle formation via association with dynamin-2 (19). Recent studies suggest that cortactin regulates NADPH oxidase activation and reactive oxygen species formation by association with p47 phox (20). Thus, cortactin exerts multifunctional roles in cellular behavior underscoring the importance in defining mechanisms for its regulation. Both tyrosine and serine phosphorylation of cortactin affect actin polymerization and cell migration (5,(21)(22)(23)(24). Src kinase catalyzes Tyr 421 , Tyr 466 , and Tyr 482 phosphorylation of cortactin; these modifications reduce F-actin cross-linking activity of cortactin (25). However, several studies have suggested that tyrosine phosphorylation of cortactin by Src kinase enhances actin assembly (26 -28). Head et al. showed that tyrosine phosphorylated cortactin is localized with F-actin in lamellipodia and podosomes (28). In vascular smooth muscle cells, tyrosine phosphorylation of cortactin is involved in the stability and turnover of podosomes (29). Tyrosine phosphorylation of cortactin significantly increases its association with myosin light chain kinase in pulmonary endothelial cells (8,18). Serine phosphorylation of cortactin is mediated by extracellular signal-regulated kinases (ERKs) (23,24,30) and other serine/threonine kinases such as Pak1 (31). Cortactin serine phosphorylation (at Ser 405 and Ser 418 ) by ERK promotes actin polymerization and tumor cell movement (24,32). In addition, serine phosphorylation of cortactin binds focal adhesion kinase, leading to its activation to control the level of cell scattering (22). As phosphorylation of proteins regulates their sta-bility, these studies raise the possibility that stress kinases could modulate cortactin concentrations in cells.
Ubiquitination regulates protein stability and involves the sequential modification of the targeted proteins by the action of an E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and E3 ubiquitin-protein ligase (33). Phosphorylation is a molecular signature that often leads to recruitment of the ubiquitination E3 ligase complex to a target protein (34 -36). Several studies have shown that calpain 2 regulates cortactin degradation (37,38); however, cortactin degradation through the ubiquitin proteolytic system has not been studied. Here, we show for the first time that ␤-Trcp, 2 an E3 ligase component, is sufficient to mediate elimination of cortactin by the ubiquitin-proteasome system. Further, ERK-dependent serine phosphorylation of cortactin is essential for cortactin ubiquitination and degradation in response to lipopolysaccharide (LPS). Hence, these results provide evidence that cortactin protein stability is regulated by the combinatorial activities of ERK and ␤-Trcp as key bioeffectors controlling epithelial barrier function.
Immunoblotting and Immunoprecipitation-Cell lysates were subjected to SDS-PAGE, electrotransferred to membranes, and immunoblotted. For immunoprecipitation, equal amounts of cell lysates (1,000 g) were incubated with 2 g/ml specific primary antibodies overnight at 4°C followed by the addition of 40 l of protein A/G-agarose for 2 h at 4°C. The immunoprecipitated complex was washed three times with 1% Triton X-100 in PBS and analyzed by Western blotting with an enhanced ECL system.
Immunostaining-MLE12 cells were plated on 35-mm MetTek glass-bottom culture dishes. After treatments as indicated, cells were fixed in 3.7% formaldehyde for 20 min, followed by permeabilization with 0.1% Triton X-100 for 2 min. Cells were incubated with a 1:200 dilution of antibodies, followed by a 1:200 dilution of fluorescence-conjugated secondary antibody sequentially for immunostaining. Immunofluorescent cell imaging was performed on a Nikon confocal microscope.
Epithelial Permeability Assay with Measuring Dextran Leak-MLE12 cells were plated at 100% confluence on permeable inserts containing 0.4-m pores. After addition of FITC-dextran (4-kDa) on the upper chamber for 20 h, fluorescence levels in the upper medium and bottom medium were measured by a fluorescence microplate reader with excitation at 488 nm and emission at 520 nm.
Measurement of Transepithelial Resistance (TER)-MLE12 cells were grown to confluence on Transwells with 0.4-m pores. TER was measured by EVOM-Epithelial Voltohmeter according to the manufacturer's instruction (World Precision Instruments Inc, Aston, Stevenage, UK).
Statistics-All results were subjected to statistical analysis using one-way ANOVA and, wherever appropriate, analyzed by Student-Newman-Keuls test. Data are expressed as mean Ϯ S.D. of triplicate samples from at least three independent experiments, and values that were p Ͻ 0.05 were considered statistically significant.

Cortactin Degradation Is Mediated by Ubiquitin-Proteasome
System-To investigate the mechanisms of cortactin degradation, we first examined the stability of cortactin in MLE12 cells. Treatment with protein synthesis inhibitor, CHX, revealed that cortactin half-life (t1 ⁄ 2 ) is ϳ 8.9 h (Fig. 1, A and B). To identify which pathway is involved in cortactin degradation, MLE12 cells were treated with proteasomal (MG-132) or lysosomal (leupeptin) inhibitors prior to CHX treatment. MG-132, not leupeptin, blocked cortactin degradation. In addition, inhibition of the proteasome system by MG-132 induced the accumulation of cortactin in a time-dependent manner ( These results indicate that cortactin is degraded by the ubiquitin-proteasome machinery in lung epithelia.

LPS Induces Cortactin Degradation in Lung Epithelial Cells and Lung
Tissues-Lung epithelium is the first site for pathogen contact. We have shown that LPS potently induces cytokine release (39) and epithelial barrier disruption (40,41). LPS treatment also induced cortactin degradation in a time- (Fig. 2, A and B) and dose- (Fig. 2, C and D) dependent fashion. Cortactin is localized in the cytoplasm and within the leading edge of migratory cells. LPS treatment (20 g/ml, 8 h) significantly reduced fluorescence immunostaining of cortactin in both the cytoplasm and leading edge of MLE12 cells (Fig. 2E). Further, cortactin expression in lung tissues from LPS (1 and 5 mg/kg, 20 h) challenged mice was examined. Intratracheal administration of LPS, compared with control, significantly induced cortactin degradation in mouse lung tissue (Fig. 2, F and G). These results demonstrate that LPS induces cortactin degradation.
Lys 79 Is a Molecular Site Regulating Cortactin Stability-We substituted several candidate lysine (K) residues of cortactin with Arg (R). Of several mutants tested, only cortactin K79R exhibited protein stability in response to CHX treatment (Fig. 3,  A and B). Further, we examined whether the cortactin K79R mutant exhibits resistance to LPS treatment. LPS (20 g/ml, 8 h) reduced levels of a cortactin wild type and a cortactin K314R mutant, whereas cortactin K79R mutant levels remained unchanged in response to LPS treatment (Fig. 3, C and D), suggesting that Lys 79 is a critical residue for regulating cortactin stability.
LPS Induces Cortactin Serine Phosphorylation via ERK Activity-Protein phosphorylation serves as a key molecular signal for the ubiquitination of proteins targeted by Skp-Cul-lin1-F box (SCF)-based E3 ligases (34 -36). To investigate whether LPS-induced cortactin degradation is regulated by its phosphorylation, first we examined serine phosphorylation of cortactin in response to LPS treatment. MLE12 cells were treated with LPS for 1-4 h, serine-phosphorylated proteins were immunoprecipitated with an antibody to panphosphoserine, followed by immunoblotting with cortactin antibody. Fig.  4A shows that LPS-induced serine phosphorylation in a timedependent manner. LPS had no effect on tyrosine phosphorylation of cortactin (data not shown). Consistent with prior studies (42), LPS induced phosphorylation of ERK in a timedependent manner (Fig. 4A). Further, we found that the LPS treatment promoted association of cortactin with ERK (Fig.  4B). Inhibition of ERK activation by PD98059 attenuated LPSinduced serine phosphorylation of cortactin (Fig. 4C). These results indicate that LPS induces cortactin serine phosphorylation via enhancing ERK activity.

ERK Regulates Cortactin Ubiquitination and Degradation-
To investigate whether the serine phosphorylation regulates stability of cortactin, we first examined the role of serine phosphorylation in LPS-induced ubiquitination of cortactin. MLE12 cells were treated with LPS (20 g/ml, 6 h) in the presence or absence of PD98059. Ubiquitination of cortactin was examined by immunoprecipitation with an anti-ubiquitin antibody, followed by immunoblotting with a cortactin antibody. Fig. 5A shows that LPS induced association of ubiquitin with cortactin, whereas PD98059 attenuated this interaction. Further,  we examined the effect of PD98059 on LPS-induced degradation of cortactin. PD98059 pretreatment attenuated the LPS-stimulated decrease in cortactin levels (Fig. 5, B and C) and cellular content as demonstrated by immunostaining (Fig. 5, D  and E), whereas p38 MAPK inhibitor SB203580 pretreatment had no effect on cortactin level (Fig. 5, B and C). Ser 405 and Ser 418 are ERK-mediated phosphorylation sites on cortactin (24,30). Furthermore, the serine phosphorylation sites mutant, cortactin S405A/S418A , were generated. Cortactin S405A/S418A levels remained stable when measured in the CHX-chase assay (Fig. 5, F and G) and LPS treatment (Fig. 5, H and I). These results suggest that serine phosphorylation of cortactin by ERK promotes LPS-induced ubiquitination and degradation.
␤-Trcp Promotes Cortactin Degradation-The SCF E3 ligase complex recognizes and binds specific substrates (43). A key subunit that recognizes substrates within the SCF complex is  the F box protein. To investigate which F box protein targets cortactin for degradation, we overexpressed over 20 F box proteins and among them, only ␤-Trcp overexpression reduced cortactin expression (data not shown). In addition, overexpression of V5-tagged ␤-Trcp reduced cortactin expression in a dose-dependent manner (Fig. 6, A and B). To investigate the role of ERK-mediated phosphorylation of cortactin in ␤-Trcp-driven cortactin degradation, cells were transfected with V5-tagged ␤-Trcp plasmid, followed by incubation with PD98059 for additional 24 h. ␤-Trcp overexpression reduced immunoreactive cortactin levels, an effect antagonized by PD98059 treatment of cells (Fig. 6, C  and D). Further, MLE12 cells were transfected with V5-tagged ␤-Trcp, followed by the addition of PD98059 in the presence of MG-132. The association of overexpressed V5-tagged ␤-Trcp and cortactin was examined by co-immunoprecipitation. Fig.  6E shows that V5-tagged ␤-Trcp was bound to cortactin, whereas PD98059 attenuated the interaction. Because Ser 405 and Ser 418 are ERK-mediated phosphorylation sites, we investigated the interaction between V5-tagged ␤-Trcp and myctagged cortactin S405A/S418A in the presence of MG-132. Fig. 6F shows that V5-tagged ␤-Trcp interacted with cortactin wild type, but not with cortactin S405A/S418A variant. Furthermore, immunofluorescence staining shows that V5-tagged ␤-Trcp (red color) and myc-tagged cortactin wild type (green color), but not cortactin S405A/S418A (green color), co-localized in the cytoplasm of MLE12 cells (Fig. 6G). Thus, the results demon-strate that ERK activation regulates ␤-Trcp-mediated cortactin degradation.
Cortactin Stability Regulates Lung Epithelial Barrier Function-Cortactin is critical for maintaining endothelial barrier integrity because its depletion results in a marked loss of cell-to-cell integrity (8,14). Here, we investigated the role of cortactin in lung epithelial integrity. Dextran leaks, TER, and E-cadherin accumulation at cell-cell contacts were determined as the index of lung epithelial barrier integrity. Down-regulation of cortactin by shcortactin increased dextran leak (Fig. 7A), as well as reduced TER (Fig. 7B). Overexpression of ubiquitin acceptor site mutant cortactin K79R rescued the effects of shcortactin (Fig. 7, A and B). The effect of shcortactin on cortactin expression was confirmed by Western blotting (Fig. 7C). Overexpression of cortactin reduced the dextran leak in MLE12 cells (Fig. 7D) and increased E-cadherin accumulation at cell-cell contacts (Fig. 7E), suggesting that the overexpression of cortactin increases lung epithelial integrity. Co-transfection of a cortactin plasmid with V5-tagged ␤-Trcp attenuated cortactinmediated lung epithelial barrier enhancement (Fig. 7, D and E). Further, overexpression of a plasmid encoding cortactin K79R produced similar effects as wild type cortactin by enhancing lung epithelial integrity; importantly, co-overexpression of ␤-Trcp had no effect on cortactin K79R -mediated lung epithelial barrier function (Fig. 7, D and E). We have shown that LPS treatment reduces lung epithelial integrity (40,41). Here, we showed that LPS increased dextran leak in MLE12 cells (Fig.  7F). Furthermore, down-regulation of ␤-Trcp by sh␤-Trcp attenuated LPS-induced dextran leak (Fig. 7F) and E-cadherin mislocalization in the cytoplasm of MLE12 cells (Fig. 7G). These results suggest a regulatory role for SCF ␤-Trcp in modulating cytoskeletal function by targeting cortactin for degradation.

DISCUSSION
Cortactin is a key structural element and protein kinase substrate that regulates cytoskeletal arrangement (1,2). Cortactin activity controlled by post-translational modifications, such as phosphorylation (23,24) and acetylation (44), has been studied extensively; however understanding of cortactin protein stability is limited. Here, we provide evidence suggesting that cortactin degradation is regulated by the ␤-Trcp and the ubiquitin-proteasome system. Lys 79 within cortactin is essential in regulating cortactin degradation. Serine phosphorylation of cortactin by ERK facilitated cortactin ubiquitination and degradation. Further, the results support a role for the ERK-SCF ␤-Trcp -ubiquitin proteasomal pathway in modulating lung epithelial barrier integrity. These results suggest that future interventions such as use of small molecular agonists or antagonists within the ubiquitin system (e.g. SCF ␤-Trcp ) could perhaps be employed to regulate E3 ligase activity, thereby impacting cell motility.
Protein degradation is an important aspect of cell regulation. Two major systems (proteasome and lysosome systems) contribute to the vast majority of intracellular protein degradation (45). Cortactin binds to filamentous actin to act as a regulator of cytoskeleton dynamics (1, 2, 6), thus understanding cortactin stability is essential in regulating cell motility. Prior studies have demonstrated that calpain 2 mediates proteolysis of cortactin in fibroblasts (37) and platelets (38). Perrin et al. showed that calpain 2 cleaves cortactin between the actin binding repeats and the ␣-helical domain (37). The current study for the first time attempts to demonstrate that cortactin is ubiquitinated and degraded in the proteasome system, but not in the lysosomal pathway. Actin-binding proteins are not stable under certain biological conditions. Alanine-rich kinase substrate (MARCKS) and fascin, two actin-binding proteins, are ubiquitinated, displaying a rapid turnover rate after preconditioning ischemia (46). Here, cortactin displayed extended in vivo stability consistent with half-lives of structural and regulatory proteins, but destabilized in the presence of LPS.
Our mutational studies suggest that Lys 79 as an acceptor site within the first actin binding repeat appears to play a critical role in regulating cortactin stability. In contrast to cortactin wild type and other Lys to Arg mutants, the cortactin K79R variant displayed significant proteolytic resistance in the CHX chase assay or in response to LPS treatment. ␤-Trcp, a well studied SCF E3 ligase subunit, regulates cell functions by mediating intracellular protein degradation (47). ␤-Trcp targets phosphorylated IB for its degradation (48), a protein also regulated by LPS. Our prior work demonstrates that ␤-Trcp also targets acyl-coA:lysophosphatidylcholine acyltransferase 1 (Lpcat1), a phospholipid-remodeling enzyme, for disposal in lung epithelial cells (36). Hence, these observations suggest that SCF ␤-Trcp appears to exert diverse effects on fundamental processes within lung epithelium.
Protein phosphorylation provides a phosphodegron molecular signal for recruiting E3 ligase-associated proteins for ubiquitination of molecular targets (34 -36). For example, ␤-Trcp targets phosphorylated IB (48) and phosphorylated ␤-catenin (49). We have shown that Lpcat1 is serine-phosphorylated by GSK3␤, and subsequently the phosphoenzyme is targeted by ␤-Trcp for disposal (36). Serine phosphorylation of cortactin on Ser 405 and Ser 418 by ERK has been well demonstrated (21,23,24); however, its link to cortactin stability has not been investigated. ERK regulates degradation of several intracellular proteins via the ubiquitin-proteasome system, such as dual-specificity phosphatase 1 (50) and GATA3 (51). In response to LPS treatment, cortactin was serine-(not tyrosine) phosphorylated, ubiquitinated, and degraded. ERK activity plays a critical role in these reactions because inhibition of ERK activity blocked cortactin phosphorylation, ubiquitination, and subsequent degradation. Serine-phosphorylated cortactin is localized in the sites of dynamic actin assembly in tumor cells (30). It is likely that as with many proline-directed kinases, ERK assembles within a scaffolding complex with multiple effectors at a docking domain or region within sites of cortactin (52). This assembly of kinases and regulatory scaffolding molecules might then provide a phosphodegron-like signature for recruitment of SCF ␤-Trcp . Serine phosphorylation sites Ser 405 and Ser 418 and ubiquitin acceptor site Lys 79 are localized at the N terminus and the C terminus of cortactin, separately. Hence, the molecular interplay between ERK and SCF ␤-Trcp within these regions of cortactin could serve as a feedback signal to control cortactin steady-state levels to dynamically regulate barrier integrity.
As the role of cortactin in controlling lung epithelial barrier function is still unknown and SCF ␤-Trcp has multiple targets, our results do not exclude a more complex model where ERK and the ubiquitin apparatus jointly modulates multiple targets to disrupt the epithelial lining during LPS-mediated inflammation. One such potential target is E-cadherin, which interacts with cortactin, and might also be modulated by the SCF complex. Future work will focus on related cell adhesion proteins that influence stability of the epithelial barrier.