Beta2-adaptin binds actopaxin and regulates cell spreading, migration and matrix degradation.

Cell adhesion to the extracellular matrix is a key event in cell migration and invasion and endocytic trafficking of adhesion receptors and signaling proteins plays a major role in regulating these processes. Beta2-adaptin is a subunit of the AP-2 complex and is involved in clathrin-mediated endocytosis. Herein, ß2-adaptin is shown to bind to the focal adhesion protein actopaxin and localize to focal adhesions during cells spreading in an actopaxin dependent manner. Furthermore, ß2-adaptin is enriched in adhesions at the leading edge of migrating cells and depletion of ß2-adaptin by RNAi increases cell spreading and inhibits directional cell migration via a loss of cellular polarity. Knockdown of ß2-adaptin in both U2OS osteosarcoma cells and MCF10A normal breast epithelial cells promotes the formation of matrix degrading invadopodia, adhesion structures linked to invasive migration in cancer cells. These data therefore suggest that actopaxin-dependent recruitment of the AP-2 complex, via an interaction with ß2-adaptin, to focal adhesions mediates cell polarity and migration and that ß2-adaptin may control the balance between the formation of normal cell adhesions and invasive adhesion structures.

Actopaxin (α-parvin) phosphorylation is required for matrix degradation and cancer cell invasion.

Dysregulation of cell adhesion and motility is known to be an important factor in the development of tumor malignancy. Actopaxin (α-parvin) is a paxillin, integrin-linked kinase, and F-actin binding focal adhesion protein with several serine phosphorylation sites in the amino terminus that contribute to the regulation of cell spreading and migration. Here, phosphorylation of actopaxin is shown to contribute to the regulation of matrix degradation and cell invasion. Osteosarcoma cells stably expressing wild type (WT), nonphosphorylatable (Quint), and phosphomimetic (S4D/S8D) actopaxin demonstrate that actopaxin phosphorylation is necessary for efficient Src and matrix metalloproteinase-driven degradation of extracellular matrix. Rac1 was found to be required for actopaxin-induced matrix degradation whereas inhibition of myosin contractility promoted degradation in the phosphomutant-expressing Quint cells, indicating that a balance of Rho GTPase signaling and regulation of cellular tension are important for the process. Furthermore, actopaxin forms a complex with the Rac1/Cdc42 GEF ß-PIX and Rac1/Cdc42 effector PAK1, to regulate actopaxin-dependent matrix degradation. Actopaxin phosphorylation is elevated in the invasive breast cancer cell line MDA-MB-231 compared with normal breast epithelial MCF10A cells. Expression of the nonphosphorylatable Quint actopaxin in MDA-MB-231 cells inhibits cell invasion whereas overexpression of WT actopaxin promotes invasion in MCF10A cells. Taken together, this study demonstrates a new role for actopaxin phosphorylation in matrix degradation and cell invasion via regulation of Rho GTPase signaling.

The UBX protein SAKS1 negatively regulates endoplasmic reticulum-associated degradation and p97-dependent degradation.

Endoplasmic reticulum-associated degradation (ERAD) is an essential quality control process whereby misfolded proteins are exported from the endoplasmic reticulum and degraded by the proteasome in the cytosol. The ATPase p97 acts as an essential component of this process by providing the force needed for retrotranslocation and by serving as a processing station for the substrate once in the cytosol. Proteins containing the ubiquitin regulatory X (UBX) ubiquitin-like domain function as adaptors for p97 through their direct binding with the amino terminus of the ATPase. We demonstrate that the UBX protein SAKS1 is able to act as an adaptor for p97 that negatively modulates ERAD. This requires the ability of SAKS1 to bind both polyubiquitin and p97. Moreover, the association between SAKS1 and p97 is positively regulated by polyubiquitin binding of the UBX protein. SAKS1 also negatively impacts the p97-dependent processing required for degradation of a cytosolic, non-ERAD, substrate. We find SAKS1 is able to protect polyubiquitin from the activity of deubiquitinases, such as ataxin-3, that are necessary for efficient ERAD. Thus, SAKS1 inhibits protein degradation mediated by p97 complexes in the cytosol with a component of the mechanism being the ability to shield polyubiquitin chains from ubiquitin-processing factors.

The scaffolding protein EBP50 regulates microvillar assembly in a phosphorylation-dependent manner.

The mechanisms by which epithelial cells regulate the presence of microvilli on their apical surface are largely unknown. A potential regulator is EBP50/NHERF1 (ERM-binding phosphoprotein of 50 kD/Na(+)-H(+) exchanger regulatory factor), a microvillar scaffolding protein with two PDZ domains followed by a C-terminal ezrin-binding domain. Using RNAi and expression of RNAi-resistant EBP50 mutants we systematically show that EBP50 is necessary for microvillar assembly and requires that EBP50 has both a functional first PDZ domain and an ezrin-binding site. Expression of mutants mimicking Cdc2 or PKC phosphorylation are nonfunctional in microvillar assembly. Biochemical analysis reveals that these mutants are defective in PDZ1 accessibility when PDZ2 is occupied, and can be rendered functional in vivo by additional mutation of PDZ2. EBP50 is not necessary for mitotic cell microvilli, and PKC activation causes a rearrangement of microvilli on cells due to phosphorylation-dependent loss of EBP50 function. Thus, EBP50 is a critical factor that regulates microvilli assembly and whose activity is regulated by signaling pathways and occupation of its PDZ2 domain.

A regulated complex of the scaffolding proteins PDZK1 and EBP50 with ezrin contribute to microvillar organization.

PDZK1 and ezrin, radixin, moesin binding phosphoprotein 50 kDa (EBP50) are postsynaptic density 95/disc-large/zona occludens (PDZ)-domain-containing scaffolding proteins found in the apical microvilli of polarized epithelial cells. Binary interactions have been shown between the tail of PDZK1 and the PDZ domains of EBP50, as well as between EBP50 and the membrane-cytoskeletal linking protein ezrin. Here, we show that these molecules form a regulated ternary complex in vitro and in vivo. Complex formation is cooperative because ezrin positively influences the PDZK1/EBP50 interaction. Moreover, the interaction of PDZK1 with EBP50 is enhanced by the occupancy of EBP50's adjacent PDZ domain. The complex is further regulated by location, because PDZK1 shuttles from the nucleus in low confluence cells to microvilli in high confluence cells, and this regulates the formation of the PDZK1/EBP50/ezrin complex in vivo. Knockdown of EBP50 decreases the presence of microvilli, a phenotype that can be rescued by EBP50 re-expression or expression of a PDZK1 chimera that is directly targeted to ezrin. Thus, when appropriately located, PDZK1 can provide a function necessary for microvilli formation normally provided by EBP50. By entering into the ternary complex, PDZK1 can both enhance the scaffolding at the apical membrane as well as augment EBP50's role in microvilli formation.

The scaffold protein PDZK1 undergoes a head-to-tail intramolecular association that negatively regulates its interaction with EBP50.

PDZK1 (also known as CAP70, NHERF3, or NaPi-Cap1) is a scaffolding protein composed of four PDZ (Post-Synaptic Density-95, Discs Large, Zonula Occludens-1) domains followed by a short carboxyl-terminal tail. This scaffold acts as a mediator of localization and expression levels of multiple receptors in the kidney, liver, and endothelium. Here, we characterize the self-association properties of the protein. PDZK1 can undergo modest homodimerization in vivo and in vitro through self-association involving its third PDZ domain. In addition, the tail of PDZK1 interacts in an intramolecular fashion with the first PDZ domain, but this interaction does not contribute to dimer formation. The interaction between the tail of PDZK1 and its first PDZ domain induces the protein to adopt a more compact conformation. A head-to-tail association has also been reported for EBP50/NHERF1, a two-PDZ domain member of the same scaffolding protein family as PDZK1, and shown to regulate binding of target proteins to the EBP50 PDZ domains. As opposed to EBP50, the association of PDZK1 with specific ligands for its PDZ domains is unaffected by the intramolecular association, establishing a different mode of interaction among these two members of the same scaffolding family. However, the tail of PDZK1 interacts with the PDZ domains of EBP50, and this interaction is negatively regulated by the intramolecular association of PDZK1. Thus, we have uncovered a regulated association between the two PDZ-containing scaffolding molecules, PDZK1 and EBP50.

CdGAP associates with actopaxin to regulate integrin-dependent changes in cell morphology and motility.

BACKGROUND: Integrin signaling, stimulated by cell adhesion to the extracellular matrix, plays a critical role in coordinating changes in cell morphology and migration. The requisite remodeling of the cytoskeleton is controlled by the Rho family of small GTPases, which are, in turn, regulated via activation by guanine-nucleotide exchange factors (GEFs) and inactivation by GTPase-activating proteins (GAPs). However, the mechanisms contributing to the precise spatial and temporal regulation of these Rho GTPase modulators remain poorly understood. RESULTS: The Cdc42/Rac GAP CdGAP has previously been implicated as an inhibitor of growth-factor-induced lamellipodia formation. Herein, CdGAP is shown to localize to focal adhesions, potentially through its direct association with the amino terminus of actopaxin, a paxillin and actin binding protein. CdGAP activity is regulated in an adhesion-dependent manner and, through the overexpression of wild-type CdGAP and a GAP-deficient mutant, as well as RNA interference, is shown to be required for normal cell spreading, polarized lamellipodia formation, and cell migration. Introduction of an actopaxin mutant defective for CdGAP binding, or reduction of actopaxin by using RNAi, significantly attenuated these effects. CONCLUSIONS: We have established that CdGAP is an important regulator of integrin-induced Rho family signaling to the cytoskeleton and that its interaction with the focal-adhesion protein actopaxin is critical for the correct spatial and/or temporal regulation of CdGAP function. A complete understanding of the coordination of signaling events downstream of integrin engagement with the extracellular matrix will provide valuable insight into the regulation of cell migration during processes such as wound repair, development, and tumor cell metastasis.

Tyrosine-phosphorylated Hic-5 inhibits epidermal growth factor-induced lamellipodia formation.

The focal adhesion protein Hic-5, a homologue to paxillin, has been shown to be tyrosine-phosphorylated in fibroblasts in response to stimuli such as osmotic stress, serum, LPA and endothelin. However, the function of this modification to Hic-5 is unclear. Herein, we show that Hic-5 is tyrosine-phosphorylated on residues 38 and 60 following epidermal growth factor (EGF) treatment of COS-7 cells, coincident with an increase in peripheral actin reorganization. To explore the role of Hic-5 phosphorylation in this process, we introduced wild-type (WT) and mutant Hic-5 constructs into COS-7 cells and determined that EGF-induced lamellipodia formation was suppressed by WT Hic-5. This effect required localization to focal adhesions as well as phosphorylation of Hic-5 as overexpression of both a non-targeting and a non-phosphorylatable Hic-5 failed to inhibit peripheral actin reorganization. Interestingly, overexpression of non-phosphorylatable Y31/118F or WT paxillin did not affect lamellipodia formation, indicating that this effect is specific to Hic-5. The EGF-induced lamellipodia were Rac-dependent and overexpressed WT Hic-5, but not non-phosphorylatable Hic-5 inhibited Rac activation. Our data suggest that Hic-5 tyrosine phosphorylation functions to regulate signaling associated with lamellipodia formation, a process fundamental to cell motility.

Actopaxin interacts with TESK1 to regulate cell spreading on fibronectin.

The focal adhesion protein actopaxin contributes to integrin-actin associations and is involved in cell adhesion, spreading, and motility. Herein, we identify and characterize an association between actopaxin and the serine/threonine kinase testicular protein kinase 1 (TESK1), a ubiquitously expressed protein previously reported to regulate cellular spreading and focal adhesion formation via phosphorylation of cofilin. The interaction between actopaxin and TESK1 is direct and the binding sites were mapped to the carboxyl terminus of both proteins. The association between actopaxin and TESK1 is negatively regulated by adhesion to fibronectin, and a phosphomimetic actopaxin mutant that promotes cell spreading also exhibits impaired binding to TESK1. Binding of actopaxin to TESK1 inhibits TESK1 kinase activity in vitro. Expression of the carboxyl terminus of actopaxin has previously been reported to retard cell spreading. This effect was reversed following overexpression of TESK1 and was found to be dependent on an inability of actopaxin carboxyl terminus expressing cells to promote cofilin phosphorylation upon matrix adhesion and caused by retention of TESK1 by this actopaxin mutant. Thus, the association between actopaxin and TESK1, which is likely regulated by phosphorylation of actopaxin, regulates TESK1 activity and subsequent cellular spreading on fibronectin.

The integrin-linked kinase regulates cell morphology and motility in a rho-associated kinase-dependent manner.

The integrin-linked kinase (ILK) is a multidomain focal adhesion protein implicated in signal transmission from integrin and growth factor receptors. We have determined that ILK regulates U2OS osteosarcoma cell spreading and motility in a manner requiring both kinase activity and localization. Overexpression of wild-type (WT) ILK resulted in suppression of cell spreading, polarization, and motility to fibronectin. Cell lines overexpressing kinase-dead (S343A) or paxillin binding site mutant ILK proteins display inhibited haptotaxis to fibronectin. Conversely, spreading and motility was potentiated in cells expressing the "dominant negative," non-targeting, kinase-deficient E359K ILK protein. Suppression of cell spreading and motility of WT ILK U2OS cells could be rescued by treatment with the Rho-associated kinase (ROCK) inhibitor Y-27632 or introduction of dominant negative ROCK or RhoA, suggesting these cells have increased RhoA signaling. Activation of focal adhesion kinase (FAK), a negative regulator of RhoA, was reduced in WT ILK cells, whereas overexpression of FAK rescued the observed defects in spreading and cell polarity. Thus, ILK-dependent effects on ROCK and/or RhoA signaling may be mediated through FAK.

Phosphorylation of actopaxin regulates cell spreading and migration.

Actopaxin is an actin and paxillin binding protein that localizes to focal adhesions. It regulates cell spreading and is phosphorylated during mitosis. Herein, we identify a role for actopaxin phosphorylation in cell spreading and migration. Stable clones of U2OS cells expressing actopaxin wild-type (WT), nonphosphorylatable, and phosphomimetic mutants were developed to evaluate actopaxin function. All proteins targeted to focal adhesions, however the nonphosphorylatable mutant inhibited spreading whereas the phosphomimetic mutant cells spread more efficiently than WT cells. Endogenous and WT actopaxin, but not the nonphosphorylatable mutant, were phosphorylated in vivo during cell adhesion/spreading. Expression of the nonphosphorylatable actopaxin mutant significantly reduced cell migration, whereas expression of the phosphomimetic increased cell migration in scrape wound and Boyden chamber migration assays. In vitro kinase assays demonstrate that extracellular signal-regulated protein kinase phosphorylates actopaxin, and treatment of U2OS cells with the MEK1 inhibitor UO126 inhibited adhesion-induced phosphorylation of actopaxin and also inhibited cell migration.