SNX9 as an adaptor for linking synaptojanin-1 to the Cdc42 effector ACK1

Sorting nexin 9 (SNX9, also referred to as SH3PX1) is a binding partner for the non-receptor and Cdc42-associated kinase (ACK) in Drosophila and mammals. ACK1 is known to bind clathrin and influence EGF receptor endocytosis. SNX9 comprises an N-terminal Src homology domain 3 (SH3), a central PHOX homology (PX) domain, and a carboxyl-terminal coiled-coil region. In order to investigate SNX9 further we have made use of a novel in vivo biotinylation system to label various GST-SH3 domains and perform blot overlays, thereby identifying synaptojanin-1 as a partner for SNX9. Biotinylated SH3 domains were also used for specific identification of target proline-rich sequences in synaptojanin and ACK1 on synthetic peptides arrays. Direct assessment of SH3 binding efficiencies at different positions within the extensive proline-rich regions of these proteins were thus determined. While SNX9 targets a number of sequences within the proline-rich regions of synaptojanin, a single site was identified in human ACK1. By testing the association of various truncations of ACK1 with SNX9 we confirmed the dominant SNX9 binding domain in human ACK1 (residues 920-955). In the presence of SNX9 we find that synaptojanin is able to colocalize with distinct ACK1 containing vesicles, indicating that this tyrosine kinase is linked to many components involved in vesicle dynamics including clathrin, AP2 and synaptojanin-1.     

Activated Cdc42-associated kinase 1 is a component of EGF receptor signaling complex and regulates EGF receptor degradation

Cdc42-associated tyrosine kinase 1 (ACK1) is a specific down-stream effector of Cdc42, a Rho family small G-protein. Previous studies have shown that ACK1 interacts with clathrin heavy chain and is involved in clathrin-coated vesicle endocytosis. Here we report that ACK1 interacted with epidermal growth factor receptor (EGFR) upon EGF stimulation via a region at carboxy terminus that is highly homologous to Gene-33/Mig-6/RALT. The interaction of ACK1 with EGFR was dependent on the kinase activity or tyrosine phosphorylation of EGFR. Immunofluorescent staining using anti-EGFR and GFP-ACK1 indicates that ACK1 was colocalized with EGFR on EEA-1 positive vesicles upon EGF stimulation. Suppression of the expression of ACK1 by ACK-RNAi inhibited ligand-induced degradation of EGFR upon EGF stimulation, suggesting that ACK1 plays an important role in regulation of EGFR degradation in cells. Furthermore, we identified ACK1 as an ubiquitin-binding protein. Through an ubiquitin-association (Uba) domain at the carboxy terminus, ACK1 binds to both poly- and mono-ubiquitin. Overexpression of the Uba domain-deletion mutant of ACK1 blocked the ligand-dependent degradation of EGFR, suggesting that ACK1 regulates EGFR degradation via its Uba domain. Taken together, our studies suggest that ACK1 senses signal of EGF and regulates ligand-induced degradation of EGFR.     

Biochemical properties of the Cdc42-associated tyrosine kinase ACK1. Substrate specificity, authphosphorylation, and interaction with Hck

ACK1 (activated Cdc42-associated kinase 1) is a nonreceptor tyrosine kinase and the only tyrosine kinase known to interact with Cdc42. To characterize the enzymatic properties of ACK, we have expressed and purified active ACK using the baculovirus/Sf9 cell system. This ACK1 construct contains (from N to C terminus) the kinase catalytic domain, SH3 domain, and Cdc42-binding Cdc42/Rac interactive binding (CRIB) domain. We characterized the substrate specificity of ACK1 using synthetic peptides, and we show that the specificity of the ACK1 catalytic domain most closely resembles that of Abl. Purified ACK1 undergoes autophosphorylation, and autophosphorylation enhances kinase activity. We identified Tyr284 in the activation loop of ACK1 as the primary autophosphorylation site using mass spectrometry. When expressed in COS-7 cells, the Y284F mutant ACK1 showed dramatically reduced levels of tyrosine phosphorylation. Although the SH3 and CRIB domains of purified ACK1 are able to bind ligands (a polyproline peptide and Cdc42, respectively), the addition of ligands did not stimulate tyrosine kinase activity. To characterize potential interacting partners for ACK1, we screened several SH2 and SH3 domains for their ability to bind to full-length ACK1 or to the catalytic-SH3-CRIB construct. ACK1 interacts most strongly with the SH3 domains of Src family kinases (Src or Hck) via its C-terminal proline-rich domain. Co-expression of Hck with kinase-inactive ACK1(K158R) in mammalian cells resulted in tyrosine phosphorylation of ACK1, suggesting that ACK1 is a substrate for Hck. Our data suggest that Hck is a novel binding partner for ACK1 that can regulate ACK1 activity by phosphorylation.     

The Cdc42 target ACK2 directly interacts with clathrin and influences clathrin assembly

The Ras-related GTP-binding protein Cdc42 has been implicated in a diversity of biological functions including the regulation of intracellular trafficking and endocytosis. While screening for Cdc42 targets that influence these activities, we identified the protein-tyrosine kinase ACK2 (for activated Cdc42-associated kinase 2) as a new binding partner for clathrin. ACK2 binds clathrin via a domain that is conserved among a number of other clathrin-binding proteins including the arrestins and AP-2. Overexpression of ACK2 in NIH3T3 cells results in an inhibition of transferrin receptor endocytosis because of a competition between ACK2 and AP-2 for clathrin. Activated Cdc42 weakens the interaction between ACK2 and clathrin and thus reverses the ACK2-mediated inhibition of endocytosis. Overexpression of ACK2 increases the amount of clathrin present in fractions enriched in clathrin-coated vesicles. Taken together, our data suggest that ACK2 may represent a novel clathrin-assembly protein and participate in the regulation of receptor-mediated endocytosis.     

The tyrosine kinase ACK1 associates with clathrin-coated vesicles through a binding motif shared by arrestin and other adaptors

One target for the small GTPase Cdc42 is the nonreceptor tyrosine kinase activated Cdc42-associated kinase (ACK), which binds selectively to Cdc42.GTP. We report that ACK1 can associate directly with the heavy chain of clathrin. A central region in ACK1 containing a conserved motif behaves as a clathrin adaptor and competes with beta-arrestin for a common binding site on the clathrin N-terminal head domain. Overexpressed ACK1 perturbs clathrin distribution, an activity dependent on the presence of C-terminal "adaptor" sequences that are also present in the related nonkinase gene 33. ACK1 interacts with the adaptor Nck via SH3 interactions but does not form a trimeric complex with p21-activated serine/threonine kinase, which also binds Nck. Stable low level expression of green fluorescent protein-ACK1 in NIH 3T3 cells has been used to localize ACK1 to clathrin-containing vesicles. The co-localization of ACK1 in vivo with clathrin and AP-2 indicates that it participates in trafficking, underlying an ability to increase receptor-mediated transferrin uptake.     

The nonreceptor tyrosine kinase ACK2, a specific target for Cdc42 and a negative regulator of cell growth and focal adhesion complexes

ACK2 (activated Cdc42-associated tyrosine kinase-2) is a nonreceptor tyrosine kinase that is a specific target/effector for the GTP-binding protein Cdc42. Thus far the biological function of this tyrosine kinase has not been determined. Using an inducible eukaryotic expression system in fibroblasts, we demonstrate that ACK2 can strongly influence cell shape and growth as well as focal complex formation. ACK2 was found to associate with the focal adhesion complex components talin and vinculin, but not with the focal adhesion kinase (FAK), in a kinase-independent manner. The tyrosine kinase activity of FAK was also inhibited in cells overexpressing both wild-type and kinase-defective ACK2. This may be due to a competition between ACK2 and FAK for Src, which is an essential cofactor for FAK activation, as we have found that ACK2 specifically binds Src in cells. The ACK2-Src interaction appears to be mediated by the SH3 domain of Src, and the phosphorylation of ACK2 is enhanced in cells overexpressing the hyperactivated Src(Y527F) mutant. Overexpression of both wild-type and kinase-defective ACK2 also results in a severe inhibition of cell growth. In addition, ACK2 dissolves actin stress fibers and disassembles focal complexes but in a kinase-dependent manner. These results, taken together with previous studies demonstrating an association of ACK2 with integrin beta(1) (Yang, W., Lin, Q., Guan, J.-L., Cerione, R. A. (1999) J. Biol. Chem. 274, 8524-8530) and clathrin (Yang, W., Lo, C. G., Dispenza, T., and Cerione, R. A. (2001) J. Biol. Chem. 276, 17468-17473), suggest that the binding and protein tyrosine kinase activities of ACK2 coordinate changes in cell morphology and growth with the disassembly of focal adhesion sites, perhaps to organize new integrin complexes that are required for endocytosis and/or for cellular differentiation.     

The Cdc42-associated kinase ACK1 is not autoinhibited but requires Src for activation

The non-RTK (receptor tyrosine kinase) ACK1 [activated Cdc42 (cell division cycle 42)-associated kinase 1] binds a number of RTKs and is associated with their endocytosis and turnover. Its mode of activation is not well established, but models have suggested that this is an autoinhibited kinase. Point mutations in its SH3 (Src homology 3)- or EGF (epidermal growth factor)-binding domains have been reported to activate ACK1, but we find neither of the corresponding W424K or F820A mutations do so. Indeed, deletion of the various ACK1 domains C-terminal to the catalytic domain are not associated with increased activity. A previous report identified only one major tyrosine phosphorylated protein of 60 kDa co-purified with ACK1. In a screen for new SH3 partners for ACK1 we found multiple Src family kinases; of these c-Src itself binds best. The SH2 and SH3 domains of Src interact with ACK1 Tyr518 and residues 623-652 respectively. Src targets the ACK1 activation loop Tyr284, a poor autophosphorylation site. We propose that ACK1 fails to undergo significant autophosphorylation on Tyr284 in vivo because it is basophilic (whereas Src is acidophilic). Subsequent ACK1 activation downstream of receptors such as EGFR (EGF receptor) (and Src) promotes turnover of ACK1 in vivo, which is blocked by Src inhibitors, and is compromised in the Src-deficient SYF cell line. The results of the present study can explain why ACK1 is responsive to so many external stimuli including RTKs and integrin ligation, since Src kinases are commonly recruited by multiple receptor systems.     

HECT E3 Ubiquitin Ligase Nedd4-1 Ubiquitinates ACK and Regulates Epidermal Growth Factor (EGF)-Induced Degradation of EGF Receptor and ACK

ACK (activated Cdc42-associated tyrosine kinase) (also Tnk2) is an ubiquitin-binding protein and plays an important role in ligand-induced and ubiquitination-mediated degradation of epidermal growth factor receptor (EGFR). Here we report that ACK is ubiquitinated by HECT E3 ubiquitin ligase Nedd4-1 and degraded along with EGFR in response to EGF stimulation. ACK interacts with Nedd4-1 through a conserved PPXY WW-binding motif. The WW3 domain in Nedd4-1 is critical for binding to ACK. Although ACK binds to both Nedd4-1 and Nedd4-2 (also Nedd4L), Nedd4-1 is the E3 ubiquitin ligase for ubiquitination of ACK in cells. Interestingly, deletion of the sterile alpha motif (SAM) domain at the N terminus dramatically reduced the ubiquitination of ACK by Nedd4-1, while deletion of the Uba domain dramatically enhanced the ubiquitination. Use of proteasomal and lysosomal inhibitors demonstrated that EGF-induced ACK degradation is processed by lysosomes, not proteasomes. RNA interference (RNAi) knockdown of Nedd4-1, not Nedd4-2, inhibited degradation of both EGFR and ACK, and overexpression of ACK mutants that are deficient in either binding to or ubiquitination by Nedd4-1 blocked EGF-induced degradation of EGFR. Our findings suggest an essential role of Nedd4-1 in regulation of EGFR degradation through interaction with and ubiquitination of ACK.Activated Cdc42-associated tyrosine kinase (ACK) (also Tnk2) is a member of the type VIII tyrosine kinase family. Activation of ACK, including both ACK1 and ACK2, occurs in response to signaling of epidermal growth factor receptor (EGFR), platelet-derived growth factor (PDGF) receptor, insulin receptor, Gas-6 receptor (Mer), M3 muscarinic receptor, integrins, or proteoglycan (3, 7, 11, 23, 26, 30, 44, 47). In Drosophila, D-ACK mediates the function of Cdc42 in dorsal closure during embryonic development (31). The ACK homologue, Ark-1, in Caenorhabditis elegans negatively regulates EGF signaling (15).A number of studies suggest a role for ACK in EGFR degradation. ACK1 and ACK2, two alternatively spliced isoforms, possess a highly conserved clathrin-binding motif and interact with clathrin (37, 45). Overexpression of ACK2 severely impairs transferrin receptor endocytosis, causes aberrant localization of AP-2, and induces changes in clathrin assembly. Furthermore, ACK2 interacts with sorting nexin 9 (SNX9, also named SH3PX1), a member of the sorting nexin family, via its proline-rich domain 1 and phosphorylates SNX9 to facilitate the degradation of EGF receptors (22). In C. elegans, Ark-1 genetically interacts with UNC101, the homologue of mammalian clathrin-associated protein AP47, and SLI-1, the homologue of mammalian Cbl that is an E3 ubiquitin ligase for ubiquitination of EGFR, and negatively regulates EGFR signaling (15).Our previous studies showed that ACK1 interacts with EGFR upon EGF stimulation via a region at the carboxyl terminus, designated the EGFR-binding domain (EBD), which is highly homologous to the EGFR/ErbB2-binding domain of Gene-33/Mig-6/RALT (32, 43). The interaction of ACK1 with EGFR is dependent on kinase activity and tyrosine phosphorylation of EGFR. Immunofluorescent staining using anti-EGFR and GFP-ACK1 indicates that ACK1 is colocalized with EGFR on large vacuolar structures upon EGF stimulation. Suppression of the expression of ACK1 by ACK-RNA interference (RNAi) inhibits ligand-induced degradation of EGFR, suggesting that ACK1 plays an important role in the regulation of EGFR degradation in cells. Furthermore, we identified ACK1 as an ubiquitin-binding protein. Through an ubiquitin association (Uba) domain at the carboxyl terminus, ACK1 is capable of interacting with both poly- and monoubiquitin. Overexpression of an Uba domain deletion mutant of ACK1 blocked the ligand-dependent degradation of EGFR, suggesting that ACK1 regulates EGFR degradation via its Uba domain. Thus, ACK1 senses EGF signaling and regulates degradation of EGFR.EGF-induced degradation of EGFR is mediated by ubiquitination (16). The ubiquitination of EGFR is activated upon EGF stimulation by recruiting the RING family E3 ubiquitin ligase Cbl to pY1045 (20, 21). This ubiquitination functions as a sorting signal for transporting EGFR to lysosomes for degradation (14). Nedd4, the HECT domain-containing E3 ubiquitin ligase, is also involved in the regulation of EGFR trafficking by ubiquitination of endocytic or vesicle sorting proteins (28). For example, it has been observed that Nedd4 ubiquitinates Cbl, Eps15, Tsg101, Hrs, and secretory carrier membrane proteins (SCAMPs) and participates in the processes of EGFR endocytosis and degradation (1, 18, 25, 42). However, exactly how Nedd4 engages in the EGFR degradation process in response to EGF stimulation is not known.In this report, we show that EGF stimulation induces ACK degradation. This degradation is associated with ubiquitination of ACK. Nedd4-1, but not Nedd4-2, is identified as the E3 ubiquitin ligase for ubiquitination of ACK. Furthermore, EGF-induced degradation of ACK is EGFR activation dependent and processed by lysosomes. RNAi knockdown and mutational analysis demonstrated that Nedd4-1 and Nedd4-1-catalyzed ubiquitination of ACK are required for EGF-induced degradation of EGFR and ACK. Our findings suggest a new mechanism in regulation of EGFR degradation.     

Epidermal growth factor stimulation of the ACK1/Dbl pathway in a Cdc42 and Grb2-dependent manner

The tyrosine kinase ACK1 phosphorylates and activates the guanine nucleotide exchange factor Dbl, which in turn directs the Rho family GTP-binding proteins. However, the regulatory mechanism of ACK1/Dbl signaling in response to extracellular stimuli remains poorly understood. Here we describe that epidermal growth factor stimulates the ACK1/Dbl pathway, leading to actin cytoskeletal rearrangements. The role of the two ACK1-binding proteins Cdc42 and Grb2 was assessed by overexpression of the Cdc42/Rac interactive binding domain and a dominant-negative Grb2 mutant, respectively. Specific inhibition of the interaction of ACK1 with Cdc42 or Grb2 by the use of these constructs diminished tyrosine phosphorylation of both ACK1 and Dbl in response to EGF. Therefore, the activation of ACK1 and subsequent downstream signaling require both Cdc42-dependent and Grb2-dependent processes within the cell. In addition, we show that EGF transiently induces formation of the focal complex and stress fibers when ACK1 was ectopically expressed. The induction of these structures was totally sensitive to the action of botulinum toxin C from Clostridium botulinum, suggesting a pivotal role of Rho. These results provide evidence that ACK1 acts as a mediator of EGF signals to Rho family GTP-binding proteins through phosphorylation and activation of GEFs such as Dbl.     

Drosophila Ack targets its substrate, the sorting nexin DSH3PX1, to a protein complex involved in axonal guidance.

Dock, the Drosophila orthologue of Nck, is an adaptor protein that is known to function in axonal guidance paradigms in the fly including proper development of neuronal connections in photoreceptor cells and axonal tracking in Bolwig's organ. To develop a better understanding of axonal guidance at the molecular level, we purified proteins in a complex with the SH2 domain of Dock from fly Schneider 2 cells. A protein designated p145 was identified and shown to be a tyrosine kinase with sequence similarity to mammalian Cdc-42-associated tyrosine kinases. We demonstrate that Drosophila Ack (DAck) can be co-immunoprecipitated with Dock and DSH3PX1 from fly cell extracts. The domains responsible for the in vitro interaction between Drosophila Ack and Dock were identified, and direct protein-protein interactions between complex members were established. We conclude that DSH3PX1 is a substrate for DAck in vivo and in vitro and define one of the major in vitro sites of DSH3PX1 phosphorylation to be Tyr-56. Tyr-56 is located within the SH3 domain of DSH3PX1, placing it in an important position for regulating the binding of proline-rich targets. We demonstrate that Tyr-56 phosphorylation by DAck diminishes the DSH3PX1 SH3 domain interaction with the Wiskott-Aldrich Syndrome protein while enabling DSH3PX1 to associate with Dock. Furthermore, when Tyr-56 is mutated to aspartate or glutamate, the binding to Wiskott-Aldrich Syndrome protein is abrogated. These results suggest that the phosphorylation of DSH3PX1 by DAck targets this sorting nexin to a protein complex that includes Dock, an adaptor protein important for axonal guidance.     

Epidermal growth factor-dependent regulation of Cdc42 is mediated by the Src tyrosine kinase

Treatment of cells with epidermal growth factor (EGF) promotes the activation of the small GTP-binding protein Cdc42, as well as its phosphorylation in cells. The EGF-dependent phosphorylation of Cdc42 occurs at tyrosine 64 in the Switch II domain and appears to be mediated through the Src tyrosine kinase, because both the expression of a dominant-negative Src mutant (mouse Src(K297R)) and treatment of cells with the Src kinase inhibitor PP2 blocks the EGF-stimulated phosphorylation of Cdc42, whereas expression of an activated Src mutant (Src(Y529F)) promotes phosphorylation in the absence of EGF treatment. The EGF-stimulated phosphorylation of Cdc42 is not required for its activation, nor does it directly affect the interactions of activated Cdc42 with target/effector proteins including PAK, ACK, WASP, or IQGAP. However, the EGF-stimulated phosphorylation of Cdc42 is accompanied by an enhancement in the interaction of Cdc42 with the Rho-GDP dissociation inhibitor (RhoGDI). The EGF-stimulated activation of Cdc42 does require activated Src, as well as the Vav2 protein, a member of the Dbl family of guanine nucleotide exchange factors. Src catalyzes the tyrosine phosphorylation of Vav2, and overexpression of Vav2 together with activated Src (Src(Y529F)) can completely bypass the need for EGF to promote the activation of Cdc42. Thus, EGF signaling through Src appears to have dual regulatory effects on Cdc42: 1). it leads to the activation of Cdc42 as mediated by the Vav2 guanine nucleotide exchange factor, and 2). it results in the phosphorylation of Cdc42, which stimulates the binding of RhoGDI, perhaps to direct the movement of Cdc42 to a specific cellular site to trigger a signaling response, because Cdc42-RhoGDI interactions are essential for Cdc42-induced cellular transformation.     

Phosphorylation of WASP by the Cdc42-associated kinase ACK1: dual hydroxyamino acid specificity in a tyrosine kinase

ACK1 is a nonreceptor tyrosine kinase that associates specifically with Cdc42. Relatively few ACK1 substrates and interacting proteins have been identified. In this study, we demonstrated that ACK1 phosphorylates the Wiskott-Aldrich syndrome protein (WASP), a Cdc42 effector that plays an important role in the formation of new actin filaments. ACK1 and WASP interact in intact cells, and overexpression of ACK1 promotes WASP phosphorylation. Phosphorylation of WASP in vitro was enhanced by the addition of Cdc42 or phosphatidylinositol 4,5-biphosphate, presumably due to release of the autoinhibitory interactions in WASP. Surprisingly, when we mapped the sites of WASP phosphorylation, we found that ACK1 possesses significant serine kinase activity toward WASP (directed at Ser-242), as well as tyrosine kinase activity directed at Tyr-256. A serine peptide derived from the Ser-242 WASP phosphorylation site is also a substrate for ACK1. ACK1 expressed in bacteria retained its serine kinase activity, eliminating the possibility of contamination with a copurifying kinase. Serine phosphorylation of WASP enhanced the ability of WASP to stimulate actin polymerization in mammalian cell lysates. Thus, the tyrosine kinase ACK1 acts as a dual specificity kinase toward this substrate. In contrast to other dual specificity kinases that more closely resemble Ser/Thr kinases, ACK1 is a tyrosine kinase with an active site that can accommodate both types of hydroxyamino acids in substrates.     

The activation mechanism of ACK1 (activated Cdc42-associated tyrosine kinase 1)

ACK [activated Cdc42 (cell division cycle 42)-associated tyrosine kinase; also called TNK2 (tyrosine kinase, non-receptor, 2)] is activated in response to multiple cellular signals, including cell adhesion, growth factor receptors and heterotrimeric G-protein-coupled receptor signalling. However, the molecular mechanism underlying activation of ACK remains largely unclear. In the present study, we demonstrated that interaction of the SH3 (Src homology 3) domain with the EBD [EGFR (epidermal growth factor receptor)-binding domain] in ACK1 forms an auto-inhibition of the kinase activity. Release of this auto-inhibition is a key step for activation of ACK1. Mutation of the SH3 domain caused activation of ACK1, independent of cell adhesion, suggesting that cell adhesion-mediated activation of ACK1 is through releasing the auto-inhibition. A region at the N-terminus of ACK1 (Leu10-Leu14) is essential for cell adhesion-mediated activation. In the activation of ACK1 by EGFR signalling, Grb2 (growth-factor-receptor-bound protein 2) mediates the interaction of ACK1 with EGFR through binding to the EBD and activates ACK1 by releasing the auto-inhibition. Furthermore, we found that mutation of Ser445 to proline caused constitutive activation of ACK1. Taken together, our studies have revealed a novel molecular mechanism underlying activation of ACK1.     

Constitutive activated Cdc42-associated kinase (Ack) phosphorylation at arrested endocytic clathrin-coated pits of cells that lack dynamin

Clathrin-mediated endocytosis is a fundamental cellular process conserved from yeast to mammals and is an important endocytic route for the internalization of many specific cargos, including activated growth factor receptors. Here we examined changes in tyrosine phosphorylation, a representative output of growth factor receptor signaling, in cells in which endocytic clathrin-coated pits are frozen at a deeply invaginated state, that is, cells that lack dynamin (fibroblasts from dynamin 1, dynamin 2 double conditional knockout mice). The major change observed in these cells relative to wild-type cells was an increase in the phosphorylation state, and thus activation, of activated Cdc42-associated kinase (Ack), a nonreceptor tyrosine kinase. Ack is concentrated at clathrin-coated pits, and binds clathrin heavy chain via two clathrin boxes. RNA interference-based approaches and pharmacological manipulations further demonstrated that the phosphorylation of Ack requires both clathrin assembly into endocytic clathrin-coated pits and active Cdc42. These findings reveal a link between progression of clathrin-coated pits to endocytic vesicles and an activation-deactivation cycle of Ack.     

EGF receptor signaling stimulates SRC kinase phosphorylation of clathrin, influencing clathrin redistribution and EGF uptake

Epidermal growth factor (EGF) binding to its receptor causes rapid phosphorylation of the clathrin heavy chain at tyrosine 1477, which lies in a domain controlling clathrin assembly. EGF-mediated clathrin phosphorylation is followed by clathrin redistribution to the cell periphery and is the product of downstream activation of SRC kinase by EGF receptor (EGFR) signaling. In cells lacking SRC kinase, or cells treated with a specific SRC family kinase inhibitor, EGF stimulation of clathrin phosphorylation and redistribution does not occur, and EGF endocytosis is delayed. These observations demonstrate a role for SRC kinase in modification and recruitment of clathrin during ligand-induced EGFR endocytosis and thereby define a novel effector mechanism for regulation of endocytosis by receptor signaling.     

Crystal structures of the phosphorylated and unphosphorylated kinase domains of the Cdc42-associated tyrosine kinase ACK1

ACK1 is a multidomain non-receptor tyrosine kinase that is an effector of the Cdc42 GTPase. Members of the ACK family have a unique domain ordering and are the only tyrosine kinases known to interact with Cdc42. In contrast with many protein kinases, ACK1 has only a modest increase in activity upon phosphorylation. We have solved the crystal structures of the human ACK1 kinase domain in both the unphosphorylated and phosphorylated states. Comparison of these structures reveals that ACK1 adopts an activated conformation independent of phosphorylation. Furthermore, the unphosphorylated activation loop is structured, and its conformation resembles that seen in activated tyrosine kinases. In addition to the apo structure, complexes are also presented with a non-hydrolyzable nucleotide analog (adenosine 5'-(beta,gamma-methylenetriphosphate)) and with the natural product debromohymenialdisine, a general inhibitor of many protein kinases. Analysis of these structures reveals a typical kinase fold, a pre-organization into the activated conformation, and an unusual substrate-binding cleft.     

Cortactin Is a Substrate of Activated Cdc42-Associated Kinase 1 (ACK1) during Ligand-induced Epidermal Growth Factor Receptor Downregulation

Background

Epidermal growth factor receptor (EGFR) internalization following ligand binding controls EGFR downstream pathway signaling activity. Internalized EGFR is poly-ubiquitinated by Cbl to promote lysosome-mediated degradation and signal downregulation. ACK1 is a non-receptor tyrosine kinase that interacts with ubiquitinated EGFR to facilitate EGFR degradation. Dynamic reorganization of the cortical actin cytoskeleton controlled by the actin related protein (Arp)2/3 complex is important in regulating EGFR endocytosis and vesicle trafficking. How ACK1-mediated EGFR internalization cooperates with Arp2/3-based actin dynamics during EGFR downregulation is unclear.

Methodology/Principal Findings

Here we show that ACK1 directly binds and phosphorylates the Arp2/3 regulatory protein cortactin, potentially providing a direct link to Arp2/3-based actin dynamics during EGFR degradation. Co-immunoprecipitation analysis indicates that the cortactin SH3 domain is responsible for binding to ACK1. In vitro kinase assays demonstrate that ACK1 phosphorylates cortactin on key tyrosine residues that create docking sites for adaptor proteins responsible for enhancing Arp2/3 nucleation. Analysis with phosphorylation-specific antibodies determined that EGFR-induced cortactin tyrosine phosphorylation is diminished coincident with EGFR degradation, whereas ERK1/2 cortactin phosphorylation utilized in promoting activation of the Arp2/3 regulator N-WASp is sustained during EGFR downregulation. Cortactin and ACK1 localize to internalized vesicles containing EGF bound to EGFR visualized by confocal microscopy. RNA interference and rescue studies indicate that ACK1 and the cortactin SH3 domain are essential for ligand-mediated EGFR internalization.

Conclusions/Significance

Cortactin is a direct binding partner and novel substrate of ACK1. Tyrosine phosphorylation of cortactin by ACK1 creates an additional means to amplify Arp2/3 dynamics through N-WASp activation, potentially contributing to the overall necessary tensile and/or propulsive forces utilized during EGFR endocytic internalization and trafficking involved in receptor degradation.     

SNX12 role in endosome membrane transport

In this paper, we investigated the role of sorting nexin 12 (SNX12) in the endocytic pathway. SNX12 is a member of the PX domain-containing sorting nexin family and shares high homology with SNX3, which plays a central role in the formation of intralumenal vesicles within multivesicular endosomes. We found that SNX12 is expressed at very low levels compared to SNX3. SNX12 is primarily associated with early endosomes and this endosomal localization depends on the binding to 3-phosphoinositides. We find that overexpression of SNX12 prevents the detachment (or maturation) of multivesicular endosomes from early endosomes. This in turn inhibits the degradative pathway from early to late endosomes/lysosomes, much like SNX3 overexpression, without affecting endocytosis, recycling and retrograde transport. In addition, while previous studies showed that Hrs knockdown prevents EGF receptor sorting into multivesicular endosomes, we find that overexpression of SNX12 restores the sorting process in an Hrs knockdown background. Altogether, our data show that despite lower expression level, SNX12 shares redundant functions with SNX3 in the biogenesis of multivesicular endosomes.