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Originally published In Press as doi:10.1074/jbc.M701997200 on April 9, 2007

J. Biol. Chem., Vol. 282, Issue 22, 16086-16094, June 1, 2007
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Receptor-mediated Endocytosis Involves Tyrosine Phosphorylation of Cortactin*

Jianwei Zhu{ddagger}1, Dan Yu§1, Xian-Chun Zeng§, Kang Zhou||, and Xi Zhan§2

From the {ddagger}Affiliated Hospital of Nantong University, 226001 Nantong, China, the §Department of Pathology, Marlene and Stewart Greenebaum Cancer Center, and Center for Vascular and Inflammatory Disease, University of Maryland School of Medicine, Baltimore, Maryland 21201, and the ||Biochemistry, Microbiology, and Molecular Biology Doctoral Program, Pennsylvania State University, University Park, Pennsylvania 16802

Received for publication, March 7, 2007 , and in revised form, April 3, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Efficient internalization of cell surface receptors requires actin polymerization mediated by Arp2/3 complex and cortactin, a prominent substrate of the protein-tyrosine kinase Src. However, the significance of cortactin tyrosine phosphorylation in endocytosis is unknown. We found that overexpression of a cortactin mutant deficient in tyrosine phosphorylation decreased transferrin uptake. Suppression of cortactin expression by RNA interference also reduced transferrin internalization. Such inhibition was effectively rescued by overexpressing wild-type cortactin but not a cortactin mutant deficient in tyrosine phosphorylation or a mutant with deletion of the Src homology 3 domain. Likewise, purified phosphorylation-null cortactin failed to restore the formation of clathrin-coated vesicles in a cortactin-depleted cell extract. In vitro analysis revealed that Src-mediated phosphorylation enhanced the association of cortactin with dynamin-2 in a tyrosine phosphorylation-dependent manner. Quantitative analysis demonstrated that Src enhances the affinity of cortactin for dynamin-2 by more than 3-fold. On the other hand, Src-treated dynamin-2 had no effect on its interaction with cortactin. These data indicate that Src kinase is implicated in clathrin-mediated endocytosis by phosphorylation of cortactin.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Receptor-mediated endocytosis constitutes an important cellular mechanism to take nutrients into cells and transduce extracellular signals essential for cell homeostasis, proliferation, and differentiation. The best characterized internalization process so far is clathrin-mediated endocytosis, which involves concentration of membrane-associated receptors that are either unoccupied (for constitutive pathway) or occupied by their ligands (for the ligand dependent pathway) in a cage-like structure that is composed primarily of clathrin (1). Under physiological conditions, assembly of clathrin-coated pits requires other coat constituents, including monomeric assembly protein (AP-180) and heterotetrameric adaptor protein 2 (AP-2), the latter of which links the clathrin cage to the cytoplasmic domains of endocytic receptors. Invagination or scission of clathrin-coated pits into the cytosol of many types of cells also requires the function of dynamin-2, a ubiquitously expressed member of the large GTPase family that is associated with the narrow neck of clathrin-coated pits (2). Regulation of clathrin-mediated endocytosis involves activation of specific intracellular protein kinases that target certain components of the endocytic machinery. The molecules implicated in the assembly of clathrin-coated vesicles (CCV),3 including clathrin, AP-2, AP-180, dynamin and other proteins such as epsin, amphiphysin, and synaptotagmin, are known to serve as the substrates of either protein tyrosine or serine and threonine kinases (3). The mechanism of phosphorylation-regulated endocytosis appears to be complicated, as phosphorylation either facilitates or hampers clathrin-mediated endocytosis. For example, kinase CK2-mediated phosphorylation of AP-2 subunits {alpha}- and beta2-adaptin reduces the interaction of AP-2 with clathrin (4, 5), a process that is essential for the assembly of clathrin-coated vesicles. Similarly, phosphorylation of dynamin-1 and synaptojanin-1 has been reported to inhibit their binding to amphiphysin, whereas phosphorylation of amphiphysin inhibits its binding to AP-2 and clathrin (6). On the other hand, exposure of cells to protein-tyrosine kinase inhibitors profoundly attenuates ligand-induced internalization of receptors for EGF, B-cell antigen, and asialoglycoprotein and partially inhibits ligand-independent or constitutive endocytosis of the transferrin (Tfn) receptor (7–9). Although the intrinsic tyrosine kinase activity associated with receptors for polypeptide growth factors such as EGF and insulin is known to be important for their endocytosis upon occupancy by ligands (1), selective inhibition of non-receptor protein-tyrosine kinase Src also attenuates the internalization of c-Kit in hematopoietic cells (10), neural cell adhesion molecule 1 in neuroblastoma cells (11), and EGF receptor (12) and constitutive internalization of Tfn induced by oxidative stress (13) and a splicing form of cholecystokinin 2 receptor (14). Despite the compelling evidence for the role of tyrosine phosphorylation in endocytosis, the mechanism by which Src regulates endocytosis is largely unknown.

Cortactin, which contains a C-terminal SH3 domain and six and one-half 37-amino acid repeats, is an Arp2/3 complex and filamentous actin (F-actin)-binding protein (15–18) that is present in the cell periphery as well as in numerous cytoplasmic punctate structures (19). Immunostaining with endosome markers or internalized ligands such as Tfn has established that many of these puncta represent endocytic vesicles (20, 21). Suppression of cortactin expression by either microinjection of cortactin antibody (21) or RNA-mediated interference (22) considerably impairs Tfn uptake in cells. Our recent in vitro analysis of Tfn sequestration using perforated cells demonstrates that depletion of cortactin in cytosol extracts weakens the formation of clathrin-coated vesicles (22), indicating that cortactin is important for clathrin-mediated endocytosis. Conversely, depletion of cortactin in cells or overexpression of a cortactin SH3 fragment also reduces clathrin-independent endocytosis of {gamma}c cytokine receptor (23), indicating that cortactin may play a broad role in protein internalization. The mechanism for cortactin-mediated endocytosis likely involves actin cytoskeleton reorganization as cortactin is known to promote the actin polymerization mediated by Arp2/3 complex (17, 18), which yields a cortical actin meshwork enriched at the leading edge of the cell as well as that associated with endocytic vesicles. In the de novo actin assembly, the major force pushing membrane protrusion, cortactin binds tightly to Arp2/3 complex at a nascent actin branching site, thereby generating a possible inward dynamic movement for an organelle that attaches to cortactin through its SH3 domain during the process of actin assembly (22). Indeed, a major cortactin SH3-binding protein has been found to be dynamin-2 (24). Importantly, binding of cortactin to dynamin-2 is dependent upon actin polymerization itself, because it associates with dynamin-2 maximally only during the process of actin assembly (22). Therefore, coordination and dynamic interaction between cortactin and dynamin-2 may provide a mechanical force responsible for an actin assembly-driven movement of endocytic vesicles to the deep cytosol, which eventually leads to detachment of vesicles from a protrusive membrane. Consistent with this notion, a recent analysis using alternating evanescent field and epifluorescence illumination has shown that maximal invagination and scission of clathrin-coated pits coincides with recruitment of cortactin to the endocytic site (25).

Cortactin is also known to be a prominent substrate of Src protein-tyrosine kinase (19, 26), and tyrosine phosphorylation of cortactin occurs frequently in response to various extracellular stimuli including growth factors (16, 27), bacterially mediated phagocytosis (28), cell shrinkage (29), and membrane injury (30). Although previous studies have found that introduction of a cortactin mutant deficient in tyrosine phosphorylation impairs cell motility, tumor invasion, invadopodia, and podosome formation (30–33), no solid evidence for a role of phosphorylated cortactin in actin polymerization either in cells (34) or in vitro4 has been found. Therefore, the physiological role of cortactin tyrosine phosphorylation remains unknown. In this study we investigated a possible role of Src-mediated tyrosine phosphorylation of cortactin in receptor-mediated endocytosis. We found that deficiency in tyrosine phosphorylation of cortactin attenuates Tfn uptake. Furthermore, we found that Src promotes the interaction between cortactin and dynamin-2. These findings indicate a role of tyrosine phosphorylation of cortactin in the regulation of receptor-mediated endocytosis.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies and Reagents—Monoclonal cortactin antibody (4F11) was purchased from Upstate%20Biotechnology">Upstate Biotechnology Inc.; polyclonal cortactin antibody was prepared as described previously (16); human Tfn antibody was from ICN Biomedicals, Inc; antibiotin antibody and NHS-SS-biotin were from Pierce; GFP antibody was either purchased from Molecular Probes or raised against recombinant His-GFP protein; anti-Myc antibody (9E10) was from BD Biosciences; polyclonal dynamin antibody was either obtained as a gift of Dr. McNiven (Mayo Clinic) or raised against the peptide CARDVLENKLLPLRRGYIGVVNRSQKD; and polyclonal antibody specific for phosphorylated cortactin was raised against a mixture of two phosphotyrosine (pY)-containing peptides: PSSPVpYEDAASFKC and pYESAEAPGHYPAEDSTpYDEYEC.

DNA Constructs and Recombinant Proteins—Plasmid encoding GFP-tagged full-length dynamin-2 (Dyn-GFP) was a gift of Dr. McNiven. Plasmids encoding Myc-cortactin and Myc-Cort-F have been previously described (35). His-tagged and tag-free cortactin proteins were prepared as described previously (17, 36). To prepare GST-tagged dynamin-2 (GST-dynamin-2) and the proline-rich domain of dynamin-2 (GST-dynamin-2-PRD), DNA fragments were generated by PCR using GFP-dynamin-2 as the template and subcloned into pGEX-4T-2 plasmid. The resulting plasmids were transformed into bacterial BL21-CodonPlus (DE3)-RIPL (Stratagene) cells for protein expression. The primers used in PCR were as follows: 5'-GTTCTTGAATTCCCATGGGCAACCGCGGGATGGA-3' and 5'-ATTATTGCGGCCGCCTAGTCGAGCAGGGACGGCTC-3' for full-length dynamin-2; and 5'-TTACCGGTCGCCACCATGGTGAGC and 5'-TATGCGGCCGCCTAGTCGAGCAGGGACGGCTCGG-3' for PRD of dynamin-2.

Cell Culture, DNA Transfection, and Immunoprecipitation—HeLa, MDA-MB-231, and HEK-293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. 3T3-L1 cells were grown in Dulbecco's modified Eagle's medium containing 10% calf serum.

To analyze the effect of pervanadate (PV) on the interaction between cortactin and dynamin, HEK-293 cells were transiently co-transfected with Dyn-GFP and Myc-cortactin, or Myc-Cort-F. After 24 h, cells were incubated in a serum-free medium for an additional 24 h. Cells were then treated with PV (100 µm Na3VO4 and 4 mM H2O2) for 30 min and lysed in a lysis buffer (20 mM Tris, pH 7.4, 10 mM NaCl, 5 mM MgCl2, 0.2 mM EDTA, 1 mM Na3VO4, 1 mM sodium molybdate, 0.5% Triton X-100, and Roche protease inhibitor mixture tablets). The lysates were then subjected to immunoprecipitation using the appropriate antibodies as indicated in Figs. 4 and 5. The methods for immunoprecipitation and immunoblot have been described previously (37).

Analysis of the Effect of Tyrosine Phosphorylation on the Affinity of Cortactin for Dynamin-2—Cortactin was phosphorylated by Src kinase (Upstate) in a reaction buffer containing 100 mM Tris, pH 7.4, 125 mM MgCl2, 25 mM MnCl2, 2 mM EGTA, 1 mM ATP, and 0.2 mM Na3VO4 at 30 °C for 30 min, the modified condition recommended by the manufacture. To analyze the affinity of cortactin for dynamin, 18 pmol of cortactin, either phosphorylated or nonphosphorylated, was incubated with different amounts of GST-dynamin-2 immobilized on glutathione-Sepharose in buffer (20 mM Tris, pH 7.4, 10 mM NaCl, 5 mM MgCl2, 1 mM Na3VO4, 0.5% Triton X-100, and 1 mM dithiothreitol). After incubation of the mixture for 2 h, samples were centrifuged at 800 x g for 10 s, and aliquots of the supernatants were fractionated by 10% SDS-PAGE followed by immunoblot using cortactin antibody. Cortactin on the blot was detected by Kodak Image Station 2000R and quantified as the percentage of depletion. The resulting data were used to fit a rectangular hyperbola using Sigma Plots 8.0, yielding values of the equilibrium dissociation constants (Kd).

Capture Enzyme-linked Immunosorbent Assay—Cells expressing cortactin-GFP or Cort-F-GFP were incubated in serum-free Dulbecco's modified Eagle's medium containing 1% bovine serum albumin and 20 mM HEPES at 37 °C for 30 min and incubated with 10 µg/ml bio-Tfn for 30 min on ice. Endocytosis was initiated by incubating the mixture at 37 °C for 5 min and terminated by placing on ice. To measure internalization of bio-Tfn, total cell lysates were added to a 96-well ELISA (enzyme-linked immunosorbent assay) plate coated with an anti-Tfn antibody. After 12 h of incubation at 4 °C, each well was added with streptavidin-horseradish peroxidase and Roche BM blue substrate, respectively. Absorption at 450 nm was determined by a microplate reader (Anthos Labtec) and was plotted as a function of time.

Cell-free Assay for CCV Formation—The cell-free internalization assay with perforated 3T3-L1 cells was performed according to the method as described (38). 3T3-L1 cells were subjected to permeabilization by submersion in liquid nitrogen and rapid thawing at 37 °C. The cells were then scraped from culture dishes, washed in KSHM buffer (100 mM potassium acetate, 85 mM sucrose, 1 mM magnesium acetate, and 20 mM HEPES-NaOH, pH 7.4), and centrifuged at 800 x g for 3 min. The permeabilized cells were then incubated with 8 µg/ml B-SS-Tfn in KSHM buffer containing 0.8% bovine serum albumin for 20 min at 4 °C. The cell suspension (10 µl) was mixed with 300 µg of brain cytosol and an ATP-generating system in a final volume of 40 µl. Biotin-SS-Tfn internalization was initiated by incubating the cells at 37 °C for 20 min and stopped by returning them to ice. After incubation the reaction was washed with 500 µl of ice-cold KSHM buffer and spun at 10,000 x g. Cell pellet was then subjected to MesNa resistance assay as described previously (22).

RNA Interference Assay—Human cortactin small interfering RNAs (siRNAs) were prepared by an in vitro transcription procedure using T7 RNA polymerase according to the manufacture's protocol of Silencer siRNA construction kit (Ambion). The target sequence is 5'-AAGUUUGGUGUCCAGAUGGAC-3'. The control siRNA against human HS1 targets 5'-GACAUCCUCAAGAAGAAGGAG-3'. Treatment of cells with siRNAs was described previously (22).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
We previously identified residues Tyr-421, Tyr-466, and Tyr-482 of cortactin as the primary sites targeted by Src (35). To study the role of cortactin tyrosine phosphorylation in receptor-mediated endocytosis, HeLa cells were infected with a retroviral vector, MGIN (39), encoding a murine cortactin construct tagged by GFP at the C terminus (cortactin-GFP), a mutant of Y421F/Y466F/Y482F (Cort-F-GFP), and a mutation with deletion of the SH3 domain (Cort{Delta}SH3-GFP) (Fig. 1A). The expression level of cortactin GFP fusions in infected cell populations was comparable with that of endogenous cortactin as determined by immunoblot (Fig. 1B). The infected cells were exposed to biotin-Tfn on ice, and internalization of biotin-Tfn was analyzed at 5 min after shifting to 37 °C. As shown in Fig. 1C, the cells expressing cortactin-GFP internalized Tfn at an efficiency similar to the cells infected with the viral GFP vector, indicating no significant effect of overexpression of wild-type cortactin on endocytosis. However, Tfn uptake in the cells expressing Cort-F-GFP was nearly 40% less than in cells expressing the vector alone (p < 0.05) (Fig. 1C). Similarly, cells overexpressing Cort{Delta}SH3-GFP showed an impaired Tfn uptake at an efficiency 43% less than the control cells. To examine the role of Src activity in Tfn uptake, cells were also treated with PP2, a selective inhibitor of Src (40), prior to Tfn uptake analysis. PP2 treatment inhibited Tfn uptake by ~60% (p < 0.005), an extent similar to a previous report using genistein, a pan-tyrosine kinase inhibitor (7). In contrast, PP2 treatment of cells expressing Cort-F-GFP resulted in only 30% inhibition of Tfn uptake (p = 0.3).

The role of cortactin phosphorylation was further analyzed with a cell-free system in which Tfn linked with biotin through a cleavable disulfide bond (B-SS-Tfn) was applied to perforated 3T3-L1 cells (22). Under these conditions, sequestration of Tfn into deeply invaginated coated pits and internalization of coated vesicles are able to be reconstituted by incubation with exogenous cytosol (38, 41–43). B-SS-Tfn internalized into coated pits becomes resistant to MesNa-mediated cleavage of B-SS-Tfn, a property that can be used indirectly to measure the CCV formation. To analyze the activity of cortactin and its variants to rescue CCV formation in the absence of cortactin, rat brain extract devoid of cortactin was prepared by absorption using cortactin antibody-conjugated beads followed by precipitation, a process that effectively removed most of the cortactin but not the dynamin-2 or actin in the extract (Fig. 1D). The cortactin-depleted brain extract was then used to reconstitute Tfn endocytosis in perforated 3T3-L1 cells. As shown in Fig. 1E, cortactin-depleted extract (del-Cort) showed impaired CCV formation (60% less than the mock-treated extract), a result that is similar to what we had reported previously (22). The addition of recombinant wild-type cortactin considerably restored the CCV formation by nearly 80%. In contrast, adding recombinant Cort-F rescued less than 50% of the CCV formation, which is statistically lower than that of wild-type cortactin (p < 0.01), indicating that tyrosine phosphorylation of cortactin is required for a maximal assembly of coated vesicles, an essential step in the early stage of endocytosis.


Figure 1
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  		FIGURE 1.
Analysis of the activity of wild-type and mutant cortactin proteins for internalization of Tfn and formation of clathrin-coated vesicles. A, schematic presentation of the cortactin variants used in this study. B, expression of cortactin variants in HeLa cells was analyzed by immunoblotting whole cell lysates with cortactin and actin antibodies. The positions of cortactin-GFP, endogenous cortactin, and actin are indicated by arrows. a.u., artificial unit. C, cells expressing cortactin variants were grown in serum-free medium at 37 °C for 60 min and incubated with biotin-Tfn for 30 min on ice. Endocytosis was initiated at 37 °C for 5 min and terminated on ice. The treated cells were subjected to enzyme-linked immunosorbent assay-based biotin-Tfn uptake as described under "Experimental Procedures." The p value calculated by Student's t test refers to the difference between PP2- and non-PP2-treated cells. D, cortactin was depleted from rat brain extracts with beads conjugated with cortactin antibody. Specific depletion was verified by immunoblotting with either cortactin (left) or dynamin and actin antibodies (right). E, 3T3-L1 cells were permeabilized by freezing and thawing. The permeabilized cells were incubated with biotin-SS-Tfn at 4 °C for 20 min and then mixed with mock-depleted brain extracts (Mock), cortactin-depleted extracts (del-Cort), or cortactin-depleted extracts supplemented with either recombinant wild-type cortactin protein (del-Cort+cortactin) or recombinant Cort-F (del-Cort+Cort-F), respectively. The cell pellets were treated with MesNa followed by measuring of the remaining B-SS-Tfn in the lysates, which represents those trapped within coated vesicles. Data shown are the mean ± S.D. of three experiments. The p value was calculated by paired Student's t test, referring to the difference between del-Cort+cortactin and del-Cort+Cort-F.

 
To further establish the role of cortactin tyrosine phosphorylation in endocytosis within cells, expression of endogenous cortactin of human breast tumor cells (MDA-MB-231) infected by retroviruses carrying different types of murine cortactin mutants was silenced by siRNA targeting of human cortactin mRNA. As a control, virus-infected MDA-MB-231 cells were also treated with siRNA against HS1, a cortactin-related gene that is exclusively expressed in hematopoietic cells. As shown in Fig. 2A, the human cortactin siRNA inhibited cortactin expression by ~60–80% as compared with that of the cells treated with the HS1 siRNA. When biotin-Tfn uptake was analyzed, a nearly 70% reduction in endocytosis was found in the cells treated with cortactin siRNA (Fig. 2B, left), which is consistent with our previous report (22) and a report from others (23). To determine whether the decrease in Tfn endocytosis was due to the specific loss of cortactin in the siRNA-treated cells, we treated the cells expressing murine cortactin-GFP with HS1 and cortactin siRNAs, respectively. In addition to the endogenous cortactin, the human cortactin siRNA also showed a slight inhibition of the exogenous murine cortactin (Fig. 2B, right), presumably because of the 95% identity between human and mouse mRNAs in the region targeted by the siRNA. Nevertheless, the overall level of exogenous cortactin proteins was greater than or similar to that of the endogenous cortactin of the control cells. At this level cortactin siRNA failed to inhibit Tfn uptake in the cortactin-GFP-expressing cells, indicating that cortactin-GFP protein maintains its endocytic function similar to the endogenous cortactin. However, the same cortactin siRNA retained inhibitory activity for Tfn uptake in the cells expressing either mutant Cort-F-GFP or Cort{Delta}SH3-GFP (Fig. 2B).

The above data confirm that tyrosine phosphorylation of cortactin and the function of the SH3 domain are implicated in endocytosis. Because the SH3 domain is also implicated in binding to dynamin, we were prompted to examine a possible role of tyrosine phosphorylation in the regulation of the interaction of cortactin with dynamin-2. Recombinant cortactin was phosphorylated by Src in vitro for 30 min, the condition under which a maximal phosphorylation was achieved (Fig. 3A, data not shown). Under the same condition no apparent phosphorylation of Cort-F was observed (Fig. 3A), consistent with our previous finding that residues Tyr-421, Tyr-466, and Tyr-482 are the primary targets of Src (35). The interaction of phosphorylated cortactin with dynamin was analyzed by pulling down Src-treated cortactin proteins with GST-dynamin-2. As shown in Fig. 3B, GST-dynamin-2 pulled down more Src-treated cortactin than the nonphosphorylated form. On the other hand, the same peptide precipitated little Cort-F regardless of Src treatment. The pulldown analysis was also used to quantify the binding affinity for GST-dynamin-2 by measuring depletion of cortactin proteins in the supernatants after GST-dynamin-2 pulldown (Fig. 3C). This analysis demonstrates that the affinity of phosphorylated cortactin for GST-dynamin-2 is ~3.5-fold higher than nonphosphorylated form (Kd = 765 nM versus Kd = 211 nM). On the other hand, Cort-F has a much lower affinity for GST-dynamin-2 (Kd = 2.3 µM), and this affinity was not significantly changed upon Src treatment (Kd = 2.3 µm versus Kd = 2.2 µm) (Fig. 3D).


Figure 2
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  		FIGURE 2.
Cortactin mutant deficient in tyrosine phosphorylation fails to rescue uptake of Tfn in cortactin knockdown cells. A, MDA-MB-231 cells were infected with retroviruses carrying GFP or GFP-tagged murine cortactin variants (cortactin-GFP, Cort-F-GFP, and Cort{Delta}SH3-GFP). The infected cells were treated with either human cortactin (C) or HS1 (H) siRNA for 2 days. The total cell lysates were prepared and analyzed for cortactin expression by immunoblotting using monoclonal cortactin antibody. The samples were also immunoblotted by actin antibody. B, siRNA-treated cells were subjected to Tfn uptake analysis after 2 days of treatment as described under "Experimental Procedures." The data shown are the mean ± S.D. of three independent experiments. The p values of Student's t test refer to the difference between cortactin- and HS1 siRNA-treated cells (*) and the difference between cortactin-siRNA-treated cells expressing cortactin-GFP and those expressing Cort-F-GFP or Cort{Delta}SH3-GFP (**).

 
The SH3 domain of cortactin is known to bind to the PRD of dynamin (24). To examine whether the binding to the PRD was also subjected to Src-mediated regulation, GST-tagged dynamin-2 PRD peptide (GST-dynamin-2-PRD) was used to pull down cortactin. Like GST-dynamin-2, GST-dynamin-2-PRD precipitated more Src-treated wild-type cortactin proteins than Cort-F proteins (Fig. 3E). Therefore, we conclude that tyrosine phosphorylation promotes the interaction between the SH3 domain of cortactin and PRD of dynamin-2.

We also evaluated the effect of tyrosine phosphorylation on the interaction of cortactin and dynamin-2 in cells. This was done using HEK-293 cells co-expressing GFP-tagged dynamin-2 at the N terminus (GFP-dynamin-2) and Myc-tagged cortactin (Myc-cortactin) or Myc-Cort-F (Fig. 4A). Both Myc-cortactin and Myc-Cort-F were expressed as a duplet around 80–90 kDa, the characteristic behavior of cortactin in SDS-PAGE. Tyrosine phosphorylation of cortactin in the cells was induced by exposing the cells to PV (Fig. 4B). Consistent with Src treatment in vitro, PV was unable to induce phosphorylation of Myc-Cort-F (Fig. 4B). The interaction between exogenous cortactin and dynamin-2 was analyzed by immunoprecipitation. Although GFP antibody precipitated GFP-dynamin-2 equally in cells expressing either Myc-cortactin or Myc-Cort-F (Fig. 4C), considerable coprecipitation of Myc-cortactin and GFP-dynamin-2 was only observed in the cells upon treatment with PV. In contrast, Myc-Cort-F was barely detected in the pellet of cells treated with and without PV.

It is known that dynamin-2 is also a substrate of Src, and phosphorylation of dynamin-2 has been implicated in ligand-dependent receptor-mediated endocytosis (44). Therefore, the apparent increased interaction between cortactin and dynamin-2 upon PV treatment could be because of phosphorylation of dynamin-2. However, PV treatment triggered little phosphorylation of GFP-dynamin-2 under conditions in which cortactin-GFP was significantly phosphorylated (Fig. 5A). We next examined the interaction between Src-treated GST-dynamin-2 and cortactin in vitro. Although Src was able to induce marked phosphorylation of GST-dynamin-2 in vitro (Fig. 5B), phosphorylated GST-dynamin-2 did not apparently show an increase in binding to His-cortactin (Fig. 5C). Therefore, Src exerts its effect mainly through phosphorylation of cortactin rather than dynamin-2.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have provided several lines of evidence showing that Src-mediated phosphorylation of cortactin is implicated in optimal clathrin dependent endocytosis. First, overexpression of a cortactin mutant, Cort-F, which lacks three tyrosine residues targeted by Src, reduced clathrin-mediated Tfn uptake by ~40%. Second, recombinant Cort-F protein was unable to rescue effectively impaired CCV formation in a cortactin-depleted brain extract. The inability to rescue the CCV formation was unlikely due to an inappropriate structural folding associated with Cort-F, as the mutant shows a gel motility similar to the wild-type (Fig. 1A), binds to Arp2/3 complex, and promotes Arp2/3-mediated actin polymerization (data not shown). Moreover, Cort-F also failed to restore internalization of Tfn in the cell wherein cortactin expression was suppressed using siRNA. Third, treatment with a Src selective inhibitor decreased Tfn uptake by 60%. PP2 or its analogs have been used previously in examining the role of Src kinases in endocytosis of different types of receptors and revealed moderate, sometime even controversial, results (13, 46, 47). These differences may be due to variations in cell types, the presence of other types of kinases, and the conditions used in these studies. Because nonphosphorylated cortactin has a low but noticeable affinity for dynamin-2 (Fig. 3D), this low activity could provide an alternative explanation for the moderate effect of Src kinase inhibitors in these studies. Finally, interaction between cortactin and dynamin-2 was significantly enhanced upon PV treatment, a chemical mixture that provokes tyrosine phosphorylation of cortactin (Fig. 4). However, vanadate, a main component in PV, has an inhibitory activity to actomyosin-ATPase (48). Although no ATPase has been found to associate with either dynamin or cortactin, we have previously observed the complex of cortactin with actin and myosin in cells (49). Therefore, PV may also indirectly affect the interaction between dynamin and cortactin through the regulation of myosin. Further experiments are required to verify this possibility.


Figure 3
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  		FIGURE 3.
Src-mediated tyrosine phosphorylation promotes the association of cortactin with dynamin-2. A, recombinant cortactin and Cort-F were phosphorylated by incubation with Src kinase at 30 °C for 30 min. Phosphorylated cortactin proteins were verified by immunoblotting using polyclonal antibody specifically against tyrosine-phosphorylated (pY) cortactin (upper panels). The amount of cortactin proteins used in the reaction was verified by Coomassie Blue staining (lower panels). B, Src-treated cortactin proteins (18 pmol) were incubated with 5 µg of immobilized GST-dynamin-2 or GST proteins at 4 °C for 90 min and pulled down by glutathione-Sepharose beads. The pellets were immunoblotted with cortactin antibody (upper panel). Equal amounts of GST-dynamin-2 and GST proteins used in the pulldown were confirmed by Coomassie staining of the same samples resolved in a separate gel (lower panel). The positions of each type of proteins were indicated. C, Src-treated cortactin proteins were incubated with GST-dynamin-2 at concentrations ranging from 0 to 3.0 µM as indicated. After 2 h of incubation at 4 °C, the samples were precipitated by pulldown. Cortactin proteins remaining in the supernatants were determined by immunoblotting. D, the density of cortactin bands was measured and normalized to the percentage of depletion. The resulting data were used to fit a rectangular hyperbola using Sigma Plots 8.0, yielding apparent Kd values. E, interaction between Src-treated cortactin and GST-dynamin-2-PRD was analyzed by pulldown assay as described in B.

 
Tfn receptor uptake is known to be a constitutive process and independent of ligand occupancy (50). Therefore, our data indicate that tyrosine phosphorylation of cortactin plays a general role in constitutive internalization. Unlike many growth factor receptors such as that for EGF, which contains an intrinsic tyrosine kinase activity and triggers tyrosine phosphorylation upon ligand binding, Tfn receptor does not contain any kinase activity. However, an earlier study had shown that exposing cells to genistein, a pan-tyrosine kinase inhibitor, reduced Tfn internalization by nearly 50% (7), although the same treatment completely abolished EGF receptor endocytosis. This suggests that the pathway for Tfn receptor uptake is less dependent upon tyrosine kinase activity than that for EGF receptor. Although ligand-dependent endocytosis may utilize a different subpopulation of endocytosis machinery (7), evidence for such distinct endocytic apparatus remains to be found. On the other hand, it has been well described that EGF receptor and Tfn receptor share a common endocytic pathway involving CCV assembly. It is interesting to note that µ2 kinase modulates the affinity of AP-2 for the sorting motif on Tfn receptor (51) and Src regulates constitutive internalization of cholecystokinin 2 receptor (14). Therefore, our data support the view that the assembly of the components for the constitutive endocytosis is a regulated process involving both serine/threonine and tyrosine kinases. Although the physiological role of tyrosine kinases in constitutive endocytosis is unclear, it is apparent that EGF receptor-mediated endocytosis, which occurs only under certain physiological conditions suitable for epithelial cell proliferation, may require a specific cellular process allowing the endocytic machinery to operate at a maximal level and to ensure a rapid attenuation of EGF signaling (52). In contrast, rapid down-regulation of Tfn receptor, which brings an essential blood nutrient to most types of mammalian cells, may likely cause harm to cell homeostasis. In fact, internalization of Tfn receptor has evolved to a pathway that is not subject to the limitation imposed by ligand saturation. This property may make the dependence of Tfn receptor internalization on tyrosine phosphorylation less apparent under regular culture conditions. The physiological significance of tyrosine phosphorylation-regulated constitutive endocytosis may be more evident under other conditions such as allowing synchronization of Tfn receptor recycling.


Figure 4
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  		FIGURE 4.
Analysis of interaction between tyrosine-phosphorylated cortactin and dynamin-2 in cells. A, HEK-293 cells were co-transfected with either GFP-dynamin-2 and Myc-cortactin or GFP-dynamin-2 and Myc-Cort-F. Total lysates of transfected cells were immunoblotted with monoclonal Myc antibody (9E10). B, transfected cells were also serum-starved for 24 h and subsequently treated with PV for 30 min. Tyrosine-phosphorylated cortactin (pY-Myc-cortactin) was verified by immunoprecipitation (IP) and immunoblotting (IB). C, the lysates of PV-treated cells expressing Myc-cortactin and GFP-dynamin-2 were immunoprecipitated with GFP polyclonal antibody. The pellets were then immunoblotted simultaneously with Myc and GFP monoclonal antibodies.

 


Figure 5
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  		FIGURE 5.
Tyrosine phosphorylation of dynamin-2 does not contribute to its interaction with cortactin. A, HeLa cells were transiently transfected with either GFP-dynamin-2 or cortactin-GFP. After 24 h of serum starvation, cells were exposed to PV for 10 min. The cell lysates were immunoprecipitated (IP) with GFP antibody and subsequently immunoblotted (IB) with phosphotyrosine (pY) antibody. The same membrane used in the immunoblot was stripped and reblotted with GFP antibody. A weak band in lane 2 (upper panel) with a gel motility similar to cortactin-GFP was an unknown phosphotyrosyl protein by reason of nonspecific activity of the GFP antibody. B, recombinant GST-dynamin-2 was treated with Src kinase at 30 °C for 30 min. Protein phosphorylation was examined by phosphotyrosine immunoblot (upper panel). The amount of proteins used in the kinase reaction was verified by Coomassie staining on a parallel gel (lower panel). C, Src-treated GST-dynamin-2 (12 pmol) was incubated with 5 µg of His-cortactin and pulled down (PD) with nickel-nitrilotriacetic acid beads. The pellets were analyzed by immunoblotting using polyclonal dynamin-2 antibody (top). The input of His-cortactin used in the pulldown was verified by Coomassie staining (bottom).

 
Our data showing that tyrosine phosphorylation of cortactin modulates the interaction between cortactin and dynamin-2 provide a plausible mechanism for the regulation of endocytosis through phosphorylation. Because of the dynamic nature of cortactin-mediated actin polymerization and possible on-and-off of cortactin phosphorylation in cells, our finding is consistent with the dynamics of endocytic vesicles, including invagination, splitting from large vesicles, and merging and lateral movements, in which the important role of actin polymerization has been indicated (53, 54). Dynamin-2 has been reported as a substrate of Src as well (55). Therefore, it is possible that phosphorylation of dynamin-2 by Src could also play a role in the regulation of the interaction of dynamin and cortactin. Yet, PV treatment, which triggers Src activity, does not provoke a significant increase in phosphorylation of ectopically expressed dynamin-2 (Fig. 5A). In vitro, Src is able to phosphorylate dynamin-2. However, phosphorylated dynamin-2 does not show any apparent change in its affinity for cortactin (Fig. 5B). Furthermore, a peptide encoding the dynamin PRD alone binds to cortactin at a lower affinity compared with phosphorylated cortactin. Thus, it is unlikely that phosphorylation of dynamin would be a major mechanism responsible for Src regulated Tfn endocytosis. This result is also consistent with previous reports that mutations at the phosphorylation sites of Tyr-597 and Tyr-599 in dynamin do not cause significant reduction of Tfn uptake (56), although the same phosphorylation site appears to be necessary for EGF internalization and may regulate its assembly and GTPase activity (44).

The mechanism by which tyrosine phosphorylation regulates the interaction between cortactin and dynamin-2 remains to be defined. We speculate that tyrosine phosphorylation triggers a change in the configuration of the cortactin SH3 domain. One possibility is that the SH3 domain may be masked by either an intra- or intermolecular interaction. It is also possible that the full activity of the SH3 domain requires a specific configuration necessary for its targets. Such configuration may be stabilized by tyrosine phosphorylation. Previous findings that Src down-regulates the cross-linking activity of cortactin in vitro and the interaction with neural Wiskott-Aldrich syndrome protein (N-WASP) support changes in the cortactin conformation upon phosphorylation (36, 57). A recent report that a cortactin mutant with mutations at tyrosine phosphorylation sites and the SH3 domain is able to restore invadopodia formation in cortactin knockdown cells further implies a functional relationship between tyrosine phosphorylation and the SH3 domain (33). Characterization of the details of the cortactin configurational change will be essential to resolve this issue.

It is interesting to note that the interaction between cortactin and dynamin-2 is also regulated by actin polymerization (22). Thus, actin polymerization and tyrosine phosphorylation may act in concert in the regulation of endocytosis by modulating the interaction between cortactin and dynamin. Under normal physiological conditions the coordination between actin polymerization and Src may increase the dynamics of receptor-mediated endocytosis. However, it remains to be determined whether hyperphosphorylation of cortactin, such as that occurring in the Src-transforming cells, could contribute to enhanced endocytosis. Alternatively, the intensive interaction between hyperphosphorylated cortactin and dynamin may trigger the formation of a type of membrane remodeling that favors oncogenic behavior of cells other than endocytosis. Indeed, dynamin-2 has been detected in podosomes of transformed fibroblasts and osteoclasts where phosphorylated cortactin and activated Src are often present (45). Thus, tyrosine phosphorylation-regulated cortactin/dynamin interaction may provide a plausible link between endocytosis and cell transformation induced by Src.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants CA113809 and CA091984 (to X. Z.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Both authors contributed equally to this work. Back

2 To whom correspondence should be addressed: Dept. of Pathology, Center for Vascular and Inflammatory Disease, University of Maryland School of Medicine, 800 West Baltimore St., Baltimore, MD 21201. Tel.: 410-706-8228; Fax: 410-706-8234; E-mail: xzhan{at}som.umaryland.edu.

3 The abbreviations used are: CCV, clathrin-coated vesicles; Arp2/3, actin-related protein complex; B-SS-Tfn, Tfn linked with biotin through a cleavable disulfide bond; Cort, cortactin; EGF, epidermal growth factor; GFP, green fluorescent protein; GST, glutathione S-transferase; HEK, human embryonic kidney; Mes, 4-morpholineethanesulfonic acid; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4,D]pyrimidine; PRD, proline-rich domain; PV, pervanadate; SH3, Src homology 3; siRNA, small interfering RNA; Tfn, transferrin. Back

4 X. Zhan, unpublished result. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Jeff Winkles and Ying Wang for critical review of the manuscript and Dr. Mark McNiven for dynamin-2 antibodies.



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 DISCUSSION
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