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Originally published In Press as doi:10.1074/jbc.M512718200 on March 23, 2006

J. Biol. Chem., Vol. 281, Issue 20, 14429-14439, May 19, 2006
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Differential Regulation of Interleukin 5-stimulated Signaling Pathways by Dynamin*

Magdalena M. Gorska{ddagger}, Osman Cen§, Qiaoling Liang{ddagger}, Susan J. Stafford§, and Rafeul Alam{ddagger}1

From the {ddagger}Division of Allergy and Immunology, Department of Medicine, National Jewish Medical and Research Center, Denver, Colorado 80206 and the §Division of Allergy and Immunology, Department of Medicine, University of Texas Medical Branch, Galveston, Texas 77555

Received for publication, November 28, 2005 , and in revised form, March 20, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Through the yeast two-hybrid screen we have identified dynamin-2 as a molecule that interacts with the {alpha} subunit of the interleukin (IL) 5 receptor. Dynamin-2 is a GTPase that is critical for endocytosis. We have shown that dynamin-2 interacts with the IL-5 receptor-associated tyrosine kinases, Lyn and JAK2, in eosinophils. Tyrosine phosphorylation of dynamin is markedly enhanced upon IL-5 stimulation. The inhibition of tyrosine kinases results in complete abolition of ligand-induced receptor endocytosis. Inhibition of dynamin by a dominant-negative mutant or by small interfering RNA results in enhancement of IL-5-stimulated ERK1/2 signaling and cell proliferation. In contrast, the absence of a functional dynamin does not affect STAT5 or AKT phosphorylation or cell survival. Thus, we have identified specific functions for dynamin in the IL-5 signaling pathway and demonstrated its role in receptor endocytosis and termination of the ERK1/2 signaling pathway.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The eosinophil is the most prominent infiltrating cell in airway inflammation in asthma and other Th2-type immune responses (1). Although two recent trials with an anti-interleukin (IL) 52 antibody raised questions about the role of eosinophils in asthma (2, 3), subsequent studies have shown that the antibody reduces eosinophil number in the lung by about 50% (4). Therefore, these trials cannot be used to define the role of eosinophils in asthma. Recent experimental data continue to strengthen the rationale for a role for eosinophils not only in inflammation but also in immunomodulation and airway remodeling (5-7). Mice deficient in eosinophils do not develop key features of airway remodeling: subepithelial fibrosis and smooth muscle hyperplasia (6). Anti-IL-5 treatment in humans substantially decreases the amount of transforming growth factor beta-expressing eosinophils and prevents the deposition of extracellular matrix proteins in airways (8). IL-13 is a key regulator of airway hyperreactivity, and eosinophils were demonstrated to be essential for the secretion of this cytokine from T cells (5). Last but not least, in some experimental models eosinophils were shown to be a dominant source of Th2-type cytokines in the lung (9, 10).

IL-5 is one of the primary regulators of eosinophil differentiation and function (1, 11). The delineation of IL-5 signaling is critical for understanding eosinophil biology. Previous studies have demonstrated that IL-5 activates Janus and Src family tyrosine kinases, which leads to the activation of the mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling pathways and STAT family of transcription factors (11-14). Although much is known about the signal initiation and propagation by IL-5, the mechanism of signal termination is not fully understood. Cessation of signaling is essential for cellular homeostasis. Long-lasting or high-amplitude signals can be a source of pathological processes, e.g. excessive proliferation, prolonged survival, or hyperactivation of "unwanted" cell. Endocytosis of the receptor is one possible mechanism of signal termination (15). Recently we performed a yeast two-hybrid screen of a hematopoietic cell cDNA library with the transmembrane-cytoplasmic portion of the IL-5 receptor {alpha} as bait (16). Among several different molecules, the screen has identified dynamin-2, a GTPase that is critical for endocytosis. Dynamin is believed to assemble around the necks of clathrin-coated pits and assist in pinching vesicles from the plasma membrane (17). We investigated the role of dynamin in IL-5 signaling and IL-5-stimulated cell function.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies—The following antibodies were purchased from Santa Cruz Biotechnology: rabbit polyclonal anti-IL-5R{alpha}, anti-IL-3/IL-5/granulocyte-macrophage colony-stimulating factor beta chain, anti-pan-dynamin, anti-Lyn, anti-JAK2, anti-ERK1, anti-beta-tubulin, anti-phospho-STAT5 (Tyr694), and mouse monoclonal anti-dynamin-1. Mouse monoclonal anti-phospho-ERK (Thr202/Tyr204) and rabbit polyclonal-anti-phospho-AKT (Thr308) were from Cell Signaling Technology. Mouse monoclonal anti-dynamin-2 antibody was from BD Transduction Laboratories. Mouse monoclonal anti-phosphotyrosine antibody (clone 4G10) was from Upstate%20Biotechnology">Upstate Biotechnology.

Interaction of Dynamin-2 with IL-5Ra in the Yeast Two-hybrid Assay— A human fetal cDNA library (Clontech) was screened using the LexA Matchmaker Yeast Two-hybrid System (Clontech). The bait was constructed by fusing the cytosolic and transmembrane portion of human IL-5R{alpha} in-frame with the LexA DNA binding domain in the pLexA plasmid. The bait construct and the fetal human cDNA library in the pB42AD (Clontech) were cotransformed into the EGY48 yeast strain carrying the reporter plasmid p8opLacZ. The screening was performed as described (16). Plasmids from positive colonies were isolated and sequenced. To confirm the interaction of IL-5R{alpha} with dynamin-2 in yeasts, vectors pLexA-IL-5R{alpha} and/or pB42AD containing cDNA for human dynamin-2 were transfected into the yeast EGY48. Transfected yeast were examined for the activation of the reporter galactosidase gene lacZ. Transfection with dynamin-2 alone served as a negative control. Transfection with pLexA-p53 (p53) and pB42AD-SV-T (SV-T) served as a positive control.

Eosinophil Purification and Stimulation—Blood for this study was obtained from mildly allergic and healthy donors (16) and the protocol for this study was approved by the Institutional Review Board of the University of Texas Medical Branch at Galveston and the National Jewish Medical & Research Center in Denver. Eosinophils were isolated from peripheral blood as described (16). In short, the polymorphonuclear cell fraction was isolated from the buffy coat by Percoll gradient (Amersham Biosciences) centrifugation. Eosinophils were negatively selected after incubation with anti-CD16 antibody-coated microbeads and passage through a magnetic column (Miltenyi Biotec). The stimulation of eosinophils was performed as described (16).


Figure 1
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  		FIGURE 1.
IL-5R{alpha} interacts with dynamin-2. A, IL-5R{alpha} interacts with dynamin-2 in the yeast. Vectors pLexA-IL-5R{alpha} (IL-5R{alpha}) and/or pB42AD-dynamin-2 were transfected into the yeast EGY48. The interaction of IL-5R{alpha} and dynamin-2 in the yeast caused the activation of the reporter galactosidase gene lacZ (positive blue colonies). Transfection with dynamin-2 plasmid alone or pLexA-IL-5R{alpha} (IL-5R{alpha}) alone produced white colonies. Transfection with pLexA-p53 (p53) and pB42AD-SV-T (SV-T) served as a positive control for the protein/protein interaction. The interaction between p53 and SV-T is well established. B, IL-5R{alpha} interacts with dynamin-2 in eosinophils. Left panels, eosinophils were purified from the peripheral blood of healthy volunteers, incubated with or without IL-5 and lysed. As a control, lysates of IL-5R{alpha}-negative, dynamin-2-positive cell lines HEK 293 (293) and NIH 3T3 (3T3) were used. Protein concentration was determined in all lysates. The volume of samples was adjusted to contain an equal amount of protein. Lysates were immunoprecipitated (IP) with a rabbit anti-IL-5R{alpha} antibody (Ab) or control IgG (cIgG). cIgG was isolated from non-immunized rabbit. Immunoprecipitates were Western blotted (WB) with anti-dynamin-2 antibody (upper panel). To assess protein loading, membranes were reprobed with anti-IL-5R{alpha} (middle and bottom panels) antibody. IgH, immunoglobulin heavy chain. Right panels, eosinophils were incubated with or without IL-5 and lysed. Lysates were immunoprecipitated with an anti-dynamin-2 monoclonal antibody or isotype-matched mouse IgG (cIgG). Immunoprecipitates were Western blotted with an anti-IL-5R{alpha} antibody (upper panel) and, next, reprobed with the anti-dyanmin-2 antibody (middle and bottom panels). C, eosinophils were incubated with or without IL-5 and lysed. Lysates were immunoprecipitated with a mouse monoclonal anti-beta chain antibody or control isotype-matched mouse IgG (cIgG). Immunoprecipitates were Western blotted with the anti-dynamin-2 (upper panel) antibody. Membranes were reprobed with antibody againstbeta chain (beta, second panel from the top, and IgH, bottom panel), and, next, with anti-IL-5R{alpha} (third panel from the top).

 
JYTF-1 Cell Line—JYTF-1, a variant of the TF-1 cell line was kindly provided by Dr. Jeffrey Jong-Young Yen, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan. JYTF-1 cells were cultured in RPMI supplemented with 10% FBS and granulocyte-macrophage colony-stimulating factor at 2 ng/ml. Before IL-5 stimulation cells were washed and starved in RPMI containing 0.5% FBS for 24 h. Stimulation with IL-5 was performed as described (18).

Cell Lysis, Immunoprecipitation, Gel Electrophoresis, and Western Blotting—The foregoing procedures were performed as described (16). Briefly, cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (1% Nonidet P-40, 50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM Na3VO4, 1 mM NaF) for 30 min, on ice. After lysis cell debris were pelleted (7 min, 14,000 x g). For immunoprecipitation, cell lysates were preclared with Protein A/G-agarose (Santa Cruz Biotechnology) and then incubated with antibody (2 µg of antibody per 2 million cells) for 1 h followed by a 2-h incubation with Protein A/G-agarose. The pelleted immune complexes were washed in RIPA buffer 5 times. The immune complex pellets were boiled in 2x Laemmli buffer. Samples were run on polyacrylamide gels and transferred to polyvinylidene difluoride membranes at 100 V for 1.5 h. Membranes were subsequently blocked in 5% bovine serum albumin, TBS-T, incubated with primary antibody at 0.1 µg/ml for 2 h, washed, incubated with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) at 1:10000 for 30 min, washed, treated with the ECL reagent (Amersham Biosciences), and exposed to ECL film. To strip and reprobe, the membranes were incubated in the stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.7) at 55 °C for 30 min, washed, blocked, and immunoblotted with a proper antibody as indicated in the text.

Cell Surface Biotinylation and Streptavidin Pulldown—Eosinophils were stimulated with IL-5 for the times indicated in the text. Then cells were washed with ice-cold phosphate-buffered saline and incubated with Sulfo-NHS-LC-Biotin (Pierce) at 1 mg/ml in phosphate-buffered saline for 2 h on ice. Next, cells were washed in ice-cold phosphate-buffered saline + 100 mM glycine and lysed in RIPA buffer. The protein concentration of the lysates was determined and the volume of the samples was adjusted to contain equal amounts of the protein. Biotinylated surface proteins were pulled down from lysates with streptavidinagarose (Sigma) and Western blotted with anti-IL-5R{alpha}.


Figure 2
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  		FIGURE 2.
IL-5R{alpha} is internalized upon stimulation with IL-5. A, surface expression of IL-5R{alpha} in the course of IL-5 stimulation. Eosinophils were stimulated with IL-5 for the indicated lengths of time and then the cell surface was biotinylated as described under "Experimental Procedures." Cells were lysed and protein concentration was determined. The volume of samples was adjusted to contain equal amounts of protein. Biotinylated surface proteins were pulled down with streptavidin-agarose and Western blotted with anti-IL-5R{alpha} (upper panel) or anti-CD45 (lower panel). B, densitometric analysis of surface IL-5R{alpha}. IL-5R{alpha} bands or CD45 bands (A) were analyzed by densitometry (Metamorph software, version 6.2r5, Universal Imaging). The IL-5R{alpha} (or CD45) surface expression level in unstimulated cells (basal level) was arbitrary set as 100%. The surface level of the studied protein in the courseof IL-5 stimulation was expressed as a percentage of the basal level. The graph shows mean result from three independent experiments. Error bars show S.D.

 
Expression Plasmids, siRNA, and Transfection—pCB expression vectors containing human wild type dynamin-1 cDNA or K44A mutant of human dynamin-1 were a generous gift of Dr. Sandra Schmid, The Scripps Research Institute, La Jolla, CA. pMCL expression vector containing the K97M mutant of MEK1 was from Dr. Natalie G. Ahn, University of Colorado, Boulder, CO. siRNA for human dynamin-2 (siGenome SMARTpool reagent) as well as non-targeting siRNA were purchased from Dharmacon. Non-targeting siRNA was directed against firefly luciferase and was designed to contain at least 4 mismatches with all known human, mouse, and rat genes. The transfection of JYTF-1 cells with plasmids or siRNA was performed using the Amaxa electroporator according to the manufacturer's protocol. The cell line Nucleofector Kit V was used.

Staining for Fluorescent Microscopy—Paraformaldehyde-fixed JYTF-1 cells were placed on slides. Next, cells were blocked for 1 h in 5% serum in phosphate-buffered saline, 0.05% saponin, and then incubated at 4 °C overnight with mouse monoclonal anti-dynamin-2 antibody or isotype control antibody at 2 µg/ml. Alexa 488-labeled secondary antibody was used at 1 µg/ml. Cells were analyzed by fluorescent microscopy.

Thymidine Incorporation—Forty-eight hours after siRNA transfection JYTF-1 were washed and starved overnight in RPMI containing 0.5% FBS. Next, cells were placed in 96-well plates at 5 x 104 cells per well in the medium containing 10% FBS with 50 µM PD98059 or with the vehicle (Me2SO). After 30 min of incubation in 37 °C, 5% CO2, IL-5 was added to a final concentration of 1 ng/ml. 3H-Labeled thymidine was added at 1 µCi per well 10 h later and cells were harvested at 96 h after transfection. The incorporated [3H]thymidine was measured by liquid scintillation.

Survival Assay—Forty-eight hours after siRNA transfection JYTF-1 were washed and starved overnight in RPMI containing 0.5% FBS. Next, cells were placed in the medium containing 10% FBS with or without 10 ng/ml IL-5. Cells were washed 24 h later and stained with annexin V/propidium iodide according to the manufacturer's protocol (BD Pharmingen). The staining of the cells was analyzed by flow cytometry.

Statistical Analyses—Each experiment was performed at least three times. Graphs in Figs. 2B, 3D, and 3G show mean ± S.D. p < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
IL-5R{alpha} Interacts with Dynamin-2—We searched for new molecules regulating IL-5 receptor biology. The transmembrane-cytoplasmic portion of the IL-5 receptor {alpha} was fused to the DNA binding domain of LexA and used as bait in the yeast two-hybrid screen of a human fetal liver cDNA library. Dynamin-2, a molecule involved in vesicular trafficking, represented one of several IL-5R{alpha}-interacting molecules identified in the screen. Fig. 1A shows the subsequent verification of dynamin-2/IL-5R{alpha} interaction in the yeast. Transfection of yeast EGY48 with plasmids encoding the IL-5R{alpha}-LexA fusion protein (LexA binds to a promoter of the reporter gene lacZ) together with plasmid encoding dynamin 2-RNA polymerase II activator fusion protein caused the activation of the reporter galactosidase gene lacZ (positive blue colonies). Transfection with dynamin-2-RNA polymerase II activator alone or with IL-5R{alpha}-LexA alone produced negative white colonies. The interaction of p53 with SV-T in the yeast two-hybrid system served as a positive control and gave blue colonies. Next we examined if this association occurred in eosinophils. Eosinophils were isolated from human peripheral blood according to a standard protocol (12). Cells were subsequently stimulated with IL-5 or incubated in the medium alone. The immunoprecipitation of cell lysates with an anti-IL-5R{alpha} antibody followed by Western blotting with an anti-dynamin-2 antibody demonstrated the interaction of dynamin-2 with {alpha} subunit of IL-5 receptor in eosinophils (Fig. 1B, left panels). The association with IL-5R{alpha} was not dependent on stimulation with IL-5. We performed two control immunoprecipitation experiments. First, control IgG isolated from non-immunized rabbit (the immunoprecipitating anti-IL-5R{alpha} antibody was generated in the rabbit) was unable to precipitate dynamin from eosinophil lysates. Second, anti-IL-5R{alpha} antibody did not precipitate dynamin from the IL-5R{alpha}-negative cell lines, HEK 293 (kidney epithelium) and NIH 3T3 (fibroblasts). It is important to note that the expression of dynamin-2 at the protein level in 293 and 3T3 cells is very well documented (19-24). We were also able to detect IL-5R{alpha}/dynamin-2 interaction by immunoprecipitation with anti-dynamin-2 antibody followed by Western blotting with anti-IL-5R{alpha} antibody (Fig. 1B, right panels).


Figure 3
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  		FIGURE 3.
Endocytic pathway is affected by IL-5-activated tyrosine kinases. A, dynamin-2 interacts with Lyn and JAK2. Eosinophil lysates were prepared as described in the legend to Fig. 2, immunoprecipitated (IP) with anti-Lyn (left panels) or anti-JAK2 (right panels) antibodies (Ab) or control IgG (cIgG) and Western blotted (WB) with anti-dynamin-2 antibody (upper panels). Membranes were reprobed with anti-Lyn (middle and bottom left panel) or anti-JAK2 (middle and bottom right panel) antibodies to showequal protein loading. IgH, immunoglobulin heavy chain. B, dynamin-2 interacts with Lyn and JAK2 only in the presence of IL-5R{alpha}. Eosinophils were stimulated with IL-5 and lysed. Lysate was divided into two samples. One sample was depleted of IL-5R{alpha} by two sequential immunoprecipitations with an anti-IL-5R{alpha} antibody. These two IL-5R{alpha} immunoprecipitates (1st IP pellet, 2nd IP pellet) and the second post-IP supernatant (2nd IP sup) were Western blotted with anti-IL-5R{alpha} antibody and, next, reprobed with anti-dynamin-2 antibody (left panels). IL-5R{alpha}-depleted and non-depleted eosinophil lysate samples were immunoprecipitated with anti-Lyn (middle panels) and anti-JAK2 (right panels) antibodies or control IgG (cIgG). Immunoprecipitates were Western blotted with anti-dynamin-2 antibody. Membranes were reprobed with anti-Lyn (middle panels) and anti-JAK2 (right panels) antibodies. C, dynamin-2 is phosphorylated upon IL-5 stimulation. Eosinophils were stimulated with IL-5 for the indicated length of time or left unstimulated (-). Cells were lysed, immunoprecipitated (IP) with anti-dynamin-2 antibody or isotype-matched control mouse IgG (cIgG), and Western blotted with anti-phosphotyrosine antibody (upper panel). The membrane was reprobed with anti-dynamin-2 to show equal protein loading (middle and bottom panels). p-dynamin-2, phosphorylated dynamin-2; IgH, immunoglobulin heavy chain. D, the densitometric analysis of p-dynamin-2. p-dynamin-2 bands (Fig. 3C) were analyzed by densitometry as described in the legend to Fig. 2B. The phosphorylation of dynamin-2 is expressed as a-fold change over baseline (dynamin-2 phosphorylation in unstimulated cells). The graph shows mean result from three independent experiments. Error bars show S.D. E, genistein inhibits tyrosine phosphorylation of dynamin-2. Eosinophils were preincubated with genistein or vehicle, Me2SO (-), stimulated with IL-5 for 10 min and lysed. Lysates were immunoprecipitated with anti-dynamin-2 or isotype-matched control mouse IgG (cIgG) and Western blotted with anti-phosphotyrosine antibody (upper panel). The membrane was reprobed with anti-dynamin-2 to demonstrate protein loading (middle and bottom panels). F, eosinophils were preincubated with genistein or Me2SO (-), stimulated with IL-5 for 10 min, and a cell surface biotinylation experiment was performed as described in the legend to Fig. 2. Densitometric analysis of IL-5R{alpha} and CD45 bands is shown in G. The IL-5R{alpha} (or CD45) surface expression level in unstimulated cells (basal level) was arbitrary set as 100%. The surface level of the studied protein upon addition of genistein and/or IL-5 was expressed as percentage of the basal level. The graph shows mean result from three independent experiments. Error bars show S.D.

 


Figure 4
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  		FIGURE 4.
IL-5 stimulation changes cellular distribution of dynamin. JYTF1 cells were serumstarved for 24 h. Next, cells were incubated with or without IL-5 and fixed in paraformaldehyde. Fixed cells were stained with anti-dynamin-2 as a primary antibody or isotype control IgG followed by Alexa 488-labeled secondary antibody. The experiment was performed three times. The localization of dynamin-2 in vesicles and clusters upon IL-5 stimulation was seen in 80 ± 10% cells.

 
IL-5 receptor is composed of {alpha} and beta subunits. The {alpha} subunit interacts with the beta subunit only upon stimulation with IL-5. In co-immunoprecipitation experiments we demonstrated lack of dynamin-2 interaction with the beta subunit in unstimulated eosinophils (Fig. 1C). IL-5 stimulation resulted in the association of beta subunit with IL-5R{alpha} and dynamin.

IL-5 Receptor {alpha} Is Internalized upon IL-5 Stimulation—The association of IL-5 receptor with the endocytosis-regulating molecule prompted us to study receptor internalization. We asked how much of IL-5R{alpha} remained on the cell surface in the course of IL-5 stimulation. To this goal we labeled cell surface proteins of resting or IL-5-stimulated eosinophils with biotin. Next, biotinylated proteins were precipitated from cell lysates with streptavidin-agarose and Western blotted with IL-5R{alpha} antibody. The expression of IL-5R{alpha} on the cell surface in the course of IL-5 stimulation is shown in Fig. 2, A, upper panel, and B. At 10 minof IL-5 stimulation the surface level of IL-5R{alpha} reached a nadir and was equal to 39% of basal expression. After this time point, the IL-5 receptor slowly reappeared on the cell surface. In control experiments we measured the surface expression of CD45. To our knowledge there are no reports suggesting changes of CD45 surface expression in eosinophils within 60 min of IL-5 stimulation. The surface level of CD45 did not change upon IL-5 stimulation and was comparable with that in unstimulated cells (Fig. 2, A, bottom panel, and B).

Dynamin-2 Coprecipitates with Lyn and JAK2 Kinases—Next we studied whether dynamin interacted with IL-5R-bound signaling molecules. The very first step in the IL-5 signaling cascade is the activation of the Src family tyrosine kinase Lyn and the Janus family tyrosine kinase JAK2 (12, 13). These kinases are essential for eosinophil function. In coprecipitation experiments we showed dynamin interaction with both Lyn and JAK2 in eosinophils (Fig. 3A). The association of dynamin-2 with tyrosine kinases was independent of stimulation with IL-5. Next we asked if the association of dynamin-2 with the tyrosine kinases occurs in the absence of IL-5R{alpha}.To this goal IL-5-stimulated eosinophil lysates were depleted of IL-5R{alpha} (and molecules associated with this receptor) by two sequential immunoprecipitations with the anti-IL-5R{alpha} antibody. The post-immunoprecipitation supernatant was free of IL-5R{alpha} but contained dynamin-2, Lyn, and JAK2 (Fig. 3B). Coprecipitation experiments demonstrated a lack of dynamin-2 interaction with tyrosine kinases when IL-5R{alpha} (and IL-5R{alpha} interacting molecules) was removed from the cell lysate. In contrast, the interaction was detected in control, non-depleted cell lysates. These results suggest that IL-5R{alpha} acts as a signaling scaffold, which promotes dynamin/kinase interaction. In the absence of the scaffold, dynamin-2 and the tyrosine kinases do not interact.

Dynamin-2 Is Tyrosine-phosphorylated upon IL-5 Stimulation—To investigate the functional connection between dynamin and kinases we first examined the tyrosine phosphorylation of dynamin in eosinophils (Fig. 3, C and D). To this goal eosinophils were stimulated with IL-5 or incubated in the medium alone. Subsequently dynamin-2 was immunoprecipitated from cell lysates and examined by Western blotting with anti-phosphotyrosine antibody. Dynamin showed minor tyrosine phosphorylation in resting eosinophils. Upon IL-5 stimulation dynamin underwent significant phosphorylation in a time-dependent manner.

The Tyrosine Kinase Inhibitor, Genistein Blocks IL-5 Receptor {alpha} Internalization—The experiments demonstrating IL-5-inducible phosphorylation of dynamin encouraged us to study the relevance of tyrosine phosphorylation for receptor endocytosis. We examined the phosphorylation of dynamin and surface expression of IL-5R{alpha} on IL-5-stimulated eosinophils pretreated with the tyrosine kinase inhibitor genistein. Genistein completely blocked dynamin phosphorylation and the IL-5-induced receptor internalization as shown on Fig. 3, E, F, upper panel, and G. In control experiments neither IL-5 alone nor IL-5 in combination with genistein affected the surface expression of CD45. Therefore, we have demonstrated that IL-5 stimulated the signaling molecules impact upon the IL-5R endocytic pathway.

IL-5 Stimulation Causes the Redistribution of Dynamin in the Cell— To further study the influence of IL-5 on dynamin biology we examined the cellular localization of the GTPase upon IL-5 stimulation. JYTF-1 is a variant of the progenitor cell line TF-1 (18), which shows the preservation of proximal (e.g. JAK2 activation) and distal (e.g. ERK) signaling events upon IL-5 stimulation. In unstimulated JYTF-1 cells dynamin localized to the submembranous region (Fig. 4). At 10 min of IL-5 stimulation (IL-5 receptor surface expression is minimal at this time point) a significant amount of dynamin was found in vesicles and clusters distant from the cell membrane. We hypothesize that these clusters represent dynamin bound to IL-5 receptor-positive endocytic vesicles. Therefore, IL-5 stimulation caused covalent modification of dynamin as well as changes in its cellular localization.


Figure 5
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  		FIGURE 5.
Dynamin affects IL-5-induced signaling. A, overexpression of dynamin-1 wild type and the GTPase-defective (K44A) mutant of dynamin-1 in JYTF-1 cells. JYTF-1 were transfected with the empty vector (E) or vectors containing dynamin-1 wild type (W) or K44A mutant (M) cDNA. The expression of dynamin-1 at 72 h after transfection was analyzed by Western blotting with anti-dynamin-1 antibody (Ab). Anti-dynamin-1 antibody does not recognize dynamin-2 (endogenous variant of dynamin). The membrane was subsequently reprobed with anti-tubulin to show equal protein loading. B, GTPase-defective mutant of dynamin modulates IL-5-activated phosphorylation. JYTF-1 cells transfected as described in A were cytokine-and serum-starved for 24 h ("Experimental Procedures"). At 72 h after transfection, cells were stimulated with IL-5 for the indicated times and lysed. Lysates were Western blotted with anti-phosphotyrosine and reprobed with anti-phospho-STAT5 (p-STAT5, Tyr694) and next, with anti-phospho-AKT (p-AKT, Thr308). Next, the membrane was reprobed with an anti-tubulin antibody to demonstrate equal protein loading. C, GTPase-defective mutant of dynamin causes the prolongation of IL-5-induced ERK activation. Lysates of transfected JYTF-1 cells were prepared as in B and Western blotted with anti-phospho-ERK (p-ERK, Thr202/Tyr204). The membrane was reprobed with anti-ERK antibody to demonstrate equal protein loading.

 
Inhibition of Dynamin GTPase Activity Potentiates IL-5-induced Signaling Events—Next we examined if dynamin affected IL-5 receptor signal transduction. The GTPase defective mutant of dynamin-1 (K44A) or wild type dynamin-1 (WT) were transiently expressed in the JYTF-1 cell line. GTPase activity of dynamin is essential for endocytosis (15). Dynamin-1 is a variant of dynamin, which is selectively expressed in neurons (25). The K44A mutant of dynamin-1 is widely used to block dynamin-2-dependent endocytosis in various cell types (15, 26-29). Fig. 5A demonstrates the overexpression of dynamin WT or K44A mutant at 72 h after transfection. Cells overexpressing dynamin variants as well as empty vector-transfected cells were subsequently examined for tyrosine phosphorylation, STAT, AKT, and ERK phosphorylation (Fig. 5, B and C). Overexpression of the K44A dynamin mutant resulted in the prolongation of ERK1/2 phosphorylation (Thr202/Tyr204). In contrast to empty vector-transfected cells, K44A-expressing cells showed significant ERK1/2 activation at the 30- and 60-min time points of IL-5 stimulation. Overexpression of wild type dynamin shortened the length of ERK1/2 activation. Interestingly, phosphorylation of AKT (Thr308) and STAT5 (Tyr694) was not affected by dynamin. Therefore, dynamin affects only select components of the IL-5 signaling pathway.

Inhibition of Dynamin Expression Potentiates IL-5-induced ERK1/2 Activation—We wondered if depletion of the entire dynamin molecule will mimic the K44A dynamin effect on ERK activation. To this goal JYTF-1 cells were separately transfected with dynamin-2 siRNA or non-targeting siRNA. Non-targeting siRNA was directed against firefly luciferase and did not have homology with any human gene. The expression of dynamin in targeting siRNA-transfected cells was minimal already at 48 h after transfection and stayed at this low level until 96 h (Fig. 6A). Similarly to GTPase defective dynamin overexpression, the inhibition of dynamin-2 expression by siRNA caused prolongation of ERK1/2 activation (Fig. 6B).


Figure 6
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  		FIGURE 6.
Dynamin siRNA mimics the K44A mutant effect on ERK activation. A, dynamin-2 siRNA substantially reduces the dynamin-2 protein level. JYTF-1 cells were transfected with dynamin-2 siRNA (D) or control, non-targeting siRNA (NT). The expression of dynamin at 48, 72, and 96 h post-transfection was monitored by Western blotting. The membrane was subsequently reprobed with anti-tubulin to show equal protein loading. B, dynamin siRNA causes the prolongation of IL-5-induced ERK activation. JYTF-1 cells transfected as in A were cytokine and serum-starved for 24 h. At 72 h after transfection, cells were stimulated with IL-5 for the indicated times and lysed. Lysates were Western blotted with anti-phospho-ERK (p-ERK, Thr202/Tyr204). The membrane was reprobed with anti-ERK antibody to demonstrate equal protein loading. Ab, antibody.

 
Inhibition of Dynamin Expression Enhances IL-5-dependent Proliferation of the Progenitor Cell Line—Next we investigated the functional relevance of dynamin for IL-5-stimulated cell function. We examined the influence of decreased dynamin expression on IL-5-stimulated cell proliferation. siRNA-transfected cells were incubated in a cytokine-free medium or medium supplemented with IL-5 and [3H]thymidine incorporation was measured (Fig. 7A, left graph). IL-5-dependent proliferation of cells transfected with dynamin siRNA was significantly increased as compared with the proliferation of non-targeting siRNA-transfected cells. Next, we asked if IL-5-stimulated JYTF-1 proliferation is dependent on the ERK pathway. The pharmacological inhibition of the ERK1/2 activator-MEK1/2 with PD98059 resulted in substantial blockade of IL-5-stimulated proliferation of non-targeting siRNA-transfected as well as dynamin siRNA-transfected cells. The degree of inhibition of proliferation by PD98059 was similar for both of these cell populations. We validated the PD98059 data using a dominant-negative (dn) form of MEK1 (K97M). To this goal JYTF-1 cells were co-transfected with dynamin-2 siRNA and the dnMEK1 vector. We performed three control transfections: dynamin-2 siRNA plus empty vector, non-targeting siRNA plus empty vector, and non-targeting siRNA plus dnMEK1 vector. Co-transfection with dnMEK1 resulted in the inhibition of IL-5-stimulated proliferation of non-targeting siRNA-transfected as well as dynamin siRNA-transfected cells (Fig. 7A, right graph). As compared with PD98059, dnMEK1 was less potent in the inhibition of cell proliferation. We attribute this discrepancy to the low transfection efficiency of JYTF-1 cells with the plasmid DNA. In parallel JYTF-1 transfection experiments with green fluorescent protein expression vector (pLEGFP-N1, Clontech) we determined the plasmid transfection efficiency to be around 50-60%. The dnMEK1 vector is larger than pLEGFP-N1 (12 versus 7 kb), which might further reduce its transfection efficiency. It is important to note that the siRNA transfection efficiency is around 85-90% as demonstrated by parallel experiments with Cy3-labeled siRNA.

These results suggest that enhanced proliferation of dynamin siRNA-transfected cells is linked to the increased activation of the ERK pathway. We also examined the influence of decreased dynamin expression on IL-5-stimulated cell survival (Fig. 7B). siRNA-transfected JYTF-1 cells were incubated in a cytokine-free medium or in medium with IL-5. After 24 h cells were stained with annexin V and propidium iodide and analyzed by flow cytometry. The percentage of apoptotic (annexin V, positive; propidium iodide, negative) and necrotic (annexin V, positive; propidium iodide, positive) cells were similar in dynamin siRNA-transfected and non-targeting siRNA-transfected cell populations. Therefore, dynamin affects IL-5-stimulated cell proliferation but not survival in this growth factor-dependent cell line.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
This study demonstrates for the first time the association of the endocytosis-regulating molecule dynamin with the {alpha} subunit of the IL-5 receptor. The study shows that there is a cross-talk between endocytosis-regulating proteins and IL-5-stimulated signaling molecules. Dynamin binds to IL-5R-interacting tyrosine kinases Lyn and JAK2. Dynamin is tyrosine phosphorylated and inhibition of tyrosine phosphorylation blocks receptor endocytosis. Dynamin is not only a target but also a regulator of tyrosine phosphorylation. Inhibition of dynamin GTPase activity leads to increased tyrosine phosphorylation and sustained activation of the ERK1/2 pathway. Dynamin inhibition does not interfere with activation of AKT and STAT signaling pathways. The study demonstrates the biological relevance of dynamin for IL-5-stimulated cell function. Dynamin is involved in the down-regulation of IL-5-induced cell proliferation. The down-modulation of cell proliferation is linked to the inhibition of the ERK pathway.

The mechanism of IL-5 receptor signal termination has not been fully explored. Lack of signal termination may lead to development of pathologic conditions. The recently constitutively activated fusion receptor tyrosine kinase FIP1L1-PDGFR{alpha} has been identified as a cause of idiopathic hypereosinophilic syndrome (30). The understanding of how the IL-5 signal is terminated may be crucial for therapy of various malignancies associated with eosinophil or B cell expansion, autoimmune and allergic disorders. Experiments performed on the TF-1 cell line suggest the involvement of proteosomal degradation of the beta chain as a mechanism of IL-5 signaling termination (31). Common beta chain was also reported to be targeted by the phosphatase SHP-1 and SOCS family member CIS1 (32, 33). SHP-1 negatively regulated IL-3-dependent proliferation of the TF1 cell line (32). CIS1 was reported to block STAT5 recruitment to the beta chain in the Ba/F3 cell line (33). Little is known about down-regulation of IL-5 signaling in eosinophils. Ligand-induced receptor endocytosis is another way of signal cessation. The association of IL-5R with the endocytic machinery was until now unknown. We demonstrated the association of IL-5R{alpha} with GTPase dynamin in yeast two-hybrid experiments as well as in eosinophils by coprecipitation. Ligand-induced endocytosis of the receptor is a multistep process. The first step involves the recruitment of the adaptor protein AP-2 to the specific motifs of the cytosolic part of receptors (YXX{Phi} motif, {Phi} is a hydrophobic residue; a dileucine-based motif or an FXNPXY motif, Ref. 34). Cell membrane-bound AP-2 recruits and promotes the polymerization of clathrin. Polymerization of clathrin causes the formation of the pit in the cell membrane (35). AP-2 also recruits some other adaptor proteins like amphiphysin. Amphiphysin recruits dynamin to the coated pit. Dynamin is believed to self-assemble into a helical collar at the neck of the clathrin-coated pit (17, 35). The GTPase activity of dynamin is necessary for collar constriction, membrane fission, and vesicle formation. Interestingly, we found the association of dynamin with the IL-5R even in the absence of IL-5 stimulation. The beta subunit of the IL-5 receptor does not bind dynamin in unstimulated eosinophils. Dynamin co-precipitates with the beta subunit upon IL-5 stimulation when beta subunit is bound to IL-5R{alpha}. This data suggests that the beta subunit does not bind dynamin directly and the beta subunit-dynamin association is mediated by the {alpha} subunit. Alternatively, the IL-5-induced change in conformation or covalent modification of the beta subunit allows the beta subunit to recruit dynamin directly. We investigated the IL-5R{alpha} surface expression after IL-5 stimulation. IL-5R{alpha} rapidly disappeared from the cell membrane. The surface expression of IL-5R{alpha} reached a nadir at 10 min and then slowly went back to the basal level. Interestingly, the IL-5R{alpha} surface expression kinetics resembles the kinetics of other dynamin-regulated receptors such as EGFR (15).


Figure 7
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  		FIGURE 7.
Dynamin affects IL-5-dependent cell function. A, dynamin-2 down-regulates IL-5-dependent cell proliferation. Left graph, cells transfected with dynamin-2 siRNA (D) or control, non-targeting siRNA (NT) were starved overnight in cytokine-free medium. Next, cells were preincubated with PD98059 (+ PD98059, hatched bars) or vehicle, Me2SO (-PD98059, open and solid bars), stimulated with IL-5 (+ IL-5, open and hatched bars) or left unstimulated (-IL-5, solid bars) for 36 h and incorporation of [3H]thymidine was measuredas described under "Experimental Procedures." Right graph, JYTF-1 cells were co-transfected with dynamin-2 siRNA (D) and vector encoding dominant-negative MEK1 (dnM). Three control transfections were performed: dynamin-2 siRNA plus empty vector (E), non-targeting siRNA (NT) plus empty vector, non-targeting siRNA plus dominant-negative MEK1 vector. Transfected cells were analyzed in the thymidine incorporation assay as described above. Solid, open, and hatched bars represent unstimulated empty vector co-transfected cells, IL-5-stimulated empty vector co-transfected cells, and IL-5-stimulated dominant-negative MEK1 co-transfected cells, respectively. B, dynamin-2 does not impact on the IL-5-dependent cell survival. siRNA-transfected cells were starved and stimulated with IL-5 as in A. Next, cells were stained with fluorescein isothiocyanate-labeled annexin V and propidium iodide and analyzed by flow cytometry. The percentage of cells in each quadrant of the flow cytometry dot plot is shown to the right of each plot.

 


Figure 8
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  		FIGURE 8.
Differential regulation of signaling pathways by dynamin-stimulated IL-5R endocytosis. Binding of IL-5 to the receptor activates ERK, AKT, and STAT signaling pathways. Subsequently, dynamin together with other vesicle-trafficking molecules trigger IL-5 receptor endocytosis. IL-5R endocytosis is involved in the termination of ERK signaling but has no effect on AKT and STAT signaling.

 
We investigated if there was any cross-talk between IL-5R-induced endocytosis and the signaling pathway. Dynamin bound tyrosine kinases Lyn and JAK2. Interestingly, this interaction happened only in the presence of IL-5R{alpha}. Dynamin underwent tyrosine phosphorylation upon IL-5 stimulation. The inhibition of tyrosine phosphorylation by genistein blocked ligand-induced receptor internalization. The importance of dynamin tyrosine phosphorylation for receptor endocytosis was previously studied. EGF stimulation of COS-7 cells resulted in the phosphorylation of dynamin on tyrosine 597 (26). Expression of the Y597F dynamin-1 mutant blocked ligand-induced EGFR internalization. The phosphorylation of dynamin-1 by c-Src was subsequently shown to induce dynamin self-assembly and increase its GTPase activity. We would like to point out that in our experiment genistein was likely to impact on several other endocytosis-regulating molecules. There are reports indicating that EGF-activated tyrosine kinases phosphorylate clathrin and regulatory proteins like Eps15 (36, 37). Phosphorylation of these proteins leads to increased endocytosis. Our results indicate that tyrosine phosphorylation is essential for cytokine receptor endocytosis. Therefore, IL-5-stimulated tyrosine kinases act as master regulators of IL-5 signaling. First, they initiate signaling. Second, by activating the receptor internalization machinery they are also capable of regulating the length and, possibly, the amplitude of the signal.

Although we have shown that dynamin physically interacts with IL-5R{alpha}, we have not studied the importance of this physical interaction per se on IL-5 signaling. Instead we focused on the impact of dynaminmediated receptor endocytosis on IL-5 receptor downstream signaling, specifically on the ERK1/2, JAK-STAT5, and AKT pathways. We recognize that many receptors undergo ligand-dependent endocytosis without having a basal interaction with dynamin. However, there are receptors, e.g. VEGFR-2/KDR and metabotropic glutamate receptor-5, that bind to dynamin under basal conditions (38, 39). The biological relevance of this basal association is not fully clear. In addition to its GTPase role in membrane fission during endocytosis, dynamin can function as a scaffold for many proteins, especially those containing BAR (Bin-Amphiphysin-Rvs) and F-BAR domains (40). There are six families of BAR domain proteins: the afaftin, PICK1 and Ica69, amphiphysin, centaurin/APPL/sorting nexins, endophyllin, and nadrin families (41). Many of these proteins are involved in membrane tubulation. Dynamin is also an important actin regulator. Through its effect on actin it actually antagonizes membrane tubulation (40). Thus, IL-5R{alpha}-associated dynamin could function as a scaffold for cytoskeleton regulators. Unlike IL-3 and the granulocyte-macrophage colony-stimulating factor, IL-5 has a nuclear localization signal and localizes to the nucleus upon binding to its receptor (42). It is interesting to note that dynamin interacts with importin-beta1 (43). Through the use of importin-beta1 and the dynamin-associated molecular complex, ErbB2 localizes to the nucleus. The basal association with dynamin could give IL-5R{alpha} a special nuclear transport mechanism that is not available to the {alpha} subunit of IL-3 or granulocyte-macrophage colony-stimulating factor receptors.

We investigated the influence of dynamin on IL-5-stimulated signaling pathways. The overexpression of the GTPase-defective mutant of dynamin resulted in the enhancement of IL-5-induced tyrosine phosphorylation and prolongation of ERK1/2 activation. Similar results were obtained with dynamin siRNA. Conversely, overexpression of wild type dynamin led to decreased ERK1/2 activation. Although our results from siRNA-transfected cells mimicked GTPase-defective mutant data, we cannot completely exclude siRNA off-target effects. In conclusion, dynamin down-regulates IL-5-induced signaling. There are reports that may provide some clues in regard to the mechanism for this phenomenon. It was previously demonstrated that a substantial amount of the Shc-Grb2-Sos complex is endocytosed with the receptor (44). The foregoing complex is important for activation of the ERK1/2 pathway. Therefore, one can speculate that endocytosis separates the Shc-Grb2-Sos complex from the membrane-bound Ras and ERK signaling ceases. On the other hand, GTPase activity of dynamin was shown to play a positive role in EGFR-stimulated ERK1/2 signaling (15). In a different report the proline-rich region but not the GTPase activity of dynamin was needed for the enhancement of TCR signaling (45). These reports together with our data suggest that a single molecule might have opposing functions dependent on its binding partner (e.g. receptor). Overexpression of wild type or the GTPase-defective mutant of dynamin had no effect on AKT and STAT5 phosphorylation. The lack of effect of dynamin on STAT5 signaling is in agreement with previous reports in which inhibition of EGFR or IL-2R receptor endocytosis had no significant effect on receptor-induced STAT3 or STAT5 phosphorylation, respectively (46, 47). Similarly, dynamin inhibition did not affect AKT phosphorylation in the insulin receptor signaling pathway (48). Interestingly, there are reports indicating that JAK binding inhibits receptor internalization (49). It is possible that STAT phosphorylation is not influenced by inhibition of endocytosis because the STAT-activating receptor does not undergo endocytosis. Fig. 8 summarizes our findings on the differential regulation of signaling pathways by dynamin-stimulated IL-5R endocytosis.

Dynamin deficiency leads to the enhancement of IL-5-induced cell proliferation. Lack of dynamin had no effect on IL-5-dependent cell survival. This is in agreement with the lack of effect of dynamin on phosphorylation of the survival promoting molecule AKT. We also showed that IL-5-stimulated JYTF-1 proliferation is strongly dependent on ERK1/2 activity. Our results suggest that the enhancement of proliferation in the absence of dynamin might be attributed to the increased activation of ERK pathway. ERK was previously demonstrated to be important for proliferation induced by beta chain-interacting cytokines (50). In contrast, ERK was reported to have only a marginal role in the prolongation of IL-5-stimulated cell survival (51). Thus, we have identified a new termination mechanism for the IL-5-induced ERK signaling pathway.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants RO1 AI50179, AI059719, and AI68088. 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 To whom correspondence should be addressed: 1400 Jackson St., Denver, CO 80206. Tel.: 303-270-2907; Fax: 303-398-1225; E-mail: alamr{at}njc.org.

2 The abbreviations used are: IL, interleukin; STAT, signal transducers and activators of transcription; ERK, extracellular signal-regulated kinase; JAK, Janus kinase; FBS, fetal bovine serum; siRNA, small interfering RNA; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; dn, dominant-negative; EGFR, epidermal growth factor receptor. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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