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The c-Cbl/CD2AP complex regulates VEGF-induced endocytosis and degradation of Flt-1 (VEGFR-1)¹

SATSUKI KOBAYASHI^*,, ASAKO SAWANO^*, YOSHIHISA NOJIMA, MASABUMI SHIBUYA^* and YOSHIRO MARU^*,,2

^* Division of Genetics, Institute of Medical Science, University of Tokyo, Tokyo;
Third Department of Internal Medicine, Gunma University School of Medicine, Gunma; and
Department of Pharmacology, Tokyo Women’s Medical University School of Medicine, Tokyo, Japan

²Correspondence: Department of Pharmacology, Tokyo Women’s Medical University School of Medicine, Shinjuku-ku, Tokyo, 162-8666, Japan. E-mail: ymaru{at}research.twmu.ac.jp

SPECIFIC AIMS

This study is aimed to uncover the molecular mechanisms that underlie the fate of Flt-1 (VEGFR-1) after VEGF stimulation.

PRINCIPAL FINDINGS

1. The vascular endothelial growth factor (VEGF) -dependent interaction between Flt-1 and c-Cbl was clearly observed in NIH3T3 fibroblasts overexpressing Flt-1 (3T3-FLT-1). When the full-length c-Cbl was transfected to 3T3-FLT-1 cells, the complex formation between CD2AP and Flt-1 was also shown to be dependent on VEGF

2. Both the Flt-1 kinase activity and tyrosine residue 1333 in Flt-1 were shown to be required for binding to c-Cbl but not to PLC-

3. A c-Cbl mutant study revealed that the SH2 domain of c-Cbl and the rest of the sequence are required for binding to VEGF-activated Flt-1 and to CD2AP, respectively

4. The second SH3 domain of CD2AP has the ability to bind to c-Cbl, which is bound to activated Flt-1

5. VEGF stimulated endocytic trafficking of Flt-1 and c-Cbl, which was impaired by a VEGF receptor kinase inhibitor SU5416 or by the tyrosine to phenylalanine mutation at 1333 (Y1333F) in Flt-1 (Fig. 1 )

View larger version (47K): [in this window] [in a new window]   Figure 1. The essential role of autophosphorylation site Y1333 in VEGF-stimulated endocytosis of Flt-1. A) CHO cells were cotransfected with the full-length c-Cbl and GFP-fused wild-type Flt-1 at the carboxyl terminus (Flt-1 WT) and stimulated by VEGF for 15 min. Cells were fixed and stained with anti-c-Cbl antibody (rhodamine). GFP signals (left panel), anti-c-Cbl immunofluorescence (middle), and merged signals (right) were observed with (+) or without (–) stimulation by VEGF. B) The Y1333 mutant of Flt-1 (Flt-1 Y1333F) was used instead of wild-type Flt-1 in the same experiment as shown in panel A. C) A VEGFR tyrosine kinase inhibitor SU5416 at 10 µM was added for 3 h before stimulation by VEGF. Note the poor formation of vesicles in panels B and C.

6. Cotransfection of c-Cbl and CD2AP gave large vesicles that contained both of the molecules, which was not observed when a CD2AP mutant was used

7. Transfection of the full-length CD2AP promoted internalization of VEGF-stimulated Flt-1 from the cell surface as well as degradation in 3T3-FLT-1 cells. CD2AP mutants and Flt-1 Y1333F inhibited those events (Fig. 2 )

View larger version (51K): [in this window] [in a new window]   Figure 2. Dominant-negative forms of CD2AP inhibit degradation of VEGF-stimulated Flt-1. A) Schematic representation of CD2AP and its mutants. The full-length CD2AP (X-CD2AP), amino-terminal half containing the three SH3 domains (X-3SH3), and carboxyl-terminal half with the proline-rich (PXXP) and coiled-coil region (X-PCC) were tagged with Xpress at the amino terminus. B) 3T3-FLT-1 cells transfected with the wild-type c-Cbl in combination with X-CD2AP (squares), X-SH3 (triangles), X-PCC (crosses), and mock (empty Xpress vector) (diamonds) were incubated at 4°C for 1 h with 2 ng/mL 125I-VEGF, then at 37°C for 2–15 min, washed with mild acidic buffers (pH 4.5), and radioactivity at the cell surface was quantitated. Cells were lysed in 1N NaOH to quantify the internalized radioactivity. The ratio of internalized radioactivity relative to that on the surface was plotted against time. C) 3T3-FLT-1 cells transfected with the wild-type c-Cbl in combination with X-CD2AP, X-SH3, or X-PCC were stimulated with VEGF at 100 ng/mL (min) and lysates were subjected to anti-Flt-1 (upper panel) and anti-actin (middle panel) Western blot. Intensities of the Flt-1 bands were quantified and expressed as % of remaining receptors for each time point (bottom panel). D) NIH3T3 cells expressing Flt-1 Y1333F at a level comparable to that in 3T3-FLT-1 cells of panel C were transfected with the wild-type c-Cbl in combination with X-CD2AP and analyzed as in panel C.

8. VEGF-stimulated Flt-1 was ubiquitinated in 3T3-FLT-1 cells, which was inhibited by c-Cbl mutants

CONCLUSIONS AND SIGNIFICANCE

c-Cbl is prebound to CD2AP in the cytoplasm. VEGF-activated Flt-1 recruits the c-Cbl/CD2AP complex at the plasma membrane. The tyrosine to phenylalanine mutation at 1333 (Y1333F) in Flt-1 impairs its binding to c-Cbl. This ternary complex is involved in the formation of endocytic vesicles where Flt-1, c-Cbl, and CD2AP are colocalized. Abrogation of the formation of this complex results in impaired internalization, ubiquitination, degradation, and endocytic vesicle formation. Our data add one example to the current view that receptor tyrosine kinases are negatively regulated by c-Cbl at least by ubiquitination and degradation with the help of CD2AP/CIN85 family of proteins (Fig. 3 ). Since Flt-1 is one of the most difficult receptors in which to examine functions under physiological circumstances, analysis of the fate of a molecule Flt-1 should help explore the fate of cells where it is expressed.

View larger version (31K): [in this window] [in a new window]   Figure 3. Schematic diagram. Interaction between Flt-1 and the c-Cbl/CD2AP complex in endocytosis. VEGF-induced autophosphorylation of Flt-1 at Y1333 recruits c-Cbl through the SH2 domain of c-Cbl. This association transmits signals to CD2AP precomplexed to c-Cbl via the second SH3 domain of CD2AP. The ternary complex formation participates in the regulation of endocytosis, degradation, and ubiquitination of Flt-1. However, the mechanism may be more complicated. CD2AP may be recruited with Flt-1 in both c-Cbl-dependent and -independent (via molecule X) manners, which is facilitated by VEGF-stimulated activation of Flt-1. Ub: ubiquitin, P: phosphate.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0767fje

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