THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 8, pp. 4570–4579, February 24, 2006

Epithelial Growth Factor-induced Phosphorylation of Caveolin 1 at Tyrosine 14 Stimulates Caveolae Formation in Epithelial Cells

Lidiya Orlichenko, Bing Huang, Eugene Krueger, and Mark A. McNiven

Caveolae are flask-shaped endocytic structures composed primarily of caveolin-1 (Cav1) and caveolin-2 (Cav2) proteins. Interestingly, a cytoplasmic accumulation of Cav1 protein does not always result in a large number of assembled caveolae organelles, suggesting a regulatory mechanism that controls caveolae assembly. In this study we report that stimulation of epithelial cells with epithelial growth factor (EGF) results in a profound increase in the number of caveolar structures at the plasma membrane. Human pancreatic tumor cells (PANC-1) and normal rat kidney cells (NRK), as a control, were treated with 30 ng/ml EGF for 0, 5, and 20 min before fixation and viewing by electron microscopy. Cells fixed without EGF treatment exhibited modest numbers of plasmamembrane- associated caveolae. Cells treated with EGF for 5 or 20 min showed an 8–10-fold increase in caveolar structures, some forming long, pronounced caveolar “towers” at the cell-cell borders. It is known that Cav1 is Src-phosphorylated on tyrosine 14 in response to EGF treatment, although the significance of this modification is unknown. We postulated that phosphorylation could provide the stimulus for caveolae assembly. To this end, we transfected cells with mutant forms of Cav1 that could not be phosphorylated (Cav1Y14F) and tested if this altered protein reduced the number of EGF-induced caveolae. We observed that EGF-stimulated PANC-1 cells expressing the mutant Cav1Y14F protein exhibited a 90–95% reduction in caveolae number compared with cells expressing wild type Cav1. This study provides novel insights into how cells regulate caveolae formation and implicates EGF-based signaling cascades in the phosphorylation of Cav1 as a stimulus for caveolae assembly.

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FIGURE 2. EGF stimulation induces a cytoplasmic redistribution of endogenous Cav1 protein along with a proliferation of caveolae-like vesicles in PANC-1. a and a , Cav1 localized predominantly along the plasma membrane at cell borders and at some cytoplasmic puncta under serum-starved conditions (arrows) as visualized by fluorescence microscopy. b and b , EGF stimulation induced a noticeable redistribution of endogenous Cav1 away from the cell periphery to a more central location (arrows). c, TEM, in the presence of ruthenium red, of the cell borders of two adjacent PANC-1 cells under low serum conditions showed few or no caveolae-like vesicles at the plasmamembrane (arrows). Cell borders between the cells are marked (CB with arrows). d, in contrast, EGF stimulation induced dramatic proliferation of caveolae-like vesicles (arrowheads) forming along areas of the plasma membrane at areas of cell-cell contact. e and e , many cells generated elaborate tubular extensions of the plasma membrane or towers (arrows) from which many putative caveolae (arrowheads) formed. Scale bars, 10 um (a, a , b, b ); 0.5 um (c and d); 0.2 um (e and e ).

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FIGURE 4. Quantitation of caveolae-like structures in PANC-1 and NRK cells after stimulation with EGF. a, quantitation of caveolae-like vesicle formation. Ruthenium red-stained vesicles along a 1-um2 area of the plasma membrane were counted as detailed under “Materials and Methods.” PANC-1 cells showed an increase of vesicular structures in only 5 min post-EGF treatment with a total 4–5-fold increase by 20 min post-EGF. In comparison, NRK cells took longer to respond to agonist but showed a greater (10 –12-fold) increase in putative caveolae by 20 min. b, quantitation of immunogold labeling of putative caveolae in resting versus EGF-stimulated cells as shown in Supplemental Fig. S1. Numbers represent the number of gold particles at the plasma membrane representing Cav1 antigen (see “Materials and Methods”). A 6–10-fold increase in labeling was observed in both cell types when examined 20 min after EGF stimulation, suggesting an increase in caveolae formation. RR, ruthenium red.

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FIGURE 6. Activation of v-Src kinase in a v-Srcts MDCK cell line induces Cav1 phosphorylation at tyrosine 14 concomitant with caveolae formation. a, Western blot (WB) analysis of immunoprecipitated (IP) Cav1 from MDCK cells expressing a temperature-sensitive v-Src protein. Immunoprecipitates from both inactive cells (40 °C) and active cells (33 °C) were blotted with the phospho-Cav1Y14 antibody and a Cav1 polyclonal to show equal loading. Cav1 was modestly phosphorylated in cells expressing inactive v-Src but highly phosphorylated in cells at the permissive temperature with active v-Src. b, low magnification TEM image of v-Srcts MDCK cells grown at the non-permissive temperature of 40 °C. Cells formed polarized monolayers at this temperature. b , higher magnification TEM image of the boxed area in b showing cell-cell interactions with few if any visible caveolae. c, low magnificationTEMimage of the cells grown at the permissive 33 °C temperature for 9 h showing compromised cellular junctions. c , high magnificationTEMimage of the boxed region in c, showing a dramatic increase in the number of caveolar vesicles formed after 9 h at the permissive temperature of 33 °C. c , additional high magnification TEM image of another cell grown at 33 °C for 9 h, also showing a marked proliferation of caveolae. d, a comparative quantitation of caveolae-like vesicles formed along the plasma membrane of v-SrctsMDCKcells grown either at the permissive or restrictive temperatures. Similar to EGF-treated cells, v-Src activation increases caveolae number by almost 4-fold. Scale bars: 2 um (b and c); 1 um (b , and c ); 0.5 um (c ).