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

Abstract

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 plasma membrane-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.

Cholesterol-sensitive Modulation of Transcytosis

“…Particularly interesting in this context is the observation that phosphorylation of Cav1 on tyrosine 14 is markedly augmented in cholesterol-depleted cells (see Figure 7 and Kim et al, 2002). This signaling event, initially shown to be activated by Src kinases, can induce aggregation, fusion, and even the biogenesis of caveolar transport vesicles (Orlichenko et al, 2006). Therefore, phosphorylated Cav1 in cholesterol-depleted cells could potentially facilitate multiple membrane transport processes, among which are those that regulate transcytosis.…”

The Caveolin-1 Connection to Cell Death and Survival

“…Phosphorylation of Caveolin-1 on Tyr-14 is also linked to augmented anchorageindependent growth and cell migration via a Grb7-dependent mechanism [57], as well as association with type-I matrix metalloproteinase [70]. Additionally, phosphorylation at this site is considered a crucial event in integrin-regulated membrane microdomain internalization [71][72], as well as EGF-induced caveolae formation [73]. Moreover, presence or absence of this site may account for functionally non-redundant roles ascribed to the two Caveolin-1 isoforms 1 and 1 in early vertebrate development [74].…”

Transit of hormonal and EGF receptor-dependent signals through cholesterol-rich membranes

“…EGFR activation may also change the raft environment by promoting the formation of oligomeric caveolin at the plasma membrane [36]. This would have the effect of drastically increasing the potential binding sites for caveolin-associated proteins, with likely dramatic effects on downstream signaling.…”

“…EGFR activation may also change the raft environment by promoting the formation of oligomeric caveolin at the plasma membrane [36]. This would have the effect of drastically …”

“…Applied to a cancer cell that accumulates cholesterol relative to corresponding normal cell types, the implications of these results are (1) that lateral diffusion of EGFR family members is increased with malignancy and (2) that cholesterol may alter the rate of formation and dissociation of receptor-associated signaling complexes. They also imply that different receptor tyrosine kinases may respond differently to changes in the cholesterol content of cell membranes.EGFR activation may also change the raft environment by promoting the formation of oligomeric caveolin at the plasma membrane [36]. This would have the effect of drastically …”