Functional analysis of caveolin tyrosine phosphorylation and the behavior of caveolin-1 (P312L), a mutant found in human breast cancers
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Caveolin is a principal marker protein of caveolae, vesicular invaginations of the plasma membrane. In addition to driving the formation of membrane invaginations, several functions were proposed for caveolin in vesicular trafficking and signal transduction processes. Here I found a novel function of caveolins mediated by tyrosine phosphorylation.;Cav-1 was first identified as a phospho-protein in Rous sarcoma virus-transformed cells. Previously, Tyr 14 was found to be the principal site for recognition by c-Src kinase; however, significance of the phosphorylation remained largely unknown. To study function of phosphorylated Cav-1, our lab generated a monoclonal antibody that recognizes only the Tyr 14 phosphorylated form of Cav-1. Using this antibody, I found that Cav-1 is a substrate of specific tyrosine kinases and that this phosphorylation occurs in a tightly regulated fashion. Also, I showed that phospho-Cav-1 is localized at focal contacts, where the majority of tyrosine-signaling events normally occur.;One of known functions of phosphorylation events is to confer binding to SH2 domain-containing proteins. Using an in vitro association approach, I showed that an SH2 domain-containing protein, Grb7, binds to Cav-1 in phosphorylated specific fashion---both in vitro and in vivo. Furthermore, I showed that binding of Grb7 to phospho-Cav-1 on Tyr 14 functionally augments both anchorage-independent growth and cell migration.;In addition to my work on caveolin phosphorylation, I studied the behavior of a naturally occurring sporadic Cav-1 mutation (P132L) that is found in ∼16% of human breast cancers. To begin to understand the role of the Cav-1 (P132L) mutation, I investigated its behavior in cultured cells. I showed that Cav-1 (P132L) was mis-folded and retained within Golgi-complex, but did not target to the plasma membrane. Moreover, when the Cav-1 (P132L) mutant was co-expressed with wild-type Cav-1, the mutant behaved in a dominant-negative fashion, leading to the intracellular retention of wild-type Cav-1.
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