Caveolin-1 was first identified as a phosphoprotein in Rous sarcoma virus (RSV)-transformed chicken embryo fibroblasts. Tyrosine 14 is now thought to be the principal site for recognition by c-Src kinase; however, little is known about this phosphorylation event. Here, we generated a monoclonal antibody (mAb) probe that recognizes only tyrosine 14-phosphorylated caveolin-1. Using this approach, we show that caveolin-1 (Y14) is a specific tyrosine kinase substrate that is constitutively phosphorylated in Src- and Abl-transformed cells and transiently phosphorylated in a regulated fashion during growth factor signaling. We also provide evidence that tyrosine-phosphorylated caveolin-1 is localized at the major sites of tyrosine-kinase signaling, i.e. focal adhesions. By analogy with other signaling events, we hypothesized that caveolin-1 could serve as a docking site for pTyr-binding molecules. In support of this hypothesis, we show that phosphorylation of caveolin-1 on tyrosine 14 confers binding to Grb7 (an SH2-domain containing protein) both in vitro and in vivo. Furthermore, we demonstrate that binding of Grb7 to tyrosine 14-phosphorylated caveolin-1 functionally augments anchorage-independent growth and epidermal growth factor (EGF)-stimulated cell migration. We discuss the possible implications of our findings in the context of signal transduction.

beta-Dystroglycan is a ubiquitously expressed integral membrane protein that undergoes tyrosine phosphorylation in an adhesion-dependent manner. Tyrosine 892 is now thought to be the principal site for recognition by the c-Src tyrosine kinase; however, little is known about the regulation of this phosphorylation event in vivo. Here, we generated a novel monoclonal antibody probe that recognizes only tyrosine 892 phosphorylated beta-dystroglycan (pY892). We show that upon tyrosine phosphorylation, beta-dystroglycan undergoes a profound change in its sub-cellular localization (e.g., from the plasma membrane to an internal membrane compartment). One possibility is that the net negative charge at position 892 causes the redistribution of beta-dystroglycan to this intracellular vesicular location. In support of this notion, mutation of tyrosine 892 to glutamate (Y892E) is sufficient to drive this intracellular localization, while other point mutants (Y892F and Y892A) remain at the plasma membrane. Interestingly, our colocalization studies with endosomal markers (EEA1, transferrin, and transferrin receptor) suggest that these phospho-beta-dystroglycan containing internal vesicles represent a subset of recycling endosomes. At the level of these internal vesicular structures, we find that tyrosine phosphorylated beta-dystroglycan is colocalized with c-Src. In addition, we demonstrate that known ligands for alpha-dystroglycan, namely, agrin and laminin, are able to induce the tyrosine phosphorylation of beta-dystroglycan. Finally, we show that tyrosine phosphorylated beta-dystroglycan is also detectable in skeletal muscle tissue lysates and is localized to an internal vesicular membrane compartment in skeletal muscle fibers in vivo. The generation of a phospho-specific beta-dystroglycan (pY892) mAb probe provides a new powerful tool for dissecting the role of dystroglycan phosphorylation in normal cellular functioning and in the pathogenesis of muscular dystrophies.

A variety of molecular influences in the extracellular matrix (ECM) interact with developing axons to guide the formation of hippocampal axon pathways. One of these influences may be chondroitin sulfate proteoglycans (CSPGs), which are known to inhibit axonal extension during development and following central nervous system injury. In this study, we examined the role of CSPGs and cell adhesion molecules in the regulation of axon tract formation during hippocampal development. We used indirect immunofluorescence to examine the developmental pattern of CSPG expression relative to axon tracts that express the cell adhesion molecule L1. Additionally, we used dissociated and explant cell cultures to examine the effects of CSPGs on hippocampal axon development in vitro. In vivo, we found that the CSPG neurocan is expressed throughout the alveus, neuropil layers, and parts of the dentate gyrus from E16 to P2. The CSPG phosphacan is expressed primarily in the neuropil layers at postnatal stages. After E18, intense labeling of neurocan was observed in regions of the alveus surrounding L1-expressing axon fascicles. In vitro, axons from brain regions that project through the alveus during development would not grow across CSPG substrata, in a concentration-dependent manner. In addition, hippocampal axons from dissociated neuron cultures only traveled across CSPG substrata as fasciculated axon bundles. These findings implicate CSPG in the regulation of axon trajectory and fasciculation during hippocampal axon tract formation.