Phosphocaveolin is Enriched at Sites of Attachment of the Actin Cytoskeleton to the Plasma Membrane

Significant data links caveolae and caveolins to the actin cytoskeleton [102,103]. In confluent, quiescent cells and in tissues, caveolin-1 is primary localized to the plasma membrane. The caveolin-1 at cell surface is tethered to the actin cytoskeleton [104,105]. Caveolin binds to actin through filamin, a protein that regulates cortical actin assembly [106]. Agents that disrupt the actin cytoskeleton cause rapid internalization of caveolin [105]. Caveolin-1 also redistributes in response to shear stress and during cell migration [30,32,107-109]. The muscle specific isoform cav-eolin-3 is also bound to actin, and loss of caveolin-3 causes a form of muscular dystrophy due to defects in the anchoring of the actin cytoskeleton to the plasma membrane [110,111].

Phosphocaveolin is ideally localized to act as the mediator between Src activation and Csk-induced inactivation in the regulation of actin assembly. Phosphocaveolin

Membrane Attachment Cytoskeletons

Fig. 6.5 Caveolin is phosphorylated at actin/plasma membrane attachment sites. Fibroblast cells plated onto fibronectin for 5 min (top) or 15 min. Actin is labeled with FITC-phalloidin (green). Phosphocaveolin is labeled with anti-PY14 antibody and Cy3-labeled secondary antibody (red). The boxed portions in the right-hand panels are shown enlarged on the left.

Fig. 6.5 Caveolin is phosphorylated at actin/plasma membrane attachment sites. Fibroblast cells plated onto fibronectin for 5 min (top) or 15 min. Actin is labeled with FITC-phalloidin (green). Phosphocaveolin is labeled with anti-PY14 antibody and Cy3-labeled secondary antibody (red). The boxed portions in the right-hand panels are shown enlarged on the left.

is highly enriched at or near focal adhesions at the ends of the actin stress fibers [18,24,27,29,32,34,35,109]. In migrating non-confluent cells, caveolin is cleared from the leading edge of the cell and is found predominantly in vesicles at the trailing edge of the cell. However, phosphocaveolin is highly enriched near forming focal adhesions at the cell edge in actively migrating cells, or in cells spreading on fibronectin. Double labeling with phalloidin showed that caveolin phosphorylation occurs at the ends of bundles of actin fibers at the edge of the cell (Fig. 6.5). The enrichment of phosphocaveolin near focal adhesions and the tight association of caveolin with cortical actin indicate that phosphorylation of caveolin may regulate the actin cytoskeleton through Csk-mediated inhibition of Src-family kinases. Consistent with a link between caveolin, Src-family kinases, and the actin cytoskeleton, integrin signaling through Fyn requires expression of caveolin-1 [62,101].

Abl in the Loop

Our data indicate that activation of Fyn in caveolae would be sufficient to induce caveolin tyrosine phosphorylation, although this phosphorylation is expected to be self-limiting and transient due to feedback inhibition through Csk. Both Abl and Fyn are required for robust, sustained caveolin phosphorylation. Our current model is that Fyn and Abl act synergistically in the phosphorylation of caveolin (Fig. 6.6). Extracellular stimuli such as insulin or stress activate Fyn in caveolae, which phosphorylates caveolin. This leads to the recruitment of Csk, attenuates Fyn, and limits the extent of phosphorylation of caveolin. At the same time, these signals activate Abl which translocates into caveolae where it also phosphorylates caveolin. Abl is not regulated by Csk, and remains active in these complexes, main

Csk Lipid Raft

Fig. 6.6 Abl kinase is required for stable phosphorylation of caveolin. Both Abl and Fyn are required for caveolin phosphorylation. Both are caveolin tyrosine kinases and directly phosphorylate it in vitro. Both are activated by oxidative stress. Fyn is resident in caveolae and may be required for the activa

Fig. 6.6 Abl kinase is required for stable phosphorylation of caveolin. Both Abl and Fyn are required for caveolin phosphorylation. Both are caveolin tyrosine kinases and directly phosphorylate it in vitro. Both are activated by oxidative stress. Fyn is resident in caveolae and may be required for the activa tion or recruitment of Abl to caveolae. Recruitment of Csk to phosphocaveolin would attenuate Fyn activity, leading to transient caveolin phosphorylation. Since Abl is not inhibited by Csk, recruitment to or activation of Abl in caveolae would induce stable phosphorylation of caveolin-1.

taining a high level of caveolin phosphorylation. In the absence of Abl, caveolin phosphorylation is limited in extent, and is transient. Recruitment/activation of Abl is required for high-level, sustained phosphorylation of caveolin-1, and sustained inhibition of Src-family kinases. This represents a novel mechanism for the attenuation of Src-family kinase activity by Abl: phosphorylation of a scaffolding protein (caveolin) and recruitment of Csk. Paxillin, a substrate of both Abl and Src, is likely to organize a similar regulatory complex by binding to Abl, Src, and Csk [112]. Csk is recruited only after phosphorylation of paxillin.

Interestingly, Fyn and Abl are required for opposing stress-induced pathways in cells. Activation of Fyn is required for the induction of survival pathways [66,113], whereas activation of Abl induces cell death [114]. Activation of Fyn and Abl are also temporally different. Fyn is activated rapidly (within minutes), while Abl activation takes longer (maximum activation after 30 minutes). Low-level exposure to oxidative stress (low concentration or short duration) stimulates survival pathways, while higher exposures induce apoptotic pathways [115]. It is possible that low-level exposures stimulate only Fyn, hence survival pathways, while higher exposures are required to activate Abl and apoptotic pathways. Abl-induced recruitment of Csk to complexes containing Src-family kinases would be a mechanism to ensure that survival pathways are turned off, allowing apoptosis to proceed. Counter-regulatory effects of Abl and Fyn have been observed in a number of additional signaling pathways [84,88,89], and negative regulatory circuits involving Abl, Fyn, and their substrates have been identified genetically in second-site repressor screens in Drosophila [82,83]. Currently, an Abl-specific inhibitor STI571 (Gleevec) is used in patients to treat chronic myelogenous leukemia. Therefore, counter-regulatory complexes containing Src-family kinases and Abl have important therapeutic implications, particularly in the treatment of cancer.

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