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Caveolae are specialized invaginated domains of the plasma membrane that act as organizing centers for signaling molecules. Caveolae are formed in membranes by the caveolins, a family of three related gene products (caveolins-1, -2, and -3) [1-4]. Caveolins form homo- and hetero-oligomers that make up the characteristic striated coat of caveolae [5]. Expression of caveolin is necessary and sufficient to induce cell-surface caveolae [6-8]. In addition to their role as coat proteins that drive the invagination of caveolae, the caveolins are also cholesterol-binding proteins, and caveolae are highly enriched in cholesterol, glycolipids and sphingolipids, forming a distinctive domain in the membrane. Lipid-modified signaling molecules, including the tandemly acylated Src-family kinases, are enriched in these structures due to their affinity for the lipid composition of these domains. However, cholesterol-enriched lipid-ordered domains, or "rafts" also form independently of caveolae [9]. The caveolins direct the composition of the signaling complexes organized in caveolae by acting as scaffolding proteins that bind to specific signaling molecules, which include Gaqii, endothelial nitric oxide synthase (eNOS), and Src-family kinases [10-13].

In addition to organizing signaling complexes, the caveolins also participate directly in signaling cascades as substrates for both tyrosine and serine/threonine kinases. Caveolin-1 is phosphorylated on a single tyrosine residue (Tyr14) in the amino-terminus of the protein. This residue is constitutively phosphorylated at low levels in most cell types, and its phosphorylation increases in response to a number of stimuli, including insulin and insulin mimetics (IGF-1, sulfonylureas, phos-phoinositolglycan), angiotensin II, endothelin-1, adrenocorticotropin (ACTH), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF) (in cells expressing very high levels of the EGF receptor or expressing mutant forms of the EGF receptor) [14-27]. Phosphorylation at Tyr14 is also increased in response to cellular stresses, including shear stress, apoptotic stress, ultraviolet radiation, oxidative stress, and osmotic shock [24,28-34], in response to integrin activation (i. e., during plating and spreading on

Py14 Caveolin

Fig. 6.1 Signaling pathways leading to and from caveolin phosphorylation. Hypothesis to be explored: insulin and stress activate a caveolin-directed tyrosine kinase localized in the caveolae. Upon activation, this kinase phosphorylates caveo-lin-1 on Tyr14 (pY14). This leads to activation of downstream signaling cascades.

Fig. 6.1 Signaling pathways leading to and from caveolin phosphorylation. Hypothesis to be explored: insulin and stress activate a caveolin-directed tyrosine kinase localized in the caveolae. Upon activation, this kinase phosphorylates caveo-lin-1 on Tyr14 (pY14). This leads to activation of downstream signaling cascades.

fibronectin) [24,34], and when caveolae are triggered to internalize [35-37]. In response to some stimuli, such as insulin, oxidative stress and osmotic shock, the increase in caveolin phosphorylation is robust and sustained, whilst in response to other stimuli, such as shear stress, PDGF and VEGF, the increase is less intense and is transient. Caveolin-1 is also phosphorylated on up to five serine/threonine residues in response to various stimuli [26,31,38,39]. Caveolin-2 is phosphorylated on two tyrosine residues in its amino-terminal domain, Tyr 19 and Tyr27 [40-43]. Tyr27 in caveolin-2 falls within an amino acid sequence (YADP) that is very similar to Tyr14 in caveolin-1 (YTVP), and phosphocaveolin-2 is found in complexes with phosphocaveolin-1. Caveolin-3 lacks a site for tyrosine phosphorylation in its amino-terminal domain, and it has not been reported to be phosphorylated on either tyrosine or serine.

The goals of our research in this area have been to: 1) trace the signal transduction pathways that lead to caveolin tyrosine phosphorylation; and 2) identify signaling cascades that lie downstream of caveolin phosphorylation. These two areas will form the focus of this chapter (Fig. 6.1). In a very satisfying way, the investigations in these two areas have come together to provide a very clear picture of at least one of the roles of caveolin phosphorylation and caveolae in cells.

Signaling Pathways Leading to Caveolin Tyrosine Phosphorylation

Caveolins-1 and -2 are Phosphorylated in Response to Insulin in Adipocytes

Whilst caveolae are found in many tissues, they are particularly abundant in lung epithelial cells, endothelial cells, muscle cells, and adipocytes [1,3,4]. In adipocytes, they cover a significant fraction (ca. 30 %) of the total inner cell surface of the plasma membrane [44]. In addition, the expression of caveolins-1 and -2 increases approximately 20-fold upon adipocyte differentiation, with a concomitant 10-fold increase in cell-surface caveolae [15,45,46]. Consistent with an important role for caveolae in adipocyte function, adipose tissue is significantly disrupted in caveolin-1 knockout animals [47-49].

Early studies on isolated caveolin-enriched cell fractions had implicated caveolae in cellular signaling [50-54]. The abundance of caveolae in adipocytes and muscle - two of the major target tissues for insulin - together with their potential role in signaling, suggested that they might play a role in insulin signal transduction. To investigate this, a simple question was asked: Does stimulation of adipocytes with insulin lead to an increase in the phosphorylation of any proteins associated with caveolae? It was found that, in adipocytes, insulin stimulates the tyrosine phosphorylation of three proteins in caveolae: caveolin-1, caveolin-2, and a 29-kDa cav-eolin-associated protein [14,15]. These three proteins are found in SDS-resistant complexes and can be co-immunoprecipitated. Caveolin phosphorylation shows two additional interesting properties. The first is that it shows specificity for insulin, and does not occur in adipocytes in response to two other growth factors, PDGF and EGF, despite the expression of active receptors for all three growth factors in these cells. This is interesting because stimulation of glucose metabolism also shows specificity for insulin in these cells. Insulin stimulates glucose transport, glycogen synthesis, and lipogenesis ten to hundreds of fold, while EGF and PDGF have no effect on these processes [55]. The second interesting property of insulin-stimulated caveolin phosphorylation is that it is cell type- dependent: it occurs only in the fully differentiated 3T3-L1 adipocytes, not in the preadipocytes, despite the expression of both caveolin and active insulin receptor in both cell types. In fact, caveolin is not phosphorylated in response to insulin in fibroblast cells engineered to express high levels of the insulin receptor. Differentiation dramatically increases the insulin responsiveness of glucose metabolism in these cells [56]. Unlike phosphorylation of the caveolins, the 29-kDa caveolin-associated protein was phosphorylated in response to PDGF as well as in response to insulin, and this phosphorylation occurred in both adipocytes and preadipocytes [14,20]. At present, the identity of this protein is unknown, but this result indicates that other growth factors also signal through tyrosine phosphorylation of caveolar proteins.

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