Src-induced Phosphorylation of Caveolin-2 on Tyrosine 19

The cytoplasmically oriented monotopic integral membrane protein stomatin forms high-order oligomers and associates with lipid rafts. To characterize the domains that are involved in oligomerization and detergent-resistant membrane (DRM) association, we expressed truncation and point mutants of stomatin and analyzed their size and buoyancy by ultracentrifugation methods. A small C-terminal region of stomatin that is largely hydrophobic, Ser-Thr-Ile-Val-PhePro-Leu-Pro-Ile (residues 264 -272), proved to be crucial for oligomerization, whereas the N-terminal domain (residues 1-20) and the last 12 C-terminal amino acids (residues 276 -287) were not essential. The introduction of alanine substitutions in the region 264 -272 resulted in the appearance of monomers. Remarkably, only three of these residues, Ile-ValPhe (residues 266 -268), were found to be indispensable for the DRM association. Interestingly, the exchange of Pro-269 and to some extent the residues 270 -272, which are essential for oligomerization, did not affect the DRM association of stomatin. This suggests that the formation of oligomers is not necessary for the association of stomatin with DRMs. Internal deletions near the membrane anchoring domain resulted in the formation of intermediate size oligomers suggesting a conformational interdependence of large parts of the C-terminal region. Fluorescence recovery after photobleaching analysis of the tagged, monomeric, non-DRM mutant ST-(1-262)-green fluorescent protein and wild type stomatin StomGFP showed a significantly higher lateral mobility of the truncation mutant in the plasma membrane suggesting a membrane interaction of the respective C-terminal region also in vivo.The 31-kDa integral protein stomatin was first identified as an abundant component of the human erythrocyte membrane (1-3); however, it is also widely expressed in various tissues and cell lines (1, 3-5). The primary structure of 287 amino acids (3, 6) is characterized by a highly charged 24-residue N terminus followed by a 29-residue hydrophobic sequence that is most probably associated with the membrane and the large C-terminal region containing 234 residues. Stomatin has an unusual topology (7), similar to caveolin (8), with the hydrophobic domain forming a putative "hairpin loop" in the lipid bilayer and the N and C termini facing the cytoplasm. Palmitoylation of the cysteine residues Cys-29 and Cys-86 further increases the affinity of stomatin for the membrane (9). The association of stomatin with the lipid droplet phospholipid monolayer (10)

Section: Discussionmentioning

confidence: 83%

Characterization of the Stomatin Domain Involved in Homo-oligomerization and Lipid Raft Association

Umlauf

Mairhofer

Prohaska

2006

“…It was found previously that caveolin-2 tyrosine phosphorylation by endogenous tyrosine kinases present in purified caveolin-rich domains from lung is inhibited by a synthetic myristoylated NH 2 -terminal Src peptide but not by a nonmyristoylated Src peptide or by either myristoylated or nonmyristoylated peptides from Yes (45). This may indicate that the specific myristoylated NH 2 terminus of Src (but not Yes) displaces the main caveolin-2 kinase (possibly endogenous Src) from these caveolae preparations.…”

Section: Lipid Raft Localization Of Src Is Dependent On Brain Lipids-mentioning

confidence: 95%

“…Some studies used "panSrc" antibody, or antibodies against the phosphorylated activation loop tyrosine of Src, which do not distinguish Src from other SFKs (44,45). However, Src monoclonal antibodies have been used to show ligand-stimulated Src recruitment to EphrinB1-containing microdomains in neurons (46), and specific antibodies show the coexistence of Src, Fyn, Lyn, and Yes in rafts from neuroblastoma cells (47,48).…”

mentioning

confidence: 99%

Lipid-dependent Recruitment of Neuronal Src to Lipid Rafts in the Brain

Mukherjee

Arnaud

Cooper

2003

Although most Src family tyrosine kinases are modified by palmitoylation as well as myristoylation, Src itself is only myristoylated. Dual acylation is important for attachment to liquid-ordered microdomains or lipid rafts. Accordingly, Src is excluded from lipid rafts in fibroblasts. Evidence of partial genetic redundancy between Src and Fyn for brain-specific targets suggests that these two kinases may occupy overlapping subcellular locations. Neuronal Src (NSrc), an alternative isoform of Src with a 6-amino acid insert in the Src homology 3 domain, is highly expressed in neurons. We investigated whether this structural difference in NSrc allows it to associate with lipid rafts. We found that perinatal mouse brains express predominantly NSrc, which is partly (10 -20%) in a lipid raft fraction from brain but not fibroblasts. The association of Src with brain lipid rafts does not depend on the NSrc insert but depends on the amino-terminal myristoylation signal. A crude lipid fraction from brain promotes NSrc entry into rafts in vitro. Moreover, lipid raft-localized NSrc is more catalytically active than NSrc from the soluble fraction, possibly because raft localization alters access to other tyrosine kinases and phosphatases. These findings suggest that NSrc may be involved in signaling from lipid rafts in mouse brain.The Src family of nonreceptor tyrosine kinases (SFKs) 1 is broadly expressed and involved in many cell surface receptormediated signaling cascades (for review, see Refs. 1 and 2). Elucidation of normal SFK function has been difficult because most stimuli that activate SFKs also activate non-SFK tyrosine kinases. Moreover, redundancy within the SFK family has confounded attributing a specific signaling process exclusively to one kinase. Gene disruption studies have sometimes revealed exclusive functions for specific SFKs in certain cell types, Src in osteoclasts (3) and Lck in T cells (4) for instance, but, for the most part, deficiency of one SFK is compensated by others (5).The subcellular localizations of SFKs have provided valuable clues toward understanding their functions. Src in focal adhesions plays a key role in integrin-dependent signaling events that affect cellular adhesion and motility (6 -8), and Lck is recruited to endosomes in CD2-activated T cells where it is involved in CD2 receptor internalization (9). Localization of SFKs to various subcellular locations can be affected by protein-protein interactions involving their SH3 or SH2 domains (7, 10). However, lipid-lipid interactions involving amino-terminal acyl groups on SFKs are the primary mechanism for membrane localization of SFKs (11-14), particularly localization to membrane microdomains or lipid rafts.Lipid rafts are "liquid-ordered" microdomains in cell membranes (15, 16) which have been shown to exist in live cells at 37°C (17-19). Enriched in cholesterol, sphingolipids, and phosphoinositides, these membrane microdomains contain proteins involved in vesicular trafficking and signal transduction. Lipid rafts often contain s...

“…4 and 8). Although the role of phosphorylated caveolins in signal transduction is not well understood, we hypothesize that such phosphorylation is important for maintaining caveolin hetero-and homo-oligomers (29,52) and for scaffolding and recruitment of signaling components (53,54) to caveolin-associated complexes, perhaps by stabilizing multiprotein complexes between caveolar resident and cytoskeletal proteins (i.e. tubulin and filamin), thereby optimizing cell signaling.…”

Section: Discussionmentioning

confidence: 99%

Microtubules and Actin Microfilaments Regulate Lipid Raft/Caveolae Localization of Adenylyl Cyclase Signaling Components

Head

Patel

Roth

et al. 2006

240

205

Microtubules and actin filaments regulate plasma membrane topography, but their role in compartmentation of caveolae-resident signaling components, in particular G protein-coupled receptors (GPCR) and their stimulation of cAMP production, has not been defined. We hypothesized that the microtubular and actin cytoskeletons influence the expression and function of lipid rafts/caveolae, thereby regulating the distribution of GPCR signaling components that promote cAMP formation. Depolymerization of microtubules with colchicine (Colch) or actin microfilaments with cytochalasin D (CD) dramatically reduced the amount of caveolin-3 in buoyant (sucrose density) fractions of adult rat cardiac myocytes. Colch or CD treatment led to the exclusion of caveolin-1, caveolin-2, ␤ 1 -adrenergic receptors (␤ 1 -AR), ␤ 2 -AR, G␣ s , and adenylyl cyclase (AC) 5/6 from buoyant fractions, decreasing AC 5/6 and tyrosine-phosphorylated caveolin-1 in caveolin-1 immunoprecipitates but in parallel increased isoproterenol (␤-AR agonist)-stimulated cAMP production. Incubation with Colch decreased co-localization (by immunofluorescence microscopy) of caveolin-3 and ␣-tubulin; both Colch and CD decreased co-localization of caveolin-3 and filamin (an F-actin cross-linking protein), decreased phosphorylation of caveolin-1, Src, and p38 MAPK, and reduced the number of caveolae/m of sarcolemma (determined by electron microscopy). Treatment of S49 T-lymphoma cells (which possess lipid rafts but lack caveolae) with CD or Colch redistributed a lipid raft marker (linker for activation of T cells (LAT)) and G␣ s from lipid raft domains. We conclude that microtubules and actin filaments restrict cAMP formation by regulating the localization and interaction of GPCR-G s -AC in lipid rafts/caveolae.