Shengwen Li

Identification of Peptide and Protein Ligands for the Caveolin-scaffolding Domain IMPLICATIONS FOR THE INTERACTION OF CAVEOLIN WITH CAVEOLAE-ASSOCIATED PROTEINS

Caveolin, a 21-24-kDa integral membrane protein, is a principal component of caveolae membranes. We have suggested that caveolin functions as a scaffolding protein to organize and concentrate certain caveolin-interacting proteins within caveolae membranes. In this regard, caveolin co-purifies with a variety of lipid-modified signaling molecules, including G-proteins, Src-like kinases, Ha-Ras, and eNOS. Using several independent approaches, it has been shown that a 20-amino acid membrane proximal region of the cytosolic amino-terminal domain of caveolin is sufficient to mediate these interactions. For example, this domain interacts with G-protein α subunits and Src-like kinases and can functionally suppress their activity. This caveolinderived protein domain has been termed the caveolin-scaffolding domain. However, it remains unknown how the caveolin-scaffolding domain recognizes these molecules. Here, we have used the caveolin-scaffolding domain as a receptor to select random peptide ligands from phage display libraries. These caveolin-selected peptide ligands are rich in aromatic amino acids and have a characteristic spacing in many cases. A known caveolin-interacting protein, Gi2α, was used as a ligand to further investigate the nature of this interaction. Gi2α and other G-protein α subunits contain a single region that generally resembles the sequences derived from phage display. We show that this short peptide sequence derived from Gi2α interacts directly with the caveolin-scaffolding domain and competitively inhibits the interaction of the caveolin-scaffolding domain with the appropriate region of Gi2α. This interaction is strictly dependent on the presence of aromatic residues within the peptide ligand, as replacement of these residues with alanine or glycine prevents their interaction with the caveolin-scaffolding domain. In addition, we have used this interaction to define which residues within the caveolin-scaffolding domain are critical for recognizing these peptide and protein ligands. Also, we find that the scaffolding domains of caveolins 1 and 3 both recognize the same peptide ligands, whereas the corresponding domain within caveolin-2 fails to recognize these ligands under the same conditions. These results serve to further demonstrate the specificity of this interaction. The implications of our current findings are discussed regarding other caveolin- and caveolae-associated proteins.

Dissecting the interaction between nitric oxide synthase (NOS) and caveolin. Functional significance of the nos caveolin binding domain in vivo.

Endothelial nitric oxide synthase (eNOS) is a dually acylated peripheral membrane protein that targets to the Golgi region and caveolae of endothelial cells. Recent evidence has shown that eNOS can co-precipitate with caveolin-1, the resident coat protein of caveolae, suggesting a direct interaction between these two proteins. To test this idea, we examined the interactions of eNOS with caveolin-1 in vitro and in vivo. Incubation of endothelial cell lysates or purified eNOS with glutathioneS-transferase (GST)-caveolin-1 resulted in the direct interaction of the two proteins. Utilizing a series of GST-caveolin-1 deletion mutants, we identified two cytoplasmic domains of caveolin-1 that interact with eNOS, the scaffolding domain (amino acids 61–101) and to a lesser extent the C-terminal tail (amino acids 135–178). Incubation of pure eNOS with peptides derived from the scaffolding domains of caveolin-1 and -3, but not the analogous regions from caveolin-2, resulted in inhibition of eNOS, inducible NOS (iNOS), and neuronal NOS (nNOS) activities. These results suggest a common mechanism and site of inhibition. Utilizing GST-eNOS fusions, the site of caveolin binding was localized between amino acids 310 and 570. Site-directed mutagenesis of the predicted caveolin binding motif within eNOS blocked the ability of caveolin-1 to suppress NO release in co-transfection experiments. Thus, our data demonstrate a novel functional role for caveolin-1 in mammalian cells as a potential molecular chaperone that directly inactivates NOS. This suggests that the direct binding of eNOS to caveolin-1, per se, and the functional consequences of eNOS targeting to caveolae are likely temporally and spatially distinct events that regulate NO production in endothelial cells. Additionally, the inactivation of eNOS and nNOS by the scaffolding domain of caveolin-3 suggests that eNOS in cardiac myocytes and nNOS in skeletal muscle are likely subject to negative regulation by this muscle-specific caveolin isoform.

Src Tyrosine Kinases, Gα Subunits, and H-Ras Share a Common Membrane-anchored Scaffolding Protein, Caveolin CAVEOLIN BINDING NEGATIVELY REGULATES THE AUTO-ACTIVATION OF Src TYROSINE KINASES

Caveolae are plasma membrane specializations present in most cell types. Caveolin, a 22-kDa integral membrane protein, is a principal structural and regulatory component of caveolae membranes. Previous studies have demonstrated that caveolin co-purifies with lipid modified signaling molecules, including Gα subunits, H-Ras, c-Src, and other related Src family tyrosine kinases. In addition, it has been shown that caveolin interacts directly with Gα subunits and H-Ras, preferentially recognizing the inactive conformation of these molecules. However, it is not known whether caveolin interacts directly or indirectly with Src family tyrosine kinases. Here, we examine the structural and functional interaction of caveolin with Src family tyrosine kinases. Caveolin was recombinantly expressed as a glutathione S-transferase fusion. Using an established in vitro binding assay, we find that caveolin interacts with wild-type Src (c-Src) but does not form a stable complex with mutationally activated Src (v-Src). Thus, it appears that caveolin prefers the inactive conformation of Src. Deletion mutagenesis indicates that the Src-interacting domain of caveolin is located within residues 82-101, a cytosolic membrane-proximal region of caveolin. A caveolin peptide derived from this region (residues 82-101) functionally suppressed the auto-activation of purified recombinant c-Src tyrosine kinase and Fyn, a related Src family tyrosine kinase. We further analyzed the effect of caveolin on c-Src activity in vivo by transiently co-expressing full-length caveolin and c-Src tyrosine kinase in 293T cells. Co-expression with caveolin dramatically suppressed the tyrosine kinase activity of c-Src as measured via an immune complex kinase assay. Thus, it appears that caveolin structurally and functionally interacts with wild-type c-Src via caveolin residues 82-101. Besides interacting with Src family kinases, this cytosolic caveolin domain (residues 82-101) has the following unique features. First, it is required to form multivalent homo-oligomers of caveolin. Second, it interacts with G-protein α-subunits and down-regulates their GTPase activity. Third, it binds to wild-type H-Ras. Fourth, it is membrane-proximal, suggesting that it may be involved in other potential protein-protein interactions. Thus, we have termed this 20-amino acid stretch of caveolin residues the caveolin scaffolding domain.

Flotillins/Cavatellins Are Differentially Expressed in Cells and Tissues and Form a Hetero-oligomeric Complex with Caveolins in Vivo CHARACTERIZATION AND EPITOPE-MAPPING OF A NOVEL FLOTILLIN-1 MONOCLONAL ANTIBODY PROBE

Caveolae are vesicular organelles that represent a subcompartment of the plasma membrane. Caveolins and flotillins are two families of mammalian caveolae-associated integral membrane proteins. However, it remains unknown whether flotillins interact with caveolin proteins to form a stable caveolar complex or if expression of flotillins can drive vesicle formation. Here, we examine the cell type and tissue-specific expression of the flotillin gene family. For this purpose, we generated a novel monoclonal antibody probe that recognizes only flotillin-1. A survey of cell and tissue types demonstrates that flotillins 1 and 2 have a complementary tissue distribution. At the cellular level, flotillin-2 was ubiquitously expressed, whereas flotillin-1 was most abundant in A498 kidney cells, muscle cell lines, and fibroblasts. Using three different models of cellular differentiation, we next examined the expression of flotillins 1 and 2. Taken together, our data suggest that the expression levels of flotillins 1 and 2 are independently regulated and does not strictly correlate with known expression patterns of caveolin family members. However, when caveolins and flotillins are co-expressed within the same cell, as in A498 cells, they form a stable hetero-oligomeric “caveolar complex.” In support of these observations, we show that heterologous expression of murine flotillin-1 in Sf21 insect cells using baculovirus-based vectors is sufficient to drive the formation of caveolae-like vesicles. These results suggest that flotillins may participate functionally in the formation of caveolae or caveolae-like vesicles in vivo. Thus, flotillin-1 represents a new integral membrane protein marker for the slightly larger caveolae-related domains (50–200 nm) that are observed in cell types that fail to express caveolin-1. As a consequence of these findings, we propose the term “cavatellins” be used (instead of flotillins) to describe this gene family.

Akt1 governs breast cancer progression in vivo

The serine threonine kinase Akt1 has been implicated in the control of cellular metabolism, survival and growth. Here, disruption of the ubiquitously expressed member of the Akt family of genes, Akt1, in the mouse demonstrates a requirement for Akt1 in ErbB2-induced mammary tumorigenesis. Akt1 deficiency delayed tumor growth and reduced lung metastases, correlating with a reduction in phosphorylation of the Akt1 target, tuberous sclerosis 2 (TSC2) at Ser-939. Akt1-deficient mammary epithelial tumor cells (MEC) were reduced in size and proliferative capacity, with reduced cyclin D1 and p27KIP1 abundance. Akt1 deficiency abrogated the oncogene-induced changes in polarization of MEC in three-dimensional culture and reverted oncogene-induced relocalization of the phosphorylated ezrin–radixin–moesin proteins. Akt1 increased MEC migration across an endothelial cell barrier, enhancing the persistence of migratory directionality. An unbiased proteomic analysis demonstrated Akt1 mediated MEC migration through paracrine signaling via induction of expression and secretion of CXCL16 and MIP1γ. Akt1 governs MEC polarity, migratory directionality and breast cancer onset induced by ErbB2 in vivo.

Mutational Analysis of the Properties of Caveolin-1 A NOVEL ROLE FOR THE C-TERMINAL DOMAIN IN MEDIATING HOMO-TYPIC CAVEOLIN-CAVEOLIN INTERACTIONS

Caveolin is a principal structural component of caveolae membranes in vivo Recently, a family of caveolin-related proteins has been identified; caveolin has been retermed caveolin-1. Caveolin family members share three characteristic properties: (i) detergent insolubility at low temperatures; (ii) self-oligomerization; and (iii) incorporation into low density Triton-insoluble fractions enriched in caveolae membranes. Here, we have used a deletion mutagenesis approach as a first step toward understanding which regions of caveolin-1 contribute to its unusual properties. Two caveolin-1 deletion mutants were created that lack either the C-terminal domain (Cav-1ΔC) or the N-terminal domain (Cav-1ΔN); these mutants were compared with the behavior of full-length caveolin-1 (Cav-1FL) expressed in parallel. Our results show that the N-terminal domain and membrane spanning segment are sufficient to form high molecular mass oligomers of caveolin-1. However, a complete caveolin-1 molecule is required for conveying detergent insolubility and incorporation into low density Triton-insoluble complexes. These data indicate that homo-oligomerization and an intact transmembrane are not sufficient to confer detergent insolubility, suggesting an unknown role for the C-terminal domain in this process. To better understand the role of the C-terminal domain, this region of caveolin-1 (residues 135-178) was expressed as a glutathione S-transferase fusion protein in Escherichia coli Purified recombinant glutathione S-transferase-C-Cav-1 was found to stably interact with full-length caveolin-1 but not with the two caveolin-1 deletion mutants. These results suggest that the C-terminal domain interacts with both the N-terminal and C-terminal domains of an adjacent caveolin-1 homo-oligomer. This appears to be a specific homo-typic interaction, because the C-terminal domain of caveolin-1 failed to interact with full-length forms of caveolin-2 and caveolin-3. Homo-typic interaction of the C-terminal domain with an adjacent homo-oligomer could provide a mechanism for clustering caveolin-1 homo-oligomers while excluding other caveolin family members. This type of lateral segregation event could promote caveolae membrane formation and contribute to the detergent insolubility of caveolins-1, −2, and −3.

Baculovirus-based Expression of Mammalian Caveolin in Sf21 Insect Cells A MODEL SYSTEM FOR THE BIOCHEMICAL AND MORPHOLOGICAL STUDY OF CAVEOLAE BIOGENESIS

Caveolae were originally defined morphologically as 50-100 nm noncoated vesicular organelles located at or near the plasma membrane. Caveolin, a vesicular integral membrane protein of 21 kDa, is a principal protein component of caveolae membranes in vivo. Caveolin interacts with itself to form high molecular mass oligomers, suggesting that it might play a structural role in the formation of caveolae membranes. However, it remains controversial whether recombinant expression of caveolin is necessary or sufficient to generate caveolae membranes in vivo. To directly address this issue, we have taken a different experimental approach by exploiting a heterologous expression system. Here, we have recombinantly expressed mammalian caveolin in Sf21 insect cells using baculovirus-based vectors. Two isoforms of caveolin have been identified that differ at their extreme N terminus; α-caveolin contains residues 1-178, and β-caveolin contains residues 32-178. After recombinant expression in Sf21 insect cells, both α- and β-caveolin formed SDS-resistant high molecular mass oligomers of the same size as native caveolin. Morphologically, expression of either caveolin isoform resulted in the intracellular accumulation of a homogeneous population of caveolae-sized vesicles with a diameter between 50 and 120 nm (80.3 ± 14.8 nm). This indicates that each caveolin isoform can independently generate these structures and that caveolin residues 1-31 are not required for this process. Using caveolin as a marker protein and a detergent-free procedure to purify caveolae from mammalian cells, we purified these recombinant caveolin-induced vesicles from insect cells. These purified recombinant vesicles: (i) have the same buoyant density as mammalian caveolae; (ii) appear as ∼50-100 nm membranous structures by whole-mount electron microscopy; and (iii) contain ∼95% of the recombinantly expressed caveolin protein by Western blotting. Immuno-labeling of these structures with anti-caveolin IgG confirmed that they contain caveolin. Thus, ectopic overexpression of caveolin in this heterologous system is sufficient to drive the formation of caveolae-like vesicles. Further functional analysis demonstrated that caveolin was capable of interacting with a known caveolin-interacting protein, Ha-Ras, when coexpressed in insect cells by co-infection with two recombinant baculoviruses. Taken together, our results demonstrate that baculovirus-based expression of caveolin in insect cells provides an attractive experimental system for studying the biogenesis of caveolae.

Mutational analysis of caveolin-induced vesicle formation Expression of caveolin-1 recruits caveolin-2 to caveolae membranes

Caveolae are vesicular organelles with a characteristic uniform diameter in the range of 50–100 nm. Although recombinant expression of caveolin‐1 is sufficient to drive caveolae formation, it remains unknown what controls the uniform diameter of these organelles. One hypothesis is that specific caveolin‐caveolin interactions regulate the size of caveolae, as caveolin‐1 undergoes two stages of self‐oligomerization. To test this hypothesis directly, we have created two caveolin‐1 deletion mutants that lack regions of caveolin‐1 that are involved in directing the self‐assembly of caveolin‐1 oligomers. More specifically, Cav‐1 Δ61–100 lacks a region of the N‐terminal domain that directs the formation of high molecular mass caveolin‐1 homo‐oligomers, while Cav‐1 ΔC lacks a complete C‐terminal domain that is required to allow caveolin homo‐oligomers to interact with each other, forming a caveolin network. It is important to note that these two mutants retain an intact transmembrane domain. Our current results show that although Cav‐1 Δ61–100 and Cav‐1 ΔC are competent to drive vesicle formation, these vesicles vary widely in their size and shape with diameters up to 500–1000 nm. In addition, caveolin‐induced vesicle formation appears to be isoform‐specific. Recombinant expression of caveolin‐2 under the same conditions failed to drive the formation of vesicles, while caveolin‐3 expression yielded caveolae‐sized vesicles. These results are consistent with the previous observation that in transformed NIH 3T3 cells that lack caveolin‐1 expression, but continue to express caveolin‐2, no morphologically distinguishable caveolae are observed. In addition, as caveolin‐2 alone exists mainly as a monomer or homo‐dimer, while caveolins 1 and 3 exist as high molecular mass homo‐oligomers, our results are consistent with the idea that the formation of high molecular mass oligomers of caveolin are required to regulate the formation of uniform caveolae‐sized vesicles. In direct support of this notion, regulated induction of caveolin‐1 expression in transformed NIH 3T3 cells was sufficient to recruit caveolin‐2 to caveolae membranes. The ability of caveolin‐1 to recruit caveolin‐2 most likely occurs through a direct interaction between caveolins 1 and 2, as caveolins 1 and 2 are normally co‐expressed and interact with each other to form high molecular mass hetero‐oligomers containing both caveolins 1 and 2.

The functional significance of nuclear receptor acetylation

The endocrine signaling governing nuclear receptor (NR) function has been known for several decades to play a crucial role in the onset and progression of several tumor types. Notably among these are the estrogen receptor (ER) in breast cancer and androgen receptor (AR) in prostate cancer. Other nuclear receptors may be involved in cancer progression including the peroxisome-proliferator activating receptor gamma (PPARgamma), which has been implicated in breast, thyroid, and colon cancers. These NR are phylogenetically conserved modular transcriptional regulators, which like histones, undergo post-translational modification by acetylation, phosphorylation and ubiquitination. Importantly, the transcriptional activity of the receptors is governed by the coactivator p300, the activity of which is thought to be rate-limiting in the activity of these receptors. Histone acetyltransferases (HATs) and histone deacetylases (HDACs), modify histones by adding or removing an acetyl group from the epsilon amino group of lysines within an evolutionarily conserved lysine motif. Histone acetylation results in changes in chromatin structure in response to specific signals. These enzymes can also directly catalyze the NRs themselves, thus modifying signals at the receptor level. The post-translational modification of NR which is regulated by hormones, alters the NR function toward a growth promoting receptor. The deacetylation of NR is mediated by TSA-sensitive and NAD-dependent deacetylases. The regulation of NR by NAD-dependent enzymes provides a direct link between intracellular metabolism and hormone signaling.

Recombinant Expression of the MAL Proteolipid, a Component of Glycolipid-enriched Membrane Microdomains, Induces the Formation of Vesicular Structures in Insect Cells

The MAL proteolipid has been identified as a component of glycolipid-enriched membrane microdomains resistant to detergent solubilization in epithelial Madin-Darby canine cells, as well as in T lymphocytes and in myelin-forming cells. To study the function of the MAL proteolipid we have ectopically expressed a tagged form of MAL in both mammalian and insect cellular backgrounds. Immunofluorescence analysis in transiently transfected COS-7 cells showed the presence of MAL in large vesicular structures, and biochemical analysis identified MAL in the fraction of membranes resistant to Triton X-100 solubilization. Electron microscopic analysis showed that the expression of MAL in Sf21 cells morphologically resulted in the intracellular accumulation of large vesicles with a diameter from 200 to greater than 700 nm that were absent in uninfected or control infected cultures. Thus, ectopic expression of MAL in this heterologous expression system was sufficient to drive the formation of vesicles with a size similar to that of the vesicles detected in mammalian cells. These vesicles were clearly different from the caveolae-like vesicles induced by caveolin expression, as evidenced by co-infection experiments using a recombinant caveolin baculovirus. Taken together, these results suggest that the MAL proteolipid might play a role as a component of the machinery of vesiculation of glycolipid-enriched membranes.