Anil Chawla

TGFβ receptor internalization into EEA1-enriched early endosomes: role in signaling to Smad2

Transforming growth factor (TGF)β is an important physiological regulator of cellular growth and differentiation. It activates a receptor threonine/serine kinase that phosphorylates the transcription factor Smad2, which then translocates into the nucleus to trigger specific transcriptional events. Here we show that activated type I and II TGFβ receptors internalize into endosomes containing the early endosomal protein EEA1. The extent of TGFβ-stimulated Smad2 phosphorylation, Smad2 nuclear translocation, and TGFβ-stimulated transcription correlated closely with the extent of internalization of the receptor. TGFβ signaling also requires SARA (Smad anchor for receptor activation), a 135-kD polypeptide that contains a FYVE Zn++ finger motif. Here we show that SARA localizes to endosomes containing EEA1, and that disruption of this localization inhibits TGFβ-induced Smad2 nuclear translocation. These results indicate that traffic of the TGFβ receptor into the endosome enables TGFβ signaling, revealing a novel function for the endosome as a compartment specialized for the amplification of certain extracellular signals.

Cidea is associated with lipid droplets and insulin sensitivity in humans

Storage of energy as triglyceride in large adipose-specific lipid droplets is a fundamental need in all mammals. Efficient sequestration of fat in adipocytes also prevents fatty acid overload in skeletal muscle and liver, which can impair insulin signaling. Here we report that the Cide domain-containing protein Cidea, previously thought to be a mitochondrial protein, colocalizes around lipid droplets with perilipin, a regulator of lipolysis. Cidea-GFP greatly enhances lipid droplet size when ectopically expressed in preadipocytes or COS cells. These results explain previous findings showing that depletion of Cidea with RNAi markedly elevates lipolysis in human adipocytes. Like perilipin, Cidea and the related lipid droplet protein Cidec/FSP27 are controlled by peroxisome proliferator-activated receptor γ (PPARγ). Treatment of lean or obese mice with the PPARγ agonist rosiglitazone markedly up-regulates Cidea expression in white adipose tissue (WAT), increasing lipid deposition. Strikingly, in both omental and s.c. WAT from BMI-matched obese humans, expression of Cidea, Cidec/FSP27, and perilipin correlates positively with insulin sensitivity (HOMA-IR index). Thus, Cidea and other lipid droplet proteins define a novel, highly regulated pathway of triglyceride deposition in human WAT. The data support a model whereby failure of this pathway results in ectopic lipid accumulation, insulin resistance, and its associated comorbidities in humans.

A functional PtdIns(3)P-binding motif.

Treating cells with the phosphatidylinositol-3-OH kinase (PI(3)K) inhibitor wortmannin causes the dissociation of the early-endosomal antigen EEA1 from early endosomes. EEA1 from cytosolic extracts binds to liposomes containing phosphatidylinositol-3-phosphate (PtdIns(3)P), the major product of PI(3)K in yeast and mammalian cells,. Here we show that a RING zinc-finger domain at the carboxy terminus of EEA1, previously identified and named the ‘FYVE’ domain, binds directly and specifically to PtdIns(3)P. This indicates that proteins containing this motif may be downstream effectors of PI(3)K in yeast and mammalian cells.

Structural Basis of 3-Phosphoinositide Recognition by Pleckstrin Homology Domains

Lipid second messengers generated by phosphoinositide (PI) 3-kinases regulate diverse cellular functions through interaction with pleckstrin homology (PH) domains in modular signaling proteins. The PH domain of Grp1, a PI 3-kinase-activated exchange factor for Arf GTPases, selectively binds phosphatidylinositol 3,4,5-trisphosphate with high affinity. We have determined the structure of the Grp1 PH domain in the unliganded form and bound to inositol 1,3,4,5-tetraphosphate. A novel mode of phosphoinositide recognition involving a 20-residue insertion within the beta6/beta7 loop explains the unusually high specificity of the Grp1 PH domain and the promiscuous 3-phosphoinositide binding typical of several PH domains including that of protein kinase B. When compared to other PH domains, general determinants of 3-phosphoinositide recognition and specificity can be deduced.

Fat-specific Protein 27, a Novel Lipid Droplet Protein That Enhances Triglyceride Storage

Fat-specific protein (FSP)27/Cidec is most highly expressed in white and brown adipose tissues and increases in abundance by over 50-fold during adipogenesis. However, its function in adipocytes has remained elusive since its discovery over 15 years ago. Here we demonstrate that FSP27/Cidec localizes to lipid droplets in cultured adipocytes and functions to promote lipid accumulation. Ectopically expressed FSP27-GFP surrounds lipid droplets in 3T3-L1 adipocytes and colocalizes with the known lipid droplet protein perilipin. Immunostaining of endogenous FSP27 in 3T3-L1 adipocytes also confirmed its presence on lipid droplets. FSP27-GFP expression also markedly increases lipid droplet size and enhances accumulation of total neutral lipids in 3T3-L1 preadipocytes as well as other cell types such as COS cells. Conversely, RNA interference-based FSP27/Cidec depletion in mature adipocytes significantly stimulates lipolysis and reduces the size of lipid droplets. These data reveal FSP27/Cidec as a novel adipocyte lipid droplet protein that negatively regulates lipolysis and promotes triglyceride accumulation.

Partial lipodystrophy and insulin resistant diabetes in a patient with a homozygous nonsense mutation in CIDEC

Lipodystrophic syndromes are characterized by adipose tissue deficiency. Although rare, they are of considerable interest as they, like obesity, typically lead to ectopic lipid accumulation, dyslipidaemia and insulin resistant diabetes. In this paper we describe a female patient with partial lipodystrophy (affecting limb, femorogluteal and subcutaneous abdominal fat), white adipocytes with multiloculated lipid droplets and insulin‐resistant diabetes, who was found to be homozygous for a premature truncation mutation in the lipid droplet protein cell death‐inducing Dffa‐like effector C (CIDEC) (E186X). The truncation disrupts the highly conserved CIDE‐C domain and the mutant protein is mistargeted and fails to increase the lipid droplet size in transfected cells. In mice, Cidec deficiency also reduces fat mass and induces the formation of white adipocytes with multilocular lipid droplets, but in contrast to our patient, Cidec null mice are protected against diet‐induced obesity and insulin resistance. In addition to describing a novel autosomal recessive form of familial partial lipodystrophy, these observations also suggest that CIDEC is required for unilocular lipid droplet formation and optimal energy storage in human fat.

Unconventional Myosin Myo1c Promotes Membrane Fusion in a Regulated Exocytic Pathway

ABSTRACT Glucose homeostasis is controlled in part by regulation of glucose uptake into muscle and adipose tissue. Intracellular membrane vesicles containing the GLUT4 glucose transporter move towards the cell cortex in response to insulin and then fuse with the plasma membrane. Here we show that the fusion step is retarded by the inhibition of phosphatidylinositol (PI) 3-kinase. Treatment of insulin-stimulated 3T3-L1 adipocytes with the PI 3-kinase inhibitor LY294002 causes the accumulation of GLUT4-containing vesicles just beneath the cell surface. This accumulation of GLUT4-containing vesicles near the plasma membrane prior to fusion requires an intact cytoskeletal network and the unconventional myosin motor Myo1c. Remarkably, enhanced Myo1c expression under these conditions causes extensive membrane ruffling and overrides the block in membrane fusion caused by LY294002, restoring the display of GLUT4 on the cell exterior. Ultrafast microscopic analysis revealed that insulin treatment leads to the mobilization of GLUT4-containing vesicles to these regions of Myo1c-induced membrane ruffles. Thus, localized membrane remodeling driven by the Myo1c motor appears to facilitate the fusion of exocytic GLUT4-containing vesicles with the adipocyte plasma membrane.

Conventional kinesin KIF5B mediates insulin‐stimulated GLUT4 movements on microtubules

Insulin stimulates glucose uptake in muscle and adipose cells by mobilizing intracellular membrane vesicles containing GLUT4 glucose transporter proteins to the plasma membrane. Here we show in live cultured adipocytes that intracellular membranes containing GLUT4–yellow fluorescent protein (YFP) move along tubulin–cyan fluorescent protein‐labeled microtubules in response to insulin by a mechanism that is insensitive to the phosphatidylinositol 3 (PI3)‐kinase inhibitor wortmannin. Insulin increased by several fold the observed frequencies, but not velocities, of long‐range movements of GLUT4–YFP on microtubules, both away from and towards the perinuclear region. Genomics screens show conventional kinesin KIF5B is highly expressed in adipocytes and this kinesin is partially co‐localized with perinuclear GLUT4. Dominant‐negative mutants of conventional kinesin light chain blocked outward GLUT4 vesicle movements and translocation of exofacial Myc‐tagged GLUT4–green fluorescent protein to the plasma membrane in response to insulin. These data reveal that insulin signaling targets the engagement or initiates the movement of GLUT4‐containing membranes on microtubules via conventional kinesin through a PI3‐kinase‐independent mechanism. This insulin signaling pathway regulating KIF5B function appears to be required for GLUT4 translocation to the plasma membrane.

Interactions of Drosophila Cbl with epidermal growth factor receptors and role of Cbl in R7 photoreceptor cell development.

The human proto-oncogene product c-Cbl and a similar protein in Caenorhabditis elegans (Sli-1) contain a proline-rich COOH-terminal region that binds Src homology 3 (SH3) domains of proteins such as the adapter Grb2. Cb1-Grb2 complexes can be recruited to tyrosine-phosphorylated epidermal growth factor (EGF) receptors through the SH2 domain of Grb2. Here we identify by molecular cloning a Drosophila cDNA encoding a protein (Drosophila Cbl [D-Cbl]) that shows high sequence similarity to the N-terminal region of human c-Cbl but lacks proline-rich sequences and fails to bind Grb2. Nonetheless, in COS-1 cells, expression of hemagglutinin epitope-tagged D-Cbl results in its coimmunoprecipitation with EGF receptors in response to EGF. EGF also caused tyrosine phosphorylation of D-Cbl in such cells, but no association of phosphatidylinositol 3-kinase was detected in assays using anti-p85 antibody. A point mutation in D-Cbl (G305E) that suppresses the negative regulation of LET-23 by the Cbl homolog Sli-1 in C. elegans prevented tyrosine phosphorylation of D-Cbl as well as binding to the liganded EGF receptor in COS-1 cells. Colocalization of EGF receptors with both endogenous c-Cbl or expressed D-Cbl in endosomes of EGF-treated COS-1 cells is also demonstrated by immunofluorescence microscopy. In lysates of adult transgenic Drosophila melanogaster, GST-DCbl binds to the tyrosine-phosphorylated 150-kDa torso-DER chimeric receptor. Expression of D-Cbl directed by the sevenless enhancer in intact Drosophila compromises severely the development of the R7 photoreceptor neuron. These data suggest that despite the lack of Grb2 binding sites, D-Cbl functions as a negative regulator of receptor tyrosine kinase signaling in the Drosophila eye by a mechanism that involves its association with EGF receptors or other tyrosine kinases.

Distinct polyphosphoinositide binding selectivities for pleckstrin homology domains of GRP1-like proteins based on diglycine versus triglycine motifs

GRP1 and the related proteins ARNO and cytohesin-1 are ARF exchange factors that contain a pleckstrin homology (PH) domain thought to target these proteins to cell membranes through binding polyphosphoinositides. Here we show the PH domains of all three proteins exhibit relatively high affinity for dioctanoyl phosphatidylinositol 3,4,5-triphosphate (PtdIns(3,4,5)P3), with KD values of 0.05, 1.6 and 1.0 μm for GRP1, ARNO, and cytohesin-1, respectively. However, the GRP1 PH domain was unique among these proteins in its striking selectivity for PtdIns(3,4,5)P3 versus phosphatidylinositol 4,5-diphosphate (PtdIns(4,5)P2), for which it exhibits about 650-fold lower apparent affinity. Addition of a glycine to the Gly274-Gly275 motif in GRP1 greatly increased its binding affinity for PtdIns(4,5)P2 with little effect on its binding to PtdIns(3,4,5)P3, while deletion of a single glycine in the corresponding triglycine motif of the ARNO PH domain markedly reduced its binding affinity for PtdIns(4,5)P2 but not for PtdIns(3,4,5)P3. In intact cells, the hemagglutinin epitope-tagged PH domain of GRP1 was recruited to ruffles in the cell surface in response to insulin, as were full-length GRP1 and cytohesin-1, but the PH domain of cytohesin-1 was not. These data indicate that the unique diglycine motif in the GRP1 PH domain, as opposed to the triglycine in ARNO and cytohesin-1, directs its remarkable PtdIns(3,4,5)P3 binding selectivity.