Emilia Galperin

Three-chromophore FRET microscopy to analyze multiprotein interactions in living cells

Nearly every major process in a cell is carried out by assemblies of multiple dynamically interacting protein molecules. To study multi-protein interactions within such molecular machineries, we have developed a fluorescence microscopy method called three-chromophore fluorescence resonance energy transfer (3-FRET). This method allows analysis of three mutually dependent energy transfer processes between the fluorescent labels, such as cyan, yellow and monomeric red fluorescent proteins. Here, we describe both theoretical and experimental approaches that discriminate the parallel versus the sequential energy transfer processes in the 3-FRET system. These approaches were established in vitro and in cultured mammalian cells, using chimeric proteins consisting of two or three fluorescent proteins linked together. The 3-FRET microscopy was further applied to the analysis of three-protein interactions in the constitutive and activation-dependent complexes in single endosomal compartments. These data highlight the potential of 3-FRET microscopy in studies of spatial and temporal regulation of signaling processes in living cells.

Visualization of Rab5 activity in living cells by FRET microscopy and influence of plasma-membrane-targeted Rab5 on clathrin-dependent endocytosis.

Rab5 is a small GTPase that controls endocytosis and early endosome dynamics. To visualize active, GTP-loaded Rab5 in living cells, we developed molecular sensors consisting of the Rab5-binding fragments of Rabaptin5 or EEA.1 fused to yellow fluorescent protein (YFP). Interaction of these sensors with GTP-bound Rab5 fused to cyan fluorescent protein (CFP) resulted in fluorescence resonance energy transfer (FRET) between CFP and YFP. Activated Rab5 was detected by FRET microscopy in endosomal compartments and often concentrated in microdomains in the endosomal membrane. Although the plasma membrane-localized activity of Rab5 was not detected by light microscopy, overexpression of a GDP-bound mutant of CFP-Rab5(S34N) inhibited internalization of the epidermal growth factor receptor by retaining receptors in clathrin-coated pits. To test whether the Rab5(S34N) mutant affects endocytosis directly at the plasma membrane, CFP-Rab5 was fused to the plasma membrane targeting sequence of K-Ras containing a CAAX motif. The resulting chimeric CFP-Rab5-CAAX was located mainly in the plasma membrane and was capable of binding GTP as judged by FRET microscopy with the Rabaptin5-based sensor. Interestingly, EEA.1 sensor did not follow activated Rab5-CAAX to the plasma membrane, suggesting that the interaction of EEA.1 with Rab5 plays a secondary role in EEA.1 targeting. Overexpression of CFP-Rab5(S34N)CAAX prevented endocytosis of receptors by retaining them in coated pits. These data suggest that the dominant-negative effect of the Rab5(S34N) mutant on the late stages of endocytosis can be mediated through the inhibition of cytosol-associated or plasma-membrane-associated rather than endosome-associated regulators of Rab proteins.

EHD3: A Protein That Resides in Recycling Tubular and Vesicular Membrane Structures and Interacts with EHD1

Here we report the characterization of an eps15 homology (EH) domain containing protein designated EHD3. EHD3 was mapped to human chromosome 2p22–23, while the murine Ehd3 homolog was mapped to chromosome 17p21. Both the human and the mouse genes contain a polymorphic (CA) repeat in their 3′UTR. One 3.6‐kb Ehd3 transcript was mainly detected in adult mouse brain and kidney and at day 7 of mouse development. On the other hand, human tissues exhibited two, 4.2‐ and 3.6‐kb, EHD3 RNA species. They were predominantly expressed in heart, brain, placenta, liver, kidney and ovary. EHD3, expressed as a green fluorescent fusion protein was localized to endocytic vesicles and to microtubule‐dependent, membrane tubules. There was a clear colocalization of EHD3‐positive structures and transferrin‐containing recycling vesicles, implying that EHD3 resides within the endocytic recycling compartment. Shuffling the N‐terminal domain of EHD1 (previously shown to reside in the transferrin‐containing, endocytic recycling compartment) with that of EHD3 resulted in a chimeric EHD protein that was localized mainly to tubules instead of the endocytic vesicles, implicating the N‐terminal domain as responsible for the tubular localization of EHD3. Mutant EHD3 forms, missing the N‐terminal or the C‐terminal domains, lost their tubular localization. Results of two‐hybrid analyses indicated that EHD1 and EHD3 interact with each other. In addition, EHD1 and EHD3 could be coimmunoprecipitated from cellular extracts, confirming the interaction implied by two‐hybrid analysis. Moreover, coexpression of EHD1 and EHD3 resulted in their colocalization in microtubule‐dependent tubules as well as in punctate forms. Based on its specific intracellular localization and its interaction with EHD1, we postulate that EHD3 localizes on endocytic tubular and vesicular structures and regulates their microtubule‐dependent movement.

Recycling to the Plasma Membrane is Delayed in EHD1 Knockout Mice

EHD1 is a member of the EHD family that contains four mammalian homologs. Among the invertebrate orthologs are a single Drosophila and Caenorhabditis elegans proteins and two plant members. They all contain three modules, a N‐terminal domain that contains nucleotide‐binding motifs, a central coiled–coil domain involved in oligomerization and a C‐terminal region that harbors the EH domain. Studies in C. elegans and EHD1 depletion by RNA interference in human cells have demonstrated that it regulates recycling of membrane proteins. We addressed the physiological role of EHD1 through its inactivation in the mouse. Ehd1 knockout mice were indistinguishable from normal mice, had a normal life span and showed no histological abnormalities. Analysis of transferrin uptake in Ehd1–/– embryonic fibroblasts demonstrated delayed recycling to the plasma membrane with accumulation of transferrin in the endocytic recycling compartment. Our results corroborate the established role of EHD1 in the exit of membrane proteins from recycling endosomes in vivo in a mouse model.

Endosomal targeting of MEK2 requires RAF, MEK kinase activity and clathrin-dependent endocytosis.

To study spatiotemporal regulation of the mitogen‐activated protein kinase (MAPK)/extracellular signal‐regulated kinase (ERK1/2) signaling cascade in living cells, a HeLa cell line in which MAPK kinase of ERK kinase (MEK) 2 (MAPK kinase) was knocked down by RNA interference and replaced with the green fluorescent protein (GFP)‐tagged MEK2 was generated. In these cells, MEK2–GFP was stably expressed at a level similar to that of the endogenous MEK2 in the parental cells. Upon activation of the EGF receptor (EGFR), a pool of MEK2–GFP was found initially translocated to the plasma membrane and then accumulated in a subset of early and late endosomes. However, activated MEK was detected only at the plasma membrane and not in endosomes. Surprisingly, MEK2–GFP endosomes did not contain active EGFR, suggesting that endosomal MEK2–GFP was separated from the upstream signaling complexes. Knockdown of clathrin by small interfering RNA (siRNA) abolished MEK2 recruitment to endosomes but resulted in increased activation of ERK without affecting the activity of MEK2–GFP. The accumulation of MEK2–GFP in endosomes was also blocked by siRNA depletion of RAF kinases and by the MEK1/2 inhibitor, UO126. We propose that the recruitment of MEK2 to endosomes can be a part of the negative feedback regulation of the EGFR–MAPK signaling pathway by endocytosis.

Shoc2 Is Targeted to Late Endosomes and Required for Erk1/2 Activation in EGF-Stimulated Cells

Shoc2 is the putative scaffold protein that interacts with RAS and RAF, and positively regulates signaling to extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). To elucidate the mechanism by which Shoc2 regulates ERK1/2 activation by the epidermal growth factor (EGF) receptor (EGFR), we studied subcellular localization of Shoc2. Upon EGFR activation, endogenous Shoc2 and red fluorescent protein tagged Shoc2 were translocated from the cytosol to a subset of late endosomes containing Rab7. The endosomal recruitment of Shoc2 was blocked by overexpression of a GDP-bound H-RAS (N17S) mutant and RNAi knockdown of clathrin, suggesting the requirement of RAS activity and clathrin-dependent endocytosis. RNAi depletion of Shoc2 strongly inhibited activation of ERK1/2 by low, physiological EGF concentrations, which was rescued by expression of wild-type recombinant Shoc2. In contrast, the Shoc2 (S2G) mutant, that is myristoylated and found in patients with the Noonan-like syndrome, did not rescue ERK1/2 activation in Shoc2-depleted cells. Shoc2 (S2G) was not located in late endosomes but was present on the plasma membrane and early endosomes. These data suggest that targeting of Shoc2 to late endosomes may facilitate EGFR-induced ERK activation under physiological conditions of cell stimulation by EGF, and therefore, may be involved in the spatiotemporal regulation of signaling through the RAS-RAF module.

Identification of factors regulating the expression of the human glucocerebrosidase gene.

Gaucher disease, the most prevalent sphingolipid disorder, is characterized by an accumulation of sphingolipids mainly in cells of the reticuloendothelial cells, and is due to decreased activity of the lysosomal enzyme glucocerebrosidase (GCase). The corresponding gene is expressed differentially, namely in different cell types there are different GCase steady-state mRNA levels. Electrophoretic mobility shift assays, DNase footprinting and chloramphenicol acetyl transferase assays were employed in order to unravel some of the transcription factors responsible for the differential expression of the glucocerebrosidase (gcs) gene. The results indicate that OCTA binding protein, AP-1, PEA3 and a CAAT binding protein participate in regulating the expression of the gcs gene. The availability of the transcription factors seems to control the level of transcription of the gcs gene.

EVALUATION AND MOLECULAR CHARACTERIZATION OF EHD1, A CANDIDATE GENE FOR BARDET-BIEDL SYNDROME 1 (BBS1)

Bardet-Biedl Syndrome (BBS) is an autosomal recessive disorder characterized by developmental abnormalities including mental retardation, obesity, retinitis pigmentosa, polydactyly, short stature, and hypogenitalism. To date, five BBS loci have been identified. BBS1, located on 11q13, is reported to be the most prevalent form of BBS in the Caucasian population. A positional cloning approach is being used to identify the gene responsible for BBS1. EHD1, a new member of the EH-domain containing proteins, was identified in this study as lying within the BBS1 disease interval. RNA analysis of many tissues revealed that expression of EHD1 is ubiquitous, with elevated levels in the testis. The genomic structure of EHD1 was elucidated by direct BAC sequencing. Following identification of the intron/exon boundaries, mutational analysis was performed by single strand conformation polymorphism and direct sequencing of affected individuals from several large kindreds linked to the BBS1 locus, as well as a cohort of unrelated probands. No disease-causing mutations were identified in this analysis, but several polymorphisms were found.

Functional Integration of the Conserved Domains of Shoc2 Scaffold

Shoc2 is a positive regulator of signaling to extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). Shoc2 is also proposed to interact with RAS and Raf-1 in order to accelerate ERK1/2 activity. To understand the mechanisms by which Shoc2 regulates ERK1/2 activation by the epidermal growth factor receptor (EGFR), we dissected the role of Shoc2 structural domains in binding to its signaling partners and its role in regulating ERK1/2 activity. Shoc2 is comprised of two main domains: the 21 leucine rich repeats (LRRs) core and the N-terminal non-LRR domain. We demonstrated that the N-terminal domain mediates Shoc2 binding to both M-Ras and Raf-1, while the C-terminal part of Shoc2 contains a late endosomal targeting motif. We found that M-Ras binding to Shoc2 is independent of its GTPase activity. While overexpression of Shoc2 did not change kinetics of ERK1/2 activity, both the N-terminal and the LRR-core domain were able to rescue ERK1/2 activity in cells depleted of Shoc2, suggesting that these Shoc2 domains are involved in modulating ERK1/2 activity.

HUWE1 Is a Molecular Link Controlling RAF-1 Activity Supported by the Shoc2 Scaffold

ABSTRACT Scaffold proteins play a critical role in controlling the activity of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway. Shoc2 is a leucine-rich repeat scaffold protein that acts as a positive modulator of ERK1/2 signaling. However, the precise mechanism by which Shoc2 modulates the activity of the ERK1/2 pathway is unclear. Here we report the identification of the E3 ubiquitin ligase HUWE1 as a binding partner and regulator of Shoc2 function. HUWE1 mediates ubiquitination and, consequently, the levels of Shoc2. Additionally, we show that both Shoc2 and HUWE1 are necessary to control the levels and ubiquitination of the Shoc2 signaling partner, RAF-1. Depletion of HUWE1 abolishes RAF-1 ubiquitination, with corresponding changes in ERK1/2 pathway activity occurring. Our results indicate that the HUWE1-mediated ubiquitination of Shoc2 is the switch that regulates the transition from an active to an inactive state of the RAF-1 kinase. Taken together, our results demonstrate that HUWE1 is a novel player involved in regulating ERK1/2 signal transmission through the Shoc2 scaffold complex.