EHD proteins: key conductors of endocytic transport

Naslavsky, Naava; Caplan, Steve

doi:10.1016/j.tcb.2010.10.003

Cited by 211 publications

(276 citation statements)

References 95 publications

Supporting

Mentioning

269

Contrasting

Unclassified

Order By: Relevance

“…This new signaling link might underlie the reported negative regulation of EGFR tyrosine kinase activity in mitosis (36). Plk1-mediated EGFR regulation might be part of a concerted mechanism because we recorded elevated phosphorylation on Eps15 and Eps15R, known EGFR substrates with negative regulatory functions in EGFR signaling (37). Eps15 and Eps15R are involved in clathrin-mediated endocytosis, similar to the inositol 5-phosphatase synaptojanin-1, for which we detected enhanced phosphorylation at the Plk1 consensus site Ser 830 (38).…”

Section: Figmentioning

confidence: 61%

Combination of Chemical Genetics and Phosphoproteomics for Kinase Signaling Analysis Enables Confident Identification of Cellular Downstream Targets

Oppermann¹,

Grundner-Culemann²,

Kumar³

et al. 2012

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

Delineation of phosphorylation-based signaling networks requires reliable data about the underlying cellular kinasesubstrate interactions. We report a chemical genetics and quantitative phosphoproteomics approach that encompasses cellular kinase activation in combination with comparative replicate mass spectrometry analyses of cells expressing either inhibitor-sensitive or resistant kinase variant. We applied this workflow to Plk1 (Polo-like kinase 1) in mitotic cells and induced cellular Plk1 activity by wash-out of the bulky kinase inhibitor 3-MB-PP1, which targets a mutant kinase version with an enlarged catalytic pocket while not interfering with wild-type Plk1. We quantified more than 20,000 distinct phosphorylation sites by SILAC, approximately half of which were measured in at least two independent experiments in cells expressing mutant and wild-type Plk1. Based on replicate phosphorylation site quantifications in both mutant and wild-type Plk1 cells, our chemical genetic proteomics concept enabled stringent comparative statistics by significance analysis of microarrays, which unveiled more than 350 cellular downstream targets of Plk1 validated by full concordance of both statistical and experimental data. Our data point to hitherto poorly characterized aspects in Plk1-controlled mitotic progression and provide a largely extended resource for functional studies. We anticipate the described strategies to be of general utility for systematic and confident identification of cellular protein kinase substrates. Molecular & Cellular Proteomics 11: 10.1074/mcp.O111.012351, 1-12, 2012.Reversible protein phosphorylation by protein kinases represents a key control mechanism in signal transmission and controls nearly all aspects of cellular physiology. Quantitative proteomics approaches that incorporate techniques such as stable isotope labeling by amino acids in cell culture (SILAC), 1 phosphopeptide fractionation and enrichment by strong cation exchange (SCX), and ion metal affinity chromatography (IMAC) together with sensitive high resolution MS analysis and automated peptide identification and quantification have made it possible to monitor phosphorylation-based signaling on a global scale (1-4). Because signaling networks are defined by the underlying kinase-substrate relationships, systematic approaches are required for the comprehensive and confident assignment of cellular kinase substrates (5). To identify cellular substrates, the catalytic activity of a kinase of interest needs to be rapidly regulated to capture a high fraction of direct phosphorylation events. This implies that protein kinase ablation by genetic knockout or RNA interference can be of limited utility, because of secondary changes that can accumulate during the time required for cellular kinase depletion (3, 5). In contrast, pharmacological interference by small molecules allows for rapid modulation of kinase activity and should therefore enable unbiased monitoring of signaling perturbations when combined with advanced MS-based proteomics. S...

show abstract

Section: Figmentioning

confidence: 61%

Combination of Chemical Genetics and Phosphoproteomics for Kinase Signaling Analysis Enables Confident Identification of Cellular Downstream Targets

Oppermann¹,

Grundner-Culemann²,

Kumar³

et al. 2012

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

show abstract

“…Whether or not the interaction between syndapin and EHD has functional significance in nerve terminals is presently not clear. We note that many other proteins interact with EHDs (Naslavsky and Caplan, 2011).…”

Section: Discussionmentioning

confidence: 89%

“…The G-domain exhibits structural similarity to the G-domain of dynamin but binds ATP instead of GTP (Daumke et al, 2007). The EH domain of EHDs preferentially binds proteins that contain acidic residues following the NPF tripeptide (Braun et al, 2005;Naslavsky and Caplan, 2011). Initial functional studies have implicated EHD as a regulator of the exit from the endocytic recycling compartment Lin et al, 2001).…”

Section: Introductionmentioning

confidence: 99%

“…Initial functional studies have implicated EHD as a regulator of the exit from the endocytic recycling compartment Lin et al, 2001). Subsequently, EHD family members have been linked to multiple steps in endosomal trafficking pathways, as well as with other functions such as non-clathrin-mediated endocytosis of growth factor receptors and cell adhesion molecules (Naslavsky and Caplan, 2011). Recently, EHD1 was detected in nerve terminals (Wei et al, 2010), suggesting a possible involvement in synaptic vesicle trafficking.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Regulation of Synaptic Vesicle Budding and Dynamin Function by an EHD ATPase

Jakobsson

Ackermann²,

Andersson³

et al. 2011

J. Neurosci.

View full text Add to dashboard Cite

Eps15 homology domain-containing proteins (EHDs) are conserved ATPases implicated in membrane remodeling. Recently, EHD1 was found to be enriched at synaptic release sites, suggesting a possible involvement in the trafficking of synaptic vesicles. We have investigated the role of an EHD1/3 ortholog (l-EHD) in the lamprey giant reticulospinal synapse. l-EHD was detected by immunogold at endocytic structures adjacent to release sites. In antibody microinjection experiments, perturbation of l-EHD inhibited synaptic vesicle endocytosis and caused accumulation of clathrin-coated pits with atypical, elongated necks. The necks were covered with helix-like material containing dynamin. To test whether l-EHD directly interferes with dynamin function, we used fluid-supported bilayers as in vitro assay. We found that l-EHD strongly inhibited vesicle budding induced by dynamin in the constant presence of GTP. l-EHD also inhibited dynamin-induced membrane tubulation in the presence of GTP␥S, a phenomenon linked with dynamin helix assembly. Our in vivo results demonstrate the involvement of l-EHD in clathrin/dynamin-dependent synaptic vesicle budding. Based on our in vitro observations, we suggest that l-EHD acts to limit the formation of long, unproductive dynamin helices, thereby promoting vesicle budding.

show abstract

“…To extend our understanding of how transport through the early endosome influences integrin function, we investigated the role of two EE/SE-associated factors: (1) AAK1L (adaptor associated kinase 1 long form; Henderson and Conner, 2007), given the growing importance of kinases in integrin recycling (Roberts et al, 2004;Woods et al, 2004) and (2) EHD3, which belongs to the Eps15 homology domain (EHD) family of proteins, which serve key roles in endosomal sorting decisions (Naslavsky and Caplan, 2011;Naslavsky et al, 2006;Naslavsky et al, 2009). We employed a live cell imaging strategy to visualize the impact of AAK1L or EHD3 depletion on integrin transport in adherent cells and provide evidence demonstrating that avb3-mediated cell adhesion is maintained by integrin recycling through an AAK1L-and EHD3-dependent recycling pathway.…”

Section: Introductionmentioning

confidence: 99%

αvβ3 integrin-mediated adhesion is regulated through an AAK1L- and EHD3-dependent rapid recycling pathway

Waxmonsky

Conner

2013

Journal of Cell Science

View full text Add to dashboard Cite

SummaryProtein transport through the endosome is critical for maintaining proper integrin cell surface integrin distribution to support cell adhesion, motility and viability. Here we employ a live-cell imaging approach to evaluate the relationship between integrin function and transport through the early endosome. We discovered that two early endosome factors, AAK1L and EHD3, are critical for avb3-integrin-mediated cell adhesion in HeLa cells. siRNA-mediated depletion of either factor delays short-loop b3 integrin recycling from the early endosome back to the cell surface. Total internal reflection fluorescence-based colocalization analysis reveals that b3 integrin transits AAK1L-and EHD3-positive endosomes near the cell surface, a subcellular location consistent with a rapid-recycling role for both factors. Moreover, structure-function analysis reveals that AAK1L kinase activity, as well as its C-terminal domain, is essential for cell adhesion maintenance. Taken together, these data reveal an important role for AAK1L and EHD3 in maintaining cell viability and adhesion by promoting avb3 integrin rapid recycling from the early endosome.

show abstract

EHD proteins: key conductors of endocytic transport

Cited by 211 publications

References 95 publications

Combination of Chemical Genetics and Phosphoproteomics for Kinase Signaling Analysis Enables Confident Identification of Cellular Downstream Targets

Combination of Chemical Genetics and Phosphoproteomics for Kinase Signaling Analysis Enables Confident Identification of Cellular Downstream Targets

Regulation of Synaptic Vesicle Budding and Dynamin Function by an EHD ATPase

αvβ3 integrin-mediated adhesion is regulated through an AAK1L- and EHD3-dependent rapid recycling pathway

Contact Info

Product

Resources

About