Journal: Scientific Reports

Article Title: Proteomics analysis identifies PEA-15 as an endosomal phosphoprotein that regulates α5β1 integrin endocytosis

doi: 10.1038/s41598-021-99348-z

Figure Lengend Snippet: Proteomics analysis reveals PEA-15 interacts with clathrin and AP-2 and integrin focal adhesion proteins. ( A ) Graphical representation of endosomal interacting partners identified in Table . PEA-15 (centered black circle) is the immunoprecipitated protein. The size of other circles is proportional to the number of peptides detected by mass spectroscopy from co-immunoprecipitated proteins. These circles are also grouped by function: adhesion (green), clathrin-mediated endocytosis (purple), caveolin-mediated endocytosis (orange), and exocytosis and golgi trafficking (blue). PEA-15 is a known regulator of integrin activity, and this is represented by a line between them. ( B ) The interaction of PEA-15 with clathrin and AP-2 was detected by a Proximity Ligation Assay (PLA). Red dots indicate interaction. Background control shows staining with PEA-15 antibodies alone. Scale bar = 25 µm, DAPI (blue) nuclear counter stain. The average number of red dots per cell was quantified using ImageJ and is shown as scatter plots with a line marking the median. Each point on the scatter plot is an average measure from a field of cells. PLA clathrin: n = 21 fields (601 cells). PLA AP-2: n = 12 fields (194 cells). PLA with antibodies only against PEA-15 was performed for the negative control. ( C ) U87MG lysate was used for immunoprecipitation of PEA-15 with anti-PEA-15 antibodies. Co-precipitated clathrin heavy chain was detected by Western blot. ( D ) Proteins that connect to PEA-15 in a cluster-filtered network of protein–protein interactions and a co-cluster correlation network as shown previously . The size and color of nodes is scaled to graph total phosphopeptides detected for each protein. White lines show proteins that co-cluster and are known to interact. Yellow lines connect positively correlating proteins; blue lines connect negatively correlating proteins.

Article Snippet: PEA-15-containing protein complexes were precipitated from whole cell lysates with anti-PEA15 antibodies (Pacific Immunology, Ramona, CA), or anti-HA antibodies (#51064-2-AP, Proteintech, Rosemont, IL) in the case of cells expressing HA-tagged PEA-15.

Techniques: Immunoprecipitation, Mass Spectrometry, Activity Assay, Proximity Ligation Assay, Staining, Negative Control, Western Blot

Journal: Scientific Reports

Article Title: Proteomics analysis identifies PEA-15 as an endosomal phosphoprotein that regulates α5β1 integrin endocytosis

doi: 10.1038/s41598-021-99348-z

Figure Lengend Snippet: PEA-15 co-traffics with Transferrin. ( A ) HeLa cells expressing GFP-tagged PEA-15 were incubated with Rhodamine-Transferrin for 2 min at 37 °C (Pulse). Non-internalized Transferrin was washed away before incubation at 37 °C in the presence of unlabeled transferrin (Chase). Immunofluorescence imaging shows the co-trafficking of GFP-PEA15 and chased Rhodamine-Transferrin to the perinuclear region. No trafficking was observed with GFP control. Scale bar in zoomed-out images = 25 µm. ( B ) Cells were processed as in Part A, but were also immunostained with antibodies against Rab5. Scale bar in zoomed-out images = 20 µm. Scale bar in zoomed-in images = 5 µm. ( C ) U87MG cells attached to fibronectin-coated glass coverslips for 30 min before co-immunostaining for endogenous PEA-15 and endogenous Rab5. Shown are maximum intensity projections from z-stacks acquired on a confocal microscope (XY) paired with orthogonal view (XZ). Arrows indicate perinuclear colocalization of PEA-15 with Rab5. ( D ) U87MG cells plated in normal growth conditions with the addition of primaquine were co-immunostained for endogenous PEA-15 and endogenous Rab5. Arrows indicate colocalization.

Article Snippet: PEA-15-containing protein complexes were precipitated from whole cell lysates with anti-PEA15 antibodies (Pacific Immunology, Ramona, CA), or anti-HA antibodies (#51064-2-AP, Proteintech, Rosemont, IL) in the case of cells expressing HA-tagged PEA-15.

Techniques: Expressing, Incubation, Immunofluorescence, Imaging, Immunostaining, Microscopy

Journal: Scientific Reports

Article Title: Proteomics analysis identifies PEA-15 as an endosomal phosphoprotein that regulates α5β1 integrin endocytosis

doi: 10.1038/s41598-021-99348-z

Figure Lengend Snippet: PEA-15 traffics to Rab5 endosomes. ( A ) HA-tagged PEA-15 was expressed in U87MG cells that were treated with primaquine or control. Cells were then fixed with paraformaldehyde and immunostained for PEA-15, Rab5 or the recycling endosome marker Rab11. Co-localization between PEA-15 and endosomal markers was measured by quantitation of the Pearson correlation using ImageJ software (* p < 0.005, n = 11 for each pairing). Enlarged vesicles co-staining for both HA-tagged PEA-15 and endosomal markers are indicated by arrows. Scale bar = 5 µm. ( B ) HA-tagged PEA-15 and mCherry-tagged Rab5 Q79L mutant were overexpressed in U87MG cells and immunostained with anti-HA antibodies for PEA-15. Co-localization of HA-PEA-15 and mCherry Rab5 Q79L occurred on ringed structures (arrows). Scale bar = 5 µm.

Article Snippet: PEA-15-containing protein complexes were precipitated from whole cell lysates with anti-PEA15 antibodies (Pacific Immunology, Ramona, CA), or anti-HA antibodies (#51064-2-AP, Proteintech, Rosemont, IL) in the case of cells expressing HA-tagged PEA-15.

Techniques: Marker, Quantitation Assay, Software, Staining, Mutagenesis

Journal: Scientific Reports

Article Title: Proteomics analysis identifies PEA-15 as an endosomal phosphoprotein that regulates α5β1 integrin endocytosis

doi: 10.1038/s41598-021-99348-z

Figure Lengend Snippet: PEA-15 is required for cell migration. ( A ) A pool of shRNAs were used to generate stable knock down of PEA-15 expression in U87MG. PEA-15 KD and scramble control spheroid invasion into a 3D matrix was then followed over time (Scale bar = 500 µm). Invasion by the cell mass was quantified with ImageJ (* p < 0.05, n = 3). ( B ) The viability of scramble and PEA-15 KD cells was measured by XTT assay (signal normalized to background, n = 3, no significant differences). ( C ) ECIS chambers were used to measure wound recovery overtime in KD cells transfected with wild type PEA-15, the S104A mutant, or the S116A mutant. Rescue of migration in KD cells was only detected in cells transfected with wild type PEA-15 (WT vs S104A: * p < 0.005, n = 4; WT vs S116A: * p < 0.005, n = 4).

Article Snippet: PEA-15-containing protein complexes were precipitated from whole cell lysates with anti-PEA15 antibodies (Pacific Immunology, Ramona, CA), or anti-HA antibodies (#51064-2-AP, Proteintech, Rosemont, IL) in the case of cells expressing HA-tagged PEA-15.

Techniques: Migration, Expressing, XTT Assay, Transfection, Mutagenesis

Journal: Scientific Reports

Article Title: Proteomics analysis identifies PEA-15 as an endosomal phosphoprotein that regulates α5β1 integrin endocytosis

doi: 10.1038/s41598-021-99348-z

Figure Lengend Snippet: PEA-15 interacts with β1 Integrin. ( A ). U87MG PEA-15 knock down cells were plated on Fibronectin-coated plates. The number of adhered cells was then quantified by crystal violet staining and is shown as absorbance at 590 nm (n = 4, p < 0.005). ( B ) Scramble and PEA-15 KD were cells grown under normal growth conditions for 24 h in plastic tissue culture dishes. Bright-field images were acquired and cell area and perimeter were recorded and used to calculate a roundness index (the dotted line indicates a value of 1, corresponding to a perfectly circular morphology). PEA-15 KD cells displayed a significantly rounder and spread morphology compared to control cells (* p < 0.005, n = 150). Scale bar = 50 µm. ( C ) Equal numbers of scramble and PEA-15 KD cells were seeded then grown to confluence in ECIS chambers. The baseline resistance of PEA-15 KD cells at confluence is significantly increased compared to scramble control (* p < 0.005, n = 4). ( D ) Recombinant β1 integrin cytoplasmic tails were incubated with lysate from HeLa cells expressing HA-tagged PEA-15. NPXA = β1 membrane proximal tyrosine mutated to alanine, αIIb = αIIb integrin cytoplasmic tail. HA-PEA-15 was detected in precipitates by Western blot. ( E ) HA-PEA-15 was expressed in PEA-15 KD cells. Cells were fixed and immunostained for HA tag and β1 integrin. Scale bar = 20 µm; zoomed scale bars = 5 µm.

Article Snippet: PEA-15-containing protein complexes were precipitated from whole cell lysates with anti-PEA15 antibodies (Pacific Immunology, Ramona, CA), or anti-HA antibodies (#51064-2-AP, Proteintech, Rosemont, IL) in the case of cells expressing HA-tagged PEA-15.

Techniques: Staining, Recombinant, Incubation, Expressing, Western Blot

Journal: Scientific Reports

Article Title: Proteomics analysis identifies PEA-15 as an endosomal phosphoprotein that regulates α5β1 integrin endocytosis

doi: 10.1038/s41598-021-99348-z

Figure Lengend Snippet: PEA-15 sorts β1 integrins to Rab5 endosomes. ( A ) Scramble and PEA-15 KD cells were allowed to adhere to FN-coated glass coverslips for 30 min. Cells were fixed and stained for β1 integrin, Rab5, and actin. Scale bar = 10 µm. Areas showing overlap between all three channels are indicated by white arrows. The Pearson correlation between β1 integrin and Rab5 endosomes was measured using ImageJ software (n = 37, * p < 0.05). ( B ) HeLa cells expressing HA-tagged PEA-15 were treated with or without GST-3FN9-11 (GST-911) and co-stained with antibodies against HA-tag and GST. Vesicular structures showing co-localized signal are indicated by blue arrows. Scale bar = 20 µm.

Article Snippet: PEA-15-containing protein complexes were precipitated from whole cell lysates with anti-PEA15 antibodies (Pacific Immunology, Ramona, CA), or anti-HA antibodies (#51064-2-AP, Proteintech, Rosemont, IL) in the case of cells expressing HA-tagged PEA-15.

Techniques: Staining, Software, Expressing

Journal: Scientific Reports

Article Title: Proteomics analysis identifies PEA-15 as an endosomal phosphoprotein that regulates α5β1 integrin endocytosis

doi: 10.1038/s41598-021-99348-z

Figure Lengend Snippet: PEA-15 regulates integrin trafficking. ( A ) Strip-resistant (internalized) biotinylated integrins were quantified by capture ELISA with antibodies against α5 integrin and expressed as a percentage of surface integrin absorbance signal. Shown is internalization after 5 min ( p = 0.2299, n = 2) and 15 min (* p < 0.05, n = 5) in the PEA-15 knock down cells compared to scramble control. Chased (recycled) biotinylated integrins were also quantified by capture ELISA. Shown is the percent α5 integrin recycled relative to pulse controls at 10 min (* p < 0.005, n = 4) and 20 min ( p = 0.5452, n = 3). Western blots show expression of α5 integrin, β1 integrin, Rab5 and Clathrin Heavy Chain, in the stable PEA-15 KD cells. ( B ) Surface biotinylated integrin α5β1 was quantified by capture ELISA (normalized absorbance to scramble control, p = 0.2439, n = 7). Degradation of surface α5 integrin was quantified over 6 h. Shown is the percent signal compared to time 0 (n = 3). ( C ) Integrin endocytosis was quantified in U87MG with two independent siRNAs (1 and 2). Shown is internalization normalized to non-target (NT) control (* p < 0.05, n = 3 for siRNA 1, n = 2 for siRNA 2). ( D ) Integrin endocytosis was measured using immunoprecipitation with antibodies against β1 integrin and detection with Western blotting with Streptavidin-HRP. Shown is a representative Western blot and internalization time course with points at 10 min ( p = 0.2425, n = 3) and 15 min (* p < 0.05, n = 3). ( E ) Surface α5β1 integrin was labeled with mouse anti-α5 antibodies in PEA-15 KD cells and scramble controls on ice. Cells were then returned to 37 °C in the presence of EGF to induce integrin trafficking, followed by fixation and immunostaining. HA-tag PEA-15 expressed in a subset of PEA-15 KD cells were co-stained with anti-HA-tag antibodies. Asterisks (*) mark transfected cells. Inset images show red-fluorescent HA-tag signal. Scale bar = 20 µm. ( F ) Integrin endocytosis was quantified in PEA-15 KD cells expressing HA-tagged PEA-15 (* p < 0.05, Scramble n = 3, PEA-15 shRNA n = 4, HA-PEA15 rescue n = 3, HA-S104A n = 3, HA-S116A n = 3).

Article Snippet: PEA-15-containing protein complexes were precipitated from whole cell lysates with anti-PEA15 antibodies (Pacific Immunology, Ramona, CA), or anti-HA antibodies (#51064-2-AP, Proteintech, Rosemont, IL) in the case of cells expressing HA-tagged PEA-15.

Techniques: Stripping Membranes, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Immunoprecipitation, Labeling, Immunostaining, Staining, Transfection, shRNA