Limits...
In human monocyte derived dendritic cells SOCS1 interacting with CYTIP induces the degradation of CYTIP by the proteasome.

Grabher D, Hofer S, Ortner D, Heufler C - PLoS ONE (2013)

Bottom Line: If dendritic cells are silenced for CYTIP they keep longer contacts with T-cells resulting in a lower T cell stimulation.We observed lower amounts of endogenous CYTIP in mature dendritic cells transfected with SOCS-1 encoding plasmid compared with untransfected dendritic cells.We here describe a newly identified binding partner of CYTIP, SOCS-1, and confirm its function in regulating the degradation of CYTIP by the proteasome.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Innsbruck Medical University, Innsbruck, Austria.

ABSTRACT
CYTIP (cytohesin interacting protein) is an intracellular molecule induced in dendritic cells during maturation. CYTIP modulates the binding intensity of the adhesion molecule LFA-1. If dendritic cells are silenced for CYTIP they keep longer contacts with T-cells resulting in a lower T cell stimulation. We identified Suppressor of cytokine signaling-1 (SOCS-1) as a binding partner for CYTIP in human monocyte derived dendritic cells. In Western blot analyses we found that CYTIP expression is down regulated at later time points, starting at about 72 hours after induction of maturation. To investigate a possible role for SOCS-1 in taking CYTIP to the degradation machinery of the cell we measured endogenous CYTIP protein levels in mature dendritic cells transfected with SOCS-1 encoding plasmid in quantitative Western blot analyses. We observed lower amounts of endogenous CYTIP in mature dendritic cells transfected with SOCS-1 encoding plasmid compared with untransfected dendritic cells. Experiments with the proteasome-inhibitor Bortezomib/Velcade® show stable CYTIP expression levels in dendritic cells. In addition, we show that CYTIP in dendritic cells matured for 48 hours is ubiquitinated and thus ready for degradation. We here describe a newly identified binding partner of CYTIP, SOCS-1, and confirm its function in regulating the degradation of CYTIP by the proteasome.

Show MeSH

Related in: MedlinePlus

Co-immunoprecipitation of CYTIP with Ubiquitin.The immunocomplex obtained by incubating 2 mg of mature dendritic cells lysate with rat polyclonal anti CYTIP antibody (1A3) was analyzed by Western blot analyses with mouse anti ubiquitin and visualized with Alexa fluor 680 goat anti mouse antibody. Ubiquitination of the CYTIP precipitate is shown (Co-IP). As a control the precipitate obtained with rat isotype control is shown.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3585367&req=5

pone-0057538-g004: Co-immunoprecipitation of CYTIP with Ubiquitin.The immunocomplex obtained by incubating 2 mg of mature dendritic cells lysate with rat polyclonal anti CYTIP antibody (1A3) was analyzed by Western blot analyses with mouse anti ubiquitin and visualized with Alexa fluor 680 goat anti mouse antibody. Ubiquitination of the CYTIP precipitate is shown (Co-IP). As a control the precipitate obtained with rat isotype control is shown.

Mentions: To gain further evidence for the hypothesis that CYTIP is taken to the proteasome for degradation by binding to SOCS-1, we investigated whether CYTIP is ubiquitinated. Ubiquitin is a ubiquitously expressed protein used to label proteins for degradation by the proteasome. We performed co-immunoprecipitation using 8 µg of a rat monoclonal anti CYTIP antibody or an isotype matched antibody as a control. In Western blot analysis with a mouse anti ubiquitin monoclonal antibody, ubiquitinated bands of approximately 50 kDa, 75 kDa, and two or three additional minor bands between 75 and 100 kDa were detected in the immunoprecipitate obtained with the anti CYTIP antibody but not with the isotype matched control antibody (Fig. 4). We assume that the bands consist of CYTIP with 2 (50 kDa), 4 (75 kDa) or more ubiquitin copies attached. This indicates that CYTIP is ubiquitinated in monocyte derived mature dendritic cells.


In human monocyte derived dendritic cells SOCS1 interacting with CYTIP induces the degradation of CYTIP by the proteasome.

Grabher D, Hofer S, Ortner D, Heufler C - PLoS ONE (2013)

Co-immunoprecipitation of CYTIP with Ubiquitin.The immunocomplex obtained by incubating 2 mg of mature dendritic cells lysate with rat polyclonal anti CYTIP antibody (1A3) was analyzed by Western blot analyses with mouse anti ubiquitin and visualized with Alexa fluor 680 goat anti mouse antibody. Ubiquitination of the CYTIP precipitate is shown (Co-IP). As a control the precipitate obtained with rat isotype control is shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585367&req=5

pone-0057538-g004: Co-immunoprecipitation of CYTIP with Ubiquitin.The immunocomplex obtained by incubating 2 mg of mature dendritic cells lysate with rat polyclonal anti CYTIP antibody (1A3) was analyzed by Western blot analyses with mouse anti ubiquitin and visualized with Alexa fluor 680 goat anti mouse antibody. Ubiquitination of the CYTIP precipitate is shown (Co-IP). As a control the precipitate obtained with rat isotype control is shown.
Mentions: To gain further evidence for the hypothesis that CYTIP is taken to the proteasome for degradation by binding to SOCS-1, we investigated whether CYTIP is ubiquitinated. Ubiquitin is a ubiquitously expressed protein used to label proteins for degradation by the proteasome. We performed co-immunoprecipitation using 8 µg of a rat monoclonal anti CYTIP antibody or an isotype matched antibody as a control. In Western blot analysis with a mouse anti ubiquitin monoclonal antibody, ubiquitinated bands of approximately 50 kDa, 75 kDa, and two or three additional minor bands between 75 and 100 kDa were detected in the immunoprecipitate obtained with the anti CYTIP antibody but not with the isotype matched control antibody (Fig. 4). We assume that the bands consist of CYTIP with 2 (50 kDa), 4 (75 kDa) or more ubiquitin copies attached. This indicates that CYTIP is ubiquitinated in monocyte derived mature dendritic cells.

Bottom Line: If dendritic cells are silenced for CYTIP they keep longer contacts with T-cells resulting in a lower T cell stimulation.We observed lower amounts of endogenous CYTIP in mature dendritic cells transfected with SOCS-1 encoding plasmid compared with untransfected dendritic cells.We here describe a newly identified binding partner of CYTIP, SOCS-1, and confirm its function in regulating the degradation of CYTIP by the proteasome.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Innsbruck Medical University, Innsbruck, Austria.

ABSTRACT
CYTIP (cytohesin interacting protein) is an intracellular molecule induced in dendritic cells during maturation. CYTIP modulates the binding intensity of the adhesion molecule LFA-1. If dendritic cells are silenced for CYTIP they keep longer contacts with T-cells resulting in a lower T cell stimulation. We identified Suppressor of cytokine signaling-1 (SOCS-1) as a binding partner for CYTIP in human monocyte derived dendritic cells. In Western blot analyses we found that CYTIP expression is down regulated at later time points, starting at about 72 hours after induction of maturation. To investigate a possible role for SOCS-1 in taking CYTIP to the degradation machinery of the cell we measured endogenous CYTIP protein levels in mature dendritic cells transfected with SOCS-1 encoding plasmid in quantitative Western blot analyses. We observed lower amounts of endogenous CYTIP in mature dendritic cells transfected with SOCS-1 encoding plasmid compared with untransfected dendritic cells. Experiments with the proteasome-inhibitor Bortezomib/Velcade® show stable CYTIP expression levels in dendritic cells. In addition, we show that CYTIP in dendritic cells matured for 48 hours is ubiquitinated and thus ready for degradation. We here describe a newly identified binding partner of CYTIP, SOCS-1, and confirm its function in regulating the degradation of CYTIP by the proteasome.

Show MeSH
Related in: MedlinePlus