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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/VelcadeĀ® 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.

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Co-immunoprecipitation of SOCS-1 with CYTIP.Rat polyclonal anti CYTIP antibody (1A3) was used to co-immunoprecipitate CYTIP and its binding partners in 2 mg of mature dendritic cells lysate. Rabbit anti human SOCS-1 polyclonal antibody was used for detection and visualized with Alexa fluor 680 goat anti rabbit antibody. SOCS-1 expression in immature (iDC) and mature dendritic cells (mDC) and in the immunoprecipitate (CO-IP) is shown. As a control, rat IgG1 Isotype control was used for co-immunoprecipitation (Isotype control). SM: size marker.
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pone-0057538-g001: Co-immunoprecipitation of SOCS-1 with CYTIP.Rat polyclonal anti CYTIP antibody (1A3) was used to co-immunoprecipitate CYTIP and its binding partners in 2 mg of mature dendritic cells lysate. Rabbit anti human SOCS-1 polyclonal antibody was used for detection and visualized with Alexa fluor 680 goat anti rabbit antibody. SOCS-1 expression in immature (iDC) and mature dendritic cells (mDC) and in the immunoprecipitate (CO-IP) is shown. As a control, rat IgG1 Isotype control was used for co-immunoprecipitation (Isotype control). SM: size marker.

Mentions: CYTIP contains several protein-protein binding sites but only cytohesin-1 [14] binding to the coiled coil domain and sorting nexin 27 (SNX 27) [15] binding to the PDZ binding motif have been characterized so far. This makes the existence of additional binding partners likely. We therefore chose to further evaluate the function of CYTIP by searching for an additional binding partner of CYTIP in mature dendritic cells. In a yeast-two-hybrid system with CYTIP as bait we screened a library derived from mature monocyte derived dendritic cells. As expected, the most frequent sequence obtained coded for cytohesin-1, which was identified as binding partner for CYTIP earlier [7]. The second most frequent sequence of the isolated clones coded for SOCS-1. To verify our result from the yeast-two-hybrid screen we co-immunoprecipitated the CYTIP-SOCS-1 complex from protein lysate derived from mature monocyte derived dendritic cells with monoclonal rat anti CYTIP antibody 1A3 and detected SOCS-1 in Western blot analyses using polyclonal rabbit anti human SOCS-1 antibody (Fig. 1 CO-IP). As a control, the co-immunoprecipitation was carried out with an isotype matched control antibody (isotype control). In addition, SOCS-1 expression in immature (iDC) and mature dendritic cells (mDC) and in the lysate used for co-immunoprecipitations is shown.


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 SOCS-1 with CYTIP.Rat polyclonal anti CYTIP antibody (1A3) was used to co-immunoprecipitate CYTIP and its binding partners in 2 mg of mature dendritic cells lysate. Rabbit anti human SOCS-1 polyclonal antibody was used for detection and visualized with Alexa fluor 680 goat anti rabbit antibody. SOCS-1 expression in immature (iDC) and mature dendritic cells (mDC) and in the immunoprecipitate (CO-IP) is shown. As a control, rat IgG1 Isotype control was used for co-immunoprecipitation (Isotype control). SM: size marker.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585367&req=5

pone-0057538-g001: Co-immunoprecipitation of SOCS-1 with CYTIP.Rat polyclonal anti CYTIP antibody (1A3) was used to co-immunoprecipitate CYTIP and its binding partners in 2 mg of mature dendritic cells lysate. Rabbit anti human SOCS-1 polyclonal antibody was used for detection and visualized with Alexa fluor 680 goat anti rabbit antibody. SOCS-1 expression in immature (iDC) and mature dendritic cells (mDC) and in the immunoprecipitate (CO-IP) is shown. As a control, rat IgG1 Isotype control was used for co-immunoprecipitation (Isotype control). SM: size marker.
Mentions: CYTIP contains several protein-protein binding sites but only cytohesin-1 [14] binding to the coiled coil domain and sorting nexin 27 (SNX 27) [15] binding to the PDZ binding motif have been characterized so far. This makes the existence of additional binding partners likely. We therefore chose to further evaluate the function of CYTIP by searching for an additional binding partner of CYTIP in mature dendritic cells. In a yeast-two-hybrid system with CYTIP as bait we screened a library derived from mature monocyte derived dendritic cells. As expected, the most frequent sequence obtained coded for cytohesin-1, which was identified as binding partner for CYTIP earlier [7]. The second most frequent sequence of the isolated clones coded for SOCS-1. To verify our result from the yeast-two-hybrid screen we co-immunoprecipitated the CYTIP-SOCS-1 complex from protein lysate derived from mature monocyte derived dendritic cells with monoclonal rat anti CYTIP antibody 1A3 and detected SOCS-1 in Western blot analyses using polyclonal rabbit anti human SOCS-1 antibody (Fig. 1 CO-IP). As a control, the co-immunoprecipitation was carried out with an isotype matched control antibody (isotype control). In addition, SOCS-1 expression in immature (iDC) and mature dendritic cells (mDC) and in the lysate used for co-immunoprecipitations is shown.

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/VelcadeĀ® 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