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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/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.

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Transfection of mature dendritic cells (mDC) with increasing amounts of SOCS-1 encoding plasmid induces a decrease of CYTIP expression.1×106 cells were transfected with the indicated amounts of SOCS-1 encoding plasmid. 16 hours later protein lysates were prepared and 40 µg of lysates were applied to each lane. CYTIP levels (39 kDa) were measured by Western blot analyses. As a control, actin expression levels were determined. Quantification of grey level pixel intensity was done with Odyssey software. One representative Western blot is shown. iDC: immature dendritic cells, mDC: mature dendritic cells, mock: transfection procedure without plasmid DNA, µg indicate the amount of plasmid used for transfection. CYTIP expression decreases significantly with SOCS-1 over expression 16 hours after transfection with 4 and 8 µg of SOCS-1 encoding plasmid.
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pone-0057538-g003: Transfection of mature dendritic cells (mDC) with increasing amounts of SOCS-1 encoding plasmid induces a decrease of CYTIP expression.1×106 cells were transfected with the indicated amounts of SOCS-1 encoding plasmid. 16 hours later protein lysates were prepared and 40 µg of lysates were applied to each lane. CYTIP levels (39 kDa) were measured by Western blot analyses. As a control, actin expression levels were determined. Quantification of grey level pixel intensity was done with Odyssey software. One representative Western blot is shown. iDC: immature dendritic cells, mDC: mature dendritic cells, mock: transfection procedure without plasmid DNA, µg indicate the amount of plasmid used for transfection. CYTIP expression decreases significantly with SOCS-1 over expression 16 hours after transfection with 4 and 8 µg of SOCS-1 encoding plasmid.

Mentions: To evaluate whether CYTIP is degraded as a consequence to its binding to SOCS-1 we measured CYTIP expression levels in mature dendritic cells transfected with increasing amounts of a SOCS-1 expression vector. If so, increasing amounts of transfected SOCS-1 should decrease the amount of CYTIP protein in dendritic cells. We used the indicated amounts of SOCS-1 encoding plasmid to transfect 1×106 mature dendritic cells each. 16 hours after transfection Western blot analysis with anti CYTIP antibody was performed. As shown in Fig. 3, in 5 independent experiments we found that CYTIP expression decreases with growing amounts of transfected SOCS-1 expression vector. Statistical analysis was performed using paired, two-tailed student’s t-test. Expression levels obtained with 4 and 8 µg of SOCS-1 plasmid DNA transfection resulted in significant decrease of CYTIP expression. One representative Western blot with immature and mature dendritic cells, a mock control and actin levels as controls 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)

Transfection of mature dendritic cells (mDC) with increasing amounts of SOCS-1 encoding plasmid induces a decrease of CYTIP expression.1×106 cells were transfected with the indicated amounts of SOCS-1 encoding plasmid. 16 hours later protein lysates were prepared and 40 µg of lysates were applied to each lane. CYTIP levels (39 kDa) were measured by Western blot analyses. As a control, actin expression levels were determined. Quantification of grey level pixel intensity was done with Odyssey software. One representative Western blot is shown. iDC: immature dendritic cells, mDC: mature dendritic cells, mock: transfection procedure without plasmid DNA, µg indicate the amount of plasmid used for transfection. CYTIP expression decreases significantly with SOCS-1 over expression 16 hours after transfection with 4 and 8 µg of SOCS-1 encoding plasmid.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585367&req=5

pone-0057538-g003: Transfection of mature dendritic cells (mDC) with increasing amounts of SOCS-1 encoding plasmid induces a decrease of CYTIP expression.1×106 cells were transfected with the indicated amounts of SOCS-1 encoding plasmid. 16 hours later protein lysates were prepared and 40 µg of lysates were applied to each lane. CYTIP levels (39 kDa) were measured by Western blot analyses. As a control, actin expression levels were determined. Quantification of grey level pixel intensity was done with Odyssey software. One representative Western blot is shown. iDC: immature dendritic cells, mDC: mature dendritic cells, mock: transfection procedure without plasmid DNA, µg indicate the amount of plasmid used for transfection. CYTIP expression decreases significantly with SOCS-1 over expression 16 hours after transfection with 4 and 8 µg of SOCS-1 encoding plasmid.
Mentions: To evaluate whether CYTIP is degraded as a consequence to its binding to SOCS-1 we measured CYTIP expression levels in mature dendritic cells transfected with increasing amounts of a SOCS-1 expression vector. If so, increasing amounts of transfected SOCS-1 should decrease the amount of CYTIP protein in dendritic cells. We used the indicated amounts of SOCS-1 encoding plasmid to transfect 1×106 mature dendritic cells each. 16 hours after transfection Western blot analysis with anti CYTIP antibody was performed. As shown in Fig. 3, in 5 independent experiments we found that CYTIP expression decreases with growing amounts of transfected SOCS-1 expression vector. Statistical analysis was performed using paired, two-tailed student’s t-test. Expression levels obtained with 4 and 8 µg of SOCS-1 plasmid DNA transfection resulted in significant decrease of CYTIP expression. One representative Western blot with immature and mature dendritic cells, a mock control and actin levels as controls 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/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