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The CacyBP/SIP protein is sumoylated in neuroblastoma NB2a cells.

Wasik U, Filipek A - Neurochem. Res. (2013)

Bottom Line: In this work we examined a post-translational modification of CacyBP/SIP which might have an effect on its function.By fractionation of NB2a cell extract we have found that, contrary to the majority of SUMO-modified proteins, sumoylated CacyBP/SIP is present in the cytoplasmic and not in the nuclear fraction.We have also established that lysine 16 is the residue which undergoes sumoylation in the CacyBP/SIP protein.

View Article: PubMed Central - PubMed

ABSTRACT
The Calcyclin binding protein and Siah-1 interacting protein (CacyBP/SIP) protein is highly expressed in mammalian brain as well as in neuroblastoma NB2a cells and pheochromocytoma PC12 cells. This protein interacts with several targets such as cytoskeletal proteins or ERK1/2 kinase and seems to be involved in many cellular processes. In this work we examined a post-translational modification of CacyBP/SIP which might have an effect on its function. Since theoretical analysis of the amino acid sequence of CacyBP/SIP indicated several lysine residues which could potentially be sumoylated we checked experimentally whether this protein might be modified by SUMO attachment. We have shown that indeed CacyBP/SIP bound the E2 SUMO ligase, Ubc9, in neuroblastoma NB2a cell extract and was sumoylated in these cells. By fractionation of NB2a cell extract we have found that, contrary to the majority of SUMO-modified proteins, sumoylated CacyBP/SIP is present in the cytoplasmic and not in the nuclear fraction. We have also established that lysine 16 is the residue which undergoes sumoylation in the CacyBP/SIP protein.

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Co-immunoprecipitation of CacyBP/SIP with Ubc9 in NB2a cell extract. Cells were co-transfected with pGW1-HA-Ubc9 and pCMV3xFLAG-CacyBP/SIP (designated as HA-Ubc9 and 3xFLAG-CacyBP/SIP) or pGW1-HA-Ubc9 and pCMV3xFLAG (designated as HA-Ubc9). Proteins from the extract were used directly for Western blot analysis (80 μg) or incubated with anti-FLAG M2 affinity resin (800 μg). Proteins co-precipitated with CacyBP/SIP-3xFLAG were examined by Western blot using anti-Ubc9 or anti-CacyBP/SIP antibodies. Western blot images representative for 3 experiments performed are shown
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Fig2: Co-immunoprecipitation of CacyBP/SIP with Ubc9 in NB2a cell extract. Cells were co-transfected with pGW1-HA-Ubc9 and pCMV3xFLAG-CacyBP/SIP (designated as HA-Ubc9 and 3xFLAG-CacyBP/SIP) or pGW1-HA-Ubc9 and pCMV3xFLAG (designated as HA-Ubc9). Proteins from the extract were used directly for Western blot analysis (80 μg) or incubated with anti-FLAG M2 affinity resin (800 μg). Proteins co-precipitated with CacyBP/SIP-3xFLAG were examined by Western blot using anti-Ubc9 or anti-CacyBP/SIP antibodies. Western blot images representative for 3 experiments performed are shown

Mentions: In order to establish whether CacyBP/SIP might be sumoylated we first checked if it bound the SUMO conjugating enzyme, Ubc9. To examine this, a co-immunoprecipitation assay was performed. Ubc9-HA and CacyBP/SIP-3xFLAG were transiently overexpressed in mouse neuroblastoma NB2a cells and proteins from the cell extract were incubated with anti-FLAG M2 affinity resin to isolate CacyBP/SIP-3xFLAG with its targets. Proteins in the elution fraction were examined by Western blot analysis for the presence of Ubc9. As shown in Fig. 2, Ubc9-HA co-precipitated with CacyBP/SIP-3xFLAG, indicating that CacyBP/SIP and Ubc9 interacted with one another and suggesting that CacyBP/SIP might undergo sumoylation.Fig. 2


The CacyBP/SIP protein is sumoylated in neuroblastoma NB2a cells.

Wasik U, Filipek A - Neurochem. Res. (2013)

Co-immunoprecipitation of CacyBP/SIP with Ubc9 in NB2a cell extract. Cells were co-transfected with pGW1-HA-Ubc9 and pCMV3xFLAG-CacyBP/SIP (designated as HA-Ubc9 and 3xFLAG-CacyBP/SIP) or pGW1-HA-Ubc9 and pCMV3xFLAG (designated as HA-Ubc9). Proteins from the extract were used directly for Western blot analysis (80 μg) or incubated with anti-FLAG M2 affinity resin (800 μg). Proteins co-precipitated with CacyBP/SIP-3xFLAG were examined by Western blot using anti-Ubc9 or anti-CacyBP/SIP antibodies. Western blot images representative for 3 experiments performed are shown
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3824344&req=5

Fig2: Co-immunoprecipitation of CacyBP/SIP with Ubc9 in NB2a cell extract. Cells were co-transfected with pGW1-HA-Ubc9 and pCMV3xFLAG-CacyBP/SIP (designated as HA-Ubc9 and 3xFLAG-CacyBP/SIP) or pGW1-HA-Ubc9 and pCMV3xFLAG (designated as HA-Ubc9). Proteins from the extract were used directly for Western blot analysis (80 μg) or incubated with anti-FLAG M2 affinity resin (800 μg). Proteins co-precipitated with CacyBP/SIP-3xFLAG were examined by Western blot using anti-Ubc9 or anti-CacyBP/SIP antibodies. Western blot images representative for 3 experiments performed are shown
Mentions: In order to establish whether CacyBP/SIP might be sumoylated we first checked if it bound the SUMO conjugating enzyme, Ubc9. To examine this, a co-immunoprecipitation assay was performed. Ubc9-HA and CacyBP/SIP-3xFLAG were transiently overexpressed in mouse neuroblastoma NB2a cells and proteins from the cell extract were incubated with anti-FLAG M2 affinity resin to isolate CacyBP/SIP-3xFLAG with its targets. Proteins in the elution fraction were examined by Western blot analysis for the presence of Ubc9. As shown in Fig. 2, Ubc9-HA co-precipitated with CacyBP/SIP-3xFLAG, indicating that CacyBP/SIP and Ubc9 interacted with one another and suggesting that CacyBP/SIP might undergo sumoylation.Fig. 2

Bottom Line: In this work we examined a post-translational modification of CacyBP/SIP which might have an effect on its function.By fractionation of NB2a cell extract we have found that, contrary to the majority of SUMO-modified proteins, sumoylated CacyBP/SIP is present in the cytoplasmic and not in the nuclear fraction.We have also established that lysine 16 is the residue which undergoes sumoylation in the CacyBP/SIP protein.

View Article: PubMed Central - PubMed

ABSTRACT
The Calcyclin binding protein and Siah-1 interacting protein (CacyBP/SIP) protein is highly expressed in mammalian brain as well as in neuroblastoma NB2a cells and pheochromocytoma PC12 cells. This protein interacts with several targets such as cytoskeletal proteins or ERK1/2 kinase and seems to be involved in many cellular processes. In this work we examined a post-translational modification of CacyBP/SIP which might have an effect on its function. Since theoretical analysis of the amino acid sequence of CacyBP/SIP indicated several lysine residues which could potentially be sumoylated we checked experimentally whether this protein might be modified by SUMO attachment. We have shown that indeed CacyBP/SIP bound the E2 SUMO ligase, Ubc9, in neuroblastoma NB2a cell extract and was sumoylated in these cells. By fractionation of NB2a cell extract we have found that, contrary to the majority of SUMO-modified proteins, sumoylated CacyBP/SIP is present in the cytoplasmic and not in the nuclear fraction. We have also established that lysine 16 is the residue which undergoes sumoylation in the CacyBP/SIP protein.

Show MeSH
Related in: MedlinePlus