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A novel protein kinase D phosphorylation site in the tumor suppressor Rab interactor 1 is critical for coordination of cell migration.

Ziegler S, Eiseler T, Scholz RP, Beck A, Link G, Hausser A - Mol. Biol. Cell (2011)

Bottom Line: PKD-mediated phosphorylation at this site was confirmed with a phospho-specific antibody and by mass spectrometry.We further provide evidence that RIN1 in vivo phosphorylation at serine 351 occurs independently of PKD.Collectively, our data identify a novel PKD signaling pathway through RIN1 and Abl kinases that is involved in the regulation of actin remodeling and cell migration.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cell Biology and Immunology, University of Stuttgart, 70569 Stuttgart, Germany Panatecs GmbH, 72070 Tübingen, Germany.

ABSTRACT
The multifunctional signal adapter protein Ras and Rab interactor 1 (RIN1) is a Ras effector protein involved in the regulation of epithelial cell processes such as cell migration and endocytosis. RIN1 signals via two downstream pathways, namely the activation of Rab5 and Abl family kinases. Protein kinase D (PKD) phosphorylates RIN1 at serine 351 in vitro, thereby regulating interaction with 14-3-3 proteins. Here, we report the identification of serine 292 in RIN1 as an in vivo PKD phosphorylation site. PKD-mediated phosphorylation at this site was confirmed with a phospho-specific antibody and by mass spectrometry. We demonstrate that phosphorylation at serine 292 controls RIN1-mediated inhibition of cell migration by modulating the activation of Abl kinases. We further provide evidence that RIN1 in vivo phosphorylation at serine 351 occurs independently of PKD. Collectively, our data identify a novel PKD signaling pathway through RIN1 and Abl kinases that is involved in the regulation of actin remodeling and cell migration.

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RIN1 and PKD1 colocalize at sites of actin remodeling. (A) COS7 cells were transfected with Flag-RIN1 WT and S292A expression plasmids, fixed and stained with Flag-specific antibody. The images shown are stacks of several confocal sections. Scale bar, 10 μm. (B) COS7 cells were transfected with Flag-RIN1 WT alone (top panel) or together with PKD1-GFP expression plasmid (bottom panel), fixed, and stained with Alexa546-coupled phalloidin together with Flag-specific antibody followed by Alexa488-coupled anti–mouse IgG (top panel) or Flag-specific antibody followed by Alexa546-coupled anti–mouse IgG (bottom panel). The images shown are stacks of several confocal sections. Scale bar, 10 μm. (C) MCF7 cells expressing GFP-tagged RIN1 were fixed and stained with the pS292-specific antibody followed by Alexa546-coupled anti–rabbit IgG together with Alexa633-coupled phalloidin (F-actin). The images shown are stacks of several confocal sections. Scale bar, 10 μm. Arrows indicate sites of actin remodeling.
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Figure 3: RIN1 and PKD1 colocalize at sites of actin remodeling. (A) COS7 cells were transfected with Flag-RIN1 WT and S292A expression plasmids, fixed and stained with Flag-specific antibody. The images shown are stacks of several confocal sections. Scale bar, 10 μm. (B) COS7 cells were transfected with Flag-RIN1 WT alone (top panel) or together with PKD1-GFP expression plasmid (bottom panel), fixed, and stained with Alexa546-coupled phalloidin together with Flag-specific antibody followed by Alexa488-coupled anti–mouse IgG (top panel) or Flag-specific antibody followed by Alexa546-coupled anti–mouse IgG (bottom panel). The images shown are stacks of several confocal sections. Scale bar, 10 μm. (C) MCF7 cells expressing GFP-tagged RIN1 were fixed and stained with the pS292-specific antibody followed by Alexa546-coupled anti–rabbit IgG together with Alexa633-coupled phalloidin (F-actin). The images shown are stacks of several confocal sections. Scale bar, 10 μm. Arrows indicate sites of actin remodeling.

Mentions: RIN1 is a cytosolic protein, recruited to the plasma membrane via interaction with active Ras (Wang et al., 2002). A portion of RIN1 is also found on Rab5-positive endosomes (Tall et al., 2001). Immunofluorescence staining of Flag-tagged RIN1 in COS7 cells verified that the protein localizes mainly in the cytosol and at distinct sites of the plasma membrane. Of note, RIN1 was also located in the nucleus (Figure 3A). Next, we analyzed whether PKD-mediated phosphorylation affects the subcellular localization of the RIN1 protein (Figure 3, A and B). Neither the exchange of serine 292 to alanine in RIN1 nor the coexpression of PKD (Figure 3B) and the expression of a phosphomimetic RIN1 S292E protein, respectively, alter the localization of the protein (Figure 3A) (unpublished data).


A novel protein kinase D phosphorylation site in the tumor suppressor Rab interactor 1 is critical for coordination of cell migration.

Ziegler S, Eiseler T, Scholz RP, Beck A, Link G, Hausser A - Mol. Biol. Cell (2011)

RIN1 and PKD1 colocalize at sites of actin remodeling. (A) COS7 cells were transfected with Flag-RIN1 WT and S292A expression plasmids, fixed and stained with Flag-specific antibody. The images shown are stacks of several confocal sections. Scale bar, 10 μm. (B) COS7 cells were transfected with Flag-RIN1 WT alone (top panel) or together with PKD1-GFP expression plasmid (bottom panel), fixed, and stained with Alexa546-coupled phalloidin together with Flag-specific antibody followed by Alexa488-coupled anti–mouse IgG (top panel) or Flag-specific antibody followed by Alexa546-coupled anti–mouse IgG (bottom panel). The images shown are stacks of several confocal sections. Scale bar, 10 μm. (C) MCF7 cells expressing GFP-tagged RIN1 were fixed and stained with the pS292-specific antibody followed by Alexa546-coupled anti–rabbit IgG together with Alexa633-coupled phalloidin (F-actin). The images shown are stacks of several confocal sections. Scale bar, 10 μm. Arrows indicate sites of actin remodeling.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 3: RIN1 and PKD1 colocalize at sites of actin remodeling. (A) COS7 cells were transfected with Flag-RIN1 WT and S292A expression plasmids, fixed and stained with Flag-specific antibody. The images shown are stacks of several confocal sections. Scale bar, 10 μm. (B) COS7 cells were transfected with Flag-RIN1 WT alone (top panel) or together with PKD1-GFP expression plasmid (bottom panel), fixed, and stained with Alexa546-coupled phalloidin together with Flag-specific antibody followed by Alexa488-coupled anti–mouse IgG (top panel) or Flag-specific antibody followed by Alexa546-coupled anti–mouse IgG (bottom panel). The images shown are stacks of several confocal sections. Scale bar, 10 μm. (C) MCF7 cells expressing GFP-tagged RIN1 were fixed and stained with the pS292-specific antibody followed by Alexa546-coupled anti–rabbit IgG together with Alexa633-coupled phalloidin (F-actin). The images shown are stacks of several confocal sections. Scale bar, 10 μm. Arrows indicate sites of actin remodeling.
Mentions: RIN1 is a cytosolic protein, recruited to the plasma membrane via interaction with active Ras (Wang et al., 2002). A portion of RIN1 is also found on Rab5-positive endosomes (Tall et al., 2001). Immunofluorescence staining of Flag-tagged RIN1 in COS7 cells verified that the protein localizes mainly in the cytosol and at distinct sites of the plasma membrane. Of note, RIN1 was also located in the nucleus (Figure 3A). Next, we analyzed whether PKD-mediated phosphorylation affects the subcellular localization of the RIN1 protein (Figure 3, A and B). Neither the exchange of serine 292 to alanine in RIN1 nor the coexpression of PKD (Figure 3B) and the expression of a phosphomimetic RIN1 S292E protein, respectively, alter the localization of the protein (Figure 3A) (unpublished data).

Bottom Line: PKD-mediated phosphorylation at this site was confirmed with a phospho-specific antibody and by mass spectrometry.We further provide evidence that RIN1 in vivo phosphorylation at serine 351 occurs independently of PKD.Collectively, our data identify a novel PKD signaling pathway through RIN1 and Abl kinases that is involved in the regulation of actin remodeling and cell migration.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cell Biology and Immunology, University of Stuttgart, 70569 Stuttgart, Germany Panatecs GmbH, 72070 Tübingen, Germany.

ABSTRACT
The multifunctional signal adapter protein Ras and Rab interactor 1 (RIN1) is a Ras effector protein involved in the regulation of epithelial cell processes such as cell migration and endocytosis. RIN1 signals via two downstream pathways, namely the activation of Rab5 and Abl family kinases. Protein kinase D (PKD) phosphorylates RIN1 at serine 351 in vitro, thereby regulating interaction with 14-3-3 proteins. Here, we report the identification of serine 292 in RIN1 as an in vivo PKD phosphorylation site. PKD-mediated phosphorylation at this site was confirmed with a phospho-specific antibody and by mass spectrometry. We demonstrate that phosphorylation at serine 292 controls RIN1-mediated inhibition of cell migration by modulating the activation of Abl kinases. We further provide evidence that RIN1 in vivo phosphorylation at serine 351 occurs independently of PKD. Collectively, our data identify a novel PKD signaling pathway through RIN1 and Abl kinases that is involved in the regulation of actin remodeling and cell migration.

Show MeSH