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A novel mutation in β integrin reveals an integrin-mediated interaction between the extracellular matrix and cki-1/p27KIP1.

Kihira S, Yu EJ, Cunningham J, Cram EJ, Lee M - PLoS ONE (2012)

Bottom Line: RNAi of unc-52/perlecan, ina-1/α integrin, pat-4/ILK, and unc-97/PINCH resulted in abnormal CKI-1::GFP localization.These data also suggest that integrin plays a major role in maintaining proper CKI-1/p27(KIP1) levels in the cell.Perturbed integrin signaling may lead to the inhibition of SCF ligase activity, mislocalization and elevation of CKI-1/p27(KIP1).

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Baylor University, Waco, Texas, United States of America.

ABSTRACT
The cell-extracellular matrix (ECM) interaction plays an essential role in maintaining tissue shapes and regulates cell behaviors such as cell adhesion, differentiation and proliferation. The mechanism by which the ECM influences the cell cycle in vivo is poorly understood. Here we demonstrate that the β integrin PAT-3 regulates the localization and expression of CKI-1, a C. elegans homologue of the cyclin dependent kinase inhibitor p27(KIP1). In nematodes expressing wild type PAT-3, CKI-1::GFP localizes primarily to nucleoli in hypodermal cells, whereas in animals expressing mutant pat-3 with a defective splice junction, CKI-1::GFP appears clumped and disorganized in nucleoplasm. RNAi analysis links cell adhesion genes to the regulation of CKI-1. RNAi of unc-52/perlecan, ina-1/α integrin, pat-4/ILK, and unc-97/PINCH resulted in abnormal CKI-1::GFP localization. Additional RNAi experiments revealed that the SCF E3 ubiquitin-ligase complex genes, skpt-1/SKP2, cul-1/CUL1 and lin-23/F-box, are required for the proper localization and expression of CKI-1, suggesting that integrin signaling and SCF E3 ligase work together to regulate the cellular distribution of CKI-1. These data also suggest that integrin plays a major role in maintaining proper CKI-1/p27(KIP1) levels in the cell. Perturbed integrin signaling may lead to the inhibition of SCF ligase activity, mislocalization and elevation of CKI-1/p27(KIP1). These results suggest that adhesion signaling is crucial for cell cycle regulation in vivo.

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Immunoblot analysis of CKI-1::GFP protein in transgenic animals.Panel A: CKI-1::GFP expression levels were assessed in the transgenic rescued lines. Top bands in lanes 1 and 2 show the relative level of UNC-54/myosin B in each sample. Bottom bands indicate the level of CKI-1::GFP. Lanes 1 and 2 represent pat-3(+) and pat-3(sp), respectively. Quantification (Table S1) using ImageJ software revealed that CKI-1::GFP level was 10-fold increased in pat-3(sp) animals compared to pat-3(+) animals. Panel B: CKI-1::GFP expression levels were assessed in pat-3(+) animals treated with RNAi directed against focal adhesion genes. Top bands represent the amount of CKI-1::GFP in extracts prepared from each RNAi condition. L4440 is a negative RNAi control. The pat-3, ina-1, unc-97, unc-52, pat-6 and let-2 RNAi caused upregulation of CKI-1::GFP, while unc-112 RNAi had no effect. Bottom bands indicate MH33 [90] levels in each lane as a loading control. Quantification (Table S1) using ImageJ software revealed that CKI-1::GFP level was increased by RNAi of pat-3, ina-1, unc-97, unc-52, pat-6, and let-2. Panel C: CKI-1::GFP expression levels were also measured in pat-3(+) animals treated with E3 ligase gene RNAi. Top bands represent the amount of CKI-1::GFP in the extracts prepared from each RNAi condition. L4440 is a negative RNAi control. The skpt-1, lin-23 and cul-1 RNAi depletions caused upregulation of CKI-1::GFP, while rbx-1 and cul-4 RNAi had no effect. Bottom bands indicate MH33 [90] levels in each lane as a loading control. Quantification (Table S1) using ImageJ software revealed that the CKI-1::GFP level was increased by RNAi of skpt-1, lin-23 and cul-1.
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pone-0042425-g003: Immunoblot analysis of CKI-1::GFP protein in transgenic animals.Panel A: CKI-1::GFP expression levels were assessed in the transgenic rescued lines. Top bands in lanes 1 and 2 show the relative level of UNC-54/myosin B in each sample. Bottom bands indicate the level of CKI-1::GFP. Lanes 1 and 2 represent pat-3(+) and pat-3(sp), respectively. Quantification (Table S1) using ImageJ software revealed that CKI-1::GFP level was 10-fold increased in pat-3(sp) animals compared to pat-3(+) animals. Panel B: CKI-1::GFP expression levels were assessed in pat-3(+) animals treated with RNAi directed against focal adhesion genes. Top bands represent the amount of CKI-1::GFP in extracts prepared from each RNAi condition. L4440 is a negative RNAi control. The pat-3, ina-1, unc-97, unc-52, pat-6 and let-2 RNAi caused upregulation of CKI-1::GFP, while unc-112 RNAi had no effect. Bottom bands indicate MH33 [90] levels in each lane as a loading control. Quantification (Table S1) using ImageJ software revealed that CKI-1::GFP level was increased by RNAi of pat-3, ina-1, unc-97, unc-52, pat-6, and let-2. Panel C: CKI-1::GFP expression levels were also measured in pat-3(+) animals treated with E3 ligase gene RNAi. Top bands represent the amount of CKI-1::GFP in the extracts prepared from each RNAi condition. L4440 is a negative RNAi control. The skpt-1, lin-23 and cul-1 RNAi depletions caused upregulation of CKI-1::GFP, while rbx-1 and cul-4 RNAi had no effect. Bottom bands indicate MH33 [90] levels in each lane as a loading control. Quantification (Table S1) using ImageJ software revealed that the CKI-1::GFP level was increased by RNAi of skpt-1, lin-23 and cul-1.

Mentions: In the pat-3(sp) rescued animals, CKI-1::GFP localization was visibly different from that seen in pat-3(+) animals. In contrast to the compact, nucleolar staining seen in pat-3(+) animals, CKI-1::GFP in pat-3(sp) was clumped and accumulated in a ring around a dark center in the nucleus (Figures 2B and 2D), suggesting mislocalization and possible exclusion from the nucleolus. In addition, the intensity of green fluorescence in pat-3(sp) was increased compared to pat-3(+). In order to test for a possible correlation between the level of CKI-1::GFP and the integrin (pat-3(+) or pat-3(sp)) expressed, we first analyzed the amount of CKI-1::GFP in the pat-3 rescued lines. Protein lysates were prepared from an equal number of L4/young adult transgenic animals and tested for CKI-1::GFP protein levels. CKI-1::GFP level in pat-3(sp) was ten fold more intense than that seen in pat-3(+) lysates (Figure 3A), suggesting that PAT-3 signaling may control CKI-1 levels.


A novel mutation in β integrin reveals an integrin-mediated interaction between the extracellular matrix and cki-1/p27KIP1.

Kihira S, Yu EJ, Cunningham J, Cram EJ, Lee M - PLoS ONE (2012)

Immunoblot analysis of CKI-1::GFP protein in transgenic animals.Panel A: CKI-1::GFP expression levels were assessed in the transgenic rescued lines. Top bands in lanes 1 and 2 show the relative level of UNC-54/myosin B in each sample. Bottom bands indicate the level of CKI-1::GFP. Lanes 1 and 2 represent pat-3(+) and pat-3(sp), respectively. Quantification (Table S1) using ImageJ software revealed that CKI-1::GFP level was 10-fold increased in pat-3(sp) animals compared to pat-3(+) animals. Panel B: CKI-1::GFP expression levels were assessed in pat-3(+) animals treated with RNAi directed against focal adhesion genes. Top bands represent the amount of CKI-1::GFP in extracts prepared from each RNAi condition. L4440 is a negative RNAi control. The pat-3, ina-1, unc-97, unc-52, pat-6 and let-2 RNAi caused upregulation of CKI-1::GFP, while unc-112 RNAi had no effect. Bottom bands indicate MH33 [90] levels in each lane as a loading control. Quantification (Table S1) using ImageJ software revealed that CKI-1::GFP level was increased by RNAi of pat-3, ina-1, unc-97, unc-52, pat-6, and let-2. Panel C: CKI-1::GFP expression levels were also measured in pat-3(+) animals treated with E3 ligase gene RNAi. Top bands represent the amount of CKI-1::GFP in the extracts prepared from each RNAi condition. L4440 is a negative RNAi control. The skpt-1, lin-23 and cul-1 RNAi depletions caused upregulation of CKI-1::GFP, while rbx-1 and cul-4 RNAi had no effect. Bottom bands indicate MH33 [90] levels in each lane as a loading control. Quantification (Table S1) using ImageJ software revealed that the CKI-1::GFP level was increased by RNAi of skpt-1, lin-23 and cul-1.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3412830&req=5

pone-0042425-g003: Immunoblot analysis of CKI-1::GFP protein in transgenic animals.Panel A: CKI-1::GFP expression levels were assessed in the transgenic rescued lines. Top bands in lanes 1 and 2 show the relative level of UNC-54/myosin B in each sample. Bottom bands indicate the level of CKI-1::GFP. Lanes 1 and 2 represent pat-3(+) and pat-3(sp), respectively. Quantification (Table S1) using ImageJ software revealed that CKI-1::GFP level was 10-fold increased in pat-3(sp) animals compared to pat-3(+) animals. Panel B: CKI-1::GFP expression levels were assessed in pat-3(+) animals treated with RNAi directed against focal adhesion genes. Top bands represent the amount of CKI-1::GFP in extracts prepared from each RNAi condition. L4440 is a negative RNAi control. The pat-3, ina-1, unc-97, unc-52, pat-6 and let-2 RNAi caused upregulation of CKI-1::GFP, while unc-112 RNAi had no effect. Bottom bands indicate MH33 [90] levels in each lane as a loading control. Quantification (Table S1) using ImageJ software revealed that CKI-1::GFP level was increased by RNAi of pat-3, ina-1, unc-97, unc-52, pat-6, and let-2. Panel C: CKI-1::GFP expression levels were also measured in pat-3(+) animals treated with E3 ligase gene RNAi. Top bands represent the amount of CKI-1::GFP in the extracts prepared from each RNAi condition. L4440 is a negative RNAi control. The skpt-1, lin-23 and cul-1 RNAi depletions caused upregulation of CKI-1::GFP, while rbx-1 and cul-4 RNAi had no effect. Bottom bands indicate MH33 [90] levels in each lane as a loading control. Quantification (Table S1) using ImageJ software revealed that the CKI-1::GFP level was increased by RNAi of skpt-1, lin-23 and cul-1.
Mentions: In the pat-3(sp) rescued animals, CKI-1::GFP localization was visibly different from that seen in pat-3(+) animals. In contrast to the compact, nucleolar staining seen in pat-3(+) animals, CKI-1::GFP in pat-3(sp) was clumped and accumulated in a ring around a dark center in the nucleus (Figures 2B and 2D), suggesting mislocalization and possible exclusion from the nucleolus. In addition, the intensity of green fluorescence in pat-3(sp) was increased compared to pat-3(+). In order to test for a possible correlation between the level of CKI-1::GFP and the integrin (pat-3(+) or pat-3(sp)) expressed, we first analyzed the amount of CKI-1::GFP in the pat-3 rescued lines. Protein lysates were prepared from an equal number of L4/young adult transgenic animals and tested for CKI-1::GFP protein levels. CKI-1::GFP level in pat-3(sp) was ten fold more intense than that seen in pat-3(+) lysates (Figure 3A), suggesting that PAT-3 signaling may control CKI-1 levels.

Bottom Line: RNAi of unc-52/perlecan, ina-1/α integrin, pat-4/ILK, and unc-97/PINCH resulted in abnormal CKI-1::GFP localization.These data also suggest that integrin plays a major role in maintaining proper CKI-1/p27(KIP1) levels in the cell.Perturbed integrin signaling may lead to the inhibition of SCF ligase activity, mislocalization and elevation of CKI-1/p27(KIP1).

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Baylor University, Waco, Texas, United States of America.

ABSTRACT
The cell-extracellular matrix (ECM) interaction plays an essential role in maintaining tissue shapes and regulates cell behaviors such as cell adhesion, differentiation and proliferation. The mechanism by which the ECM influences the cell cycle in vivo is poorly understood. Here we demonstrate that the β integrin PAT-3 regulates the localization and expression of CKI-1, a C. elegans homologue of the cyclin dependent kinase inhibitor p27(KIP1). In nematodes expressing wild type PAT-3, CKI-1::GFP localizes primarily to nucleoli in hypodermal cells, whereas in animals expressing mutant pat-3 with a defective splice junction, CKI-1::GFP appears clumped and disorganized in nucleoplasm. RNAi analysis links cell adhesion genes to the regulation of CKI-1. RNAi of unc-52/perlecan, ina-1/α integrin, pat-4/ILK, and unc-97/PINCH resulted in abnormal CKI-1::GFP localization. Additional RNAi experiments revealed that the SCF E3 ubiquitin-ligase complex genes, skpt-1/SKP2, cul-1/CUL1 and lin-23/F-box, are required for the proper localization and expression of CKI-1, suggesting that integrin signaling and SCF E3 ligase work together to regulate the cellular distribution of CKI-1. These data also suggest that integrin plays a major role in maintaining proper CKI-1/p27(KIP1) levels in the cell. Perturbed integrin signaling may lead to the inhibition of SCF ligase activity, mislocalization and elevation of CKI-1/p27(KIP1). These results suggest that adhesion signaling is crucial for cell cycle regulation in vivo.

Show MeSH
Related in: MedlinePlus