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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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RT-PCR analysis of cki-1 mRNA in cki-1::GFP transgenic and βpat-3 rescued lines lacking cki-1::GFP.Panel A: Lanes 1 and 2 display the level of mRNA in pat-3(+) and pat-3(sp) animals, respectively. The GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene, gpd-1, was used as an mRNA loading control. The level of each transcript was measured using ImageJ software. Quantification of cDNA using ImageJ software revealed that cki-1 mRNA levels were similar in all lines compared (Table S1). Panel B: cki-1 mRNA was measured in βpat-3 rescued lines lacking the CKI-1::GFP construct. Lanes 1–3 are N2, JE443 pat-3(+), and BU7221 pat-3(sp). The GAPDH gene, gpd-1, was used as an mRNA loading control. Quantification of cDNA levels using ImageJ software revealed that cki-1 mRNA levels were similar in all lines compared (Table S1).
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pone-0042425-g004: RT-PCR analysis of cki-1 mRNA in cki-1::GFP transgenic and βpat-3 rescued lines lacking cki-1::GFP.Panel A: Lanes 1 and 2 display the level of mRNA in pat-3(+) and pat-3(sp) animals, respectively. The GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene, gpd-1, was used as an mRNA loading control. The level of each transcript was measured using ImageJ software. Quantification of cDNA using ImageJ software revealed that cki-1 mRNA levels were similar in all lines compared (Table S1). Panel B: cki-1 mRNA was measured in βpat-3 rescued lines lacking the CKI-1::GFP construct. Lanes 1–3 are N2, JE443 pat-3(+), and BU7221 pat-3(sp). The GAPDH gene, gpd-1, was used as an mRNA loading control. Quantification of cDNA levels using ImageJ software revealed that cki-1 mRNA levels were similar in all lines compared (Table S1).

Mentions: Because the immunoblot results revealed that pat-3(sp) animals produced more CKI-1::GFP protein than pat-3(+), we next assessed the effect on cki-1 transcription. RNA from each rescued line was isolated and analyzed for the amount of pat-3 or cki-1 mRNA using RT-PCR (Figure 4A). We also measured the cki-1 mRNA level in BU7221, a pat-3(sp) rescued line without cki-1::GFP [34]. No significant differences were seen in any of the experiments, suggesting that pat-3(sp) does not significantly increase the level of cki-1 mRNA compared to controls (Figure 4B).


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)

RT-PCR analysis of cki-1 mRNA in cki-1::GFP transgenic and βpat-3 rescued lines lacking cki-1::GFP.Panel A: Lanes 1 and 2 display the level of mRNA in pat-3(+) and pat-3(sp) animals, respectively. The GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene, gpd-1, was used as an mRNA loading control. The level of each transcript was measured using ImageJ software. Quantification of cDNA using ImageJ software revealed that cki-1 mRNA levels were similar in all lines compared (Table S1). Panel B: cki-1 mRNA was measured in βpat-3 rescued lines lacking the CKI-1::GFP construct. Lanes 1–3 are N2, JE443 pat-3(+), and BU7221 pat-3(sp). The GAPDH gene, gpd-1, was used as an mRNA loading control. Quantification of cDNA levels using ImageJ software revealed that cki-1 mRNA levels were similar in all lines compared (Table S1).
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pone-0042425-g004: RT-PCR analysis of cki-1 mRNA in cki-1::GFP transgenic and βpat-3 rescued lines lacking cki-1::GFP.Panel A: Lanes 1 and 2 display the level of mRNA in pat-3(+) and pat-3(sp) animals, respectively. The GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene, gpd-1, was used as an mRNA loading control. The level of each transcript was measured using ImageJ software. Quantification of cDNA using ImageJ software revealed that cki-1 mRNA levels were similar in all lines compared (Table S1). Panel B: cki-1 mRNA was measured in βpat-3 rescued lines lacking the CKI-1::GFP construct. Lanes 1–3 are N2, JE443 pat-3(+), and BU7221 pat-3(sp). The GAPDH gene, gpd-1, was used as an mRNA loading control. Quantification of cDNA levels using ImageJ software revealed that cki-1 mRNA levels were similar in all lines compared (Table S1).
Mentions: Because the immunoblot results revealed that pat-3(sp) animals produced more CKI-1::GFP protein than pat-3(+), we next assessed the effect on cki-1 transcription. RNA from each rescued line was isolated and analyzed for the amount of pat-3 or cki-1 mRNA using RT-PCR (Figure 4A). We also measured the cki-1 mRNA level in BU7221, a pat-3(sp) rescued line without cki-1::GFP [34]. No significant differences were seen in any of the experiments, suggesting that pat-3(sp) does not significantly increase the level of cki-1 mRNA compared to controls (Figure 4B).

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