Limits...
Perturbing the ubiquitin pathway reveals how mitosis is hijacked to denucleate and regulate cell proliferation and differentiation in vivo.

Caceres A, Shang F, Wawrousek E, Liu Q, Avidan O, Cvekl A, Yang Y, Haririnia A, Storaska A, Fushman D, Kuszak J, Dudek E, Smith D, Taylor A - PLoS ONE (2010)

Bottom Line: Of the 7 lysines (K) least is known about effects of modification of K6.Thus the nucleus remains intact and DNAseIIβ neither gains entry to the nucleus nor degrades the DNA.These results could not be obtained using chemical proteasome inhibitors that cannot be directed to specific tissues.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Nutrition and Vision Research, US Department of Agriculture Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts, United States of America.

ABSTRACT

Background: The eye lens presents a unique opportunity to explore roles for specific molecules in cell proliferation, differentiation and development because cells remain in place throughout life and, like red blood cells and keratinocytes, they go through the most extreme differentiation, including removal of nuclei and cessation of protein synthesis. Ubiquitination controls many critical cellular processes, most of which require specific lysines on ubiquitin (Ub). Of the 7 lysines (K) least is known about effects of modification of K6.

Methodology and principal findings: We replaced K6 with tryptophan (W) because K6 is the most readily modified K and W is the most structurally similar residue to biotin. The backbone of K6W-Ub is indistinguishable from that of Wt-Ub. K6W-Ub is effectively conjugated and deconjugated but the conjugates are not degraded via the ubiquitin proteasome pathways (UPP). Expression of K6W-ubiquitin in the lens and lens cells results in accumulation of intracellular aggregates and also slows cell proliferation and the differentiation program, including expression of lens specific proteins, differentiation of epithelial cells into fibers, achieving proper fiber cell morphology, and removal of nuclei. The latter is critical for transparency, but the mechanism by which cell nuclei are removed has remained an age old enigma. This was also solved by expressing K6W-Ub. p27(kip), a UPP substrate accumulates in lenses which express K6W-Ub. This precludes phosphorylation of nuclear lamin by the mitotic kinase, a prerequisite for disassembly of the nuclear membrane. Thus the nucleus remains intact and DNAseII╬▓ neither gains entry to the nucleus nor degrades the DNA. These results could not be obtained using chemical proteasome inhibitors that cannot be directed to specific tissues.

Conclusions and significance: K6W-Ub provides a novel, genetic means to study functions of the UPP because it can be targeted to specific cells and tissues. A fully functional UPP is required to execute most stages of lens differentiation, specifically removal of cell nuclei. In the absence of a functional UPP, small aggregate prone, cataractous lenses are formed.

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K6 on Ub is required to degrade p27.(A) HLEC expressing K6W-Ub show stabilization of p27 in G2/M synchronized cells. Cells were synchronized in early mitosis phase using nocodazole. K6W-ubiquitin was expressed in HLEC via an adenoviral vector and levels of endogenous p27 were determined by western blotting. (B) Cdk/cyclin B activity is blocked in a dose-dependent manner by the addition of p27 recombinant protein in vitro. Cdk1/cyclin B activity was determined in vitro by the quantification of phosphorylated-Rb by ELISA. (C) HLEC expressing K6W-Ub show decreased phosphorylation of lamin A/C in early and late G2/M phase. Cells were synchronized at G1 phase with hydroxyurea. After release of hydroxyurea, cells were allowed to grow for 4, 12 and 16 hrs to synchronize at S, early and late G2/M phase respectively. Immunohistochemistry was done to detect levels of phosphorylated lamin A/C using anti-lamin A/C (phospho Ser 392). (D) HLEC in which K6W-Ub was expressed show significantly lower amounts of phosphorylated lamin A/C at early and late G2/M phase. Cells positive for phosphorylated lamin A/C were counted from 10 different fields from fluorescent micrographs in panel C. Positive cells were averaged and statistics were performed at p<0.05.
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pone-0013331-g005: K6 on Ub is required to degrade p27.(A) HLEC expressing K6W-Ub show stabilization of p27 in G2/M synchronized cells. Cells were synchronized in early mitosis phase using nocodazole. K6W-ubiquitin was expressed in HLEC via an adenoviral vector and levels of endogenous p27 were determined by western blotting. (B) Cdk/cyclin B activity is blocked in a dose-dependent manner by the addition of p27 recombinant protein in vitro. Cdk1/cyclin B activity was determined in vitro by the quantification of phosphorylated-Rb by ELISA. (C) HLEC expressing K6W-Ub show decreased phosphorylation of lamin A/C in early and late G2/M phase. Cells were synchronized at G1 phase with hydroxyurea. After release of hydroxyurea, cells were allowed to grow for 4, 12 and 16 hrs to synchronize at S, early and late G2/M phase respectively. Immunohistochemistry was done to detect levels of phosphorylated lamin A/C using anti-lamin A/C (phospho Ser 392). (D) HLEC in which K6W-Ub was expressed show significantly lower amounts of phosphorylated lamin A/C at early and late G2/M phase. Cells positive for phosphorylated lamin A/C were counted from 10 different fields from fluorescent micrographs in panel C. Positive cells were averaged and statistics were performed at p<0.05.

Mentions: Mitotic models were used to corroborate and extend the above results. p27 is seen to accumulate both at its native mass and as high mass conjugates in cells synchronized in the G2/M phase of the cell cycle when K6W-Ub is overexpressed, but not when cells are infected with control virus (Figure 5A). In vitro tests showed that p27 indeed inhibits the Cdk1/cyclinB complex and that its inhibition is dose dependent (Figure 5B). We then observed that for synchronized cells there are significantly higher levels of phosphorylated lamin in control-infected as compared with K6W-Ub-expressing cells (Figure 5C green, Figure 5D). This corroborates the hypothesis that to promote nuclear disassembly it is necessary at early mitosis to enhance levels of phosphorylated lamin and that that requires diminished concentrations of p27 to allow for activation of the kinase complex. Prior observation of p27-induced inhibition of Cdk1/cyclin B activity and arrest of cells at the G2/M transition are consistent with our data [35], suggesting that, as in other cells, in lens inhibition of Cdk1/cyclin B is a direct consequence of the accumulation of p27 (or other Cdk inhibitors) and that expression of K6W-Ub enhances this phenomenon. Thus, the data define a novel pathway by which nuclei can be removed during lens cell differentiation.


Perturbing the ubiquitin pathway reveals how mitosis is hijacked to denucleate and regulate cell proliferation and differentiation in vivo.

Caceres A, Shang F, Wawrousek E, Liu Q, Avidan O, Cvekl A, Yang Y, Haririnia A, Storaska A, Fushman D, Kuszak J, Dudek E, Smith D, Taylor A - PLoS ONE (2010)

K6 on Ub is required to degrade p27.(A) HLEC expressing K6W-Ub show stabilization of p27 in G2/M synchronized cells. Cells were synchronized in early mitosis phase using nocodazole. K6W-ubiquitin was expressed in HLEC via an adenoviral vector and levels of endogenous p27 were determined by western blotting. (B) Cdk/cyclin B activity is blocked in a dose-dependent manner by the addition of p27 recombinant protein in vitro. Cdk1/cyclin B activity was determined in vitro by the quantification of phosphorylated-Rb by ELISA. (C) HLEC expressing K6W-Ub show decreased phosphorylation of lamin A/C in early and late G2/M phase. Cells were synchronized at G1 phase with hydroxyurea. After release of hydroxyurea, cells were allowed to grow for 4, 12 and 16 hrs to synchronize at S, early and late G2/M phase respectively. Immunohistochemistry was done to detect levels of phosphorylated lamin A/C using anti-lamin A/C (phospho Ser 392). (D) HLEC in which K6W-Ub was expressed show significantly lower amounts of phosphorylated lamin A/C at early and late G2/M phase. Cells positive for phosphorylated lamin A/C were counted from 10 different fields from fluorescent micrographs in panel C. Positive cells were averaged and statistics were performed at p<0.05.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2958118&req=5

pone-0013331-g005: K6 on Ub is required to degrade p27.(A) HLEC expressing K6W-Ub show stabilization of p27 in G2/M synchronized cells. Cells were synchronized in early mitosis phase using nocodazole. K6W-ubiquitin was expressed in HLEC via an adenoviral vector and levels of endogenous p27 were determined by western blotting. (B) Cdk/cyclin B activity is blocked in a dose-dependent manner by the addition of p27 recombinant protein in vitro. Cdk1/cyclin B activity was determined in vitro by the quantification of phosphorylated-Rb by ELISA. (C) HLEC expressing K6W-Ub show decreased phosphorylation of lamin A/C in early and late G2/M phase. Cells were synchronized at G1 phase with hydroxyurea. After release of hydroxyurea, cells were allowed to grow for 4, 12 and 16 hrs to synchronize at S, early and late G2/M phase respectively. Immunohistochemistry was done to detect levels of phosphorylated lamin A/C using anti-lamin A/C (phospho Ser 392). (D) HLEC in which K6W-Ub was expressed show significantly lower amounts of phosphorylated lamin A/C at early and late G2/M phase. Cells positive for phosphorylated lamin A/C were counted from 10 different fields from fluorescent micrographs in panel C. Positive cells were averaged and statistics were performed at p<0.05.
Mentions: Mitotic models were used to corroborate and extend the above results. p27 is seen to accumulate both at its native mass and as high mass conjugates in cells synchronized in the G2/M phase of the cell cycle when K6W-Ub is overexpressed, but not when cells are infected with control virus (Figure 5A). In vitro tests showed that p27 indeed inhibits the Cdk1/cyclinB complex and that its inhibition is dose dependent (Figure 5B). We then observed that for synchronized cells there are significantly higher levels of phosphorylated lamin in control-infected as compared with K6W-Ub-expressing cells (Figure 5C green, Figure 5D). This corroborates the hypothesis that to promote nuclear disassembly it is necessary at early mitosis to enhance levels of phosphorylated lamin and that that requires diminished concentrations of p27 to allow for activation of the kinase complex. Prior observation of p27-induced inhibition of Cdk1/cyclin B activity and arrest of cells at the G2/M transition are consistent with our data [35], suggesting that, as in other cells, in lens inhibition of Cdk1/cyclin B is a direct consequence of the accumulation of p27 (or other Cdk inhibitors) and that expression of K6W-Ub enhances this phenomenon. Thus, the data define a novel pathway by which nuclei can be removed during lens cell differentiation.

Bottom Line: Of the 7 lysines (K) least is known about effects of modification of K6.Thus the nucleus remains intact and DNAseIIβ neither gains entry to the nucleus nor degrades the DNA.These results could not be obtained using chemical proteasome inhibitors that cannot be directed to specific tissues.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Nutrition and Vision Research, US Department of Agriculture Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts, United States of America.

ABSTRACT

Background: The eye lens presents a unique opportunity to explore roles for specific molecules in cell proliferation, differentiation and development because cells remain in place throughout life and, like red blood cells and keratinocytes, they go through the most extreme differentiation, including removal of nuclei and cessation of protein synthesis. Ubiquitination controls many critical cellular processes, most of which require specific lysines on ubiquitin (Ub). Of the 7 lysines (K) least is known about effects of modification of K6.

Methodology and principal findings: We replaced K6 with tryptophan (W) because K6 is the most readily modified K and W is the most structurally similar residue to biotin. The backbone of K6W-Ub is indistinguishable from that of Wt-Ub. K6W-Ub is effectively conjugated and deconjugated but the conjugates are not degraded via the ubiquitin proteasome pathways (UPP). Expression of K6W-ubiquitin in the lens and lens cells results in accumulation of intracellular aggregates and also slows cell proliferation and the differentiation program, including expression of lens specific proteins, differentiation of epithelial cells into fibers, achieving proper fiber cell morphology, and removal of nuclei. The latter is critical for transparency, but the mechanism by which cell nuclei are removed has remained an age old enigma. This was also solved by expressing K6W-Ub. p27(kip), a UPP substrate accumulates in lenses which express K6W-Ub. This precludes phosphorylation of nuclear lamin by the mitotic kinase, a prerequisite for disassembly of the nuclear membrane. Thus the nucleus remains intact and DNAseII╬▓ neither gains entry to the nucleus nor degrades the DNA. These results could not be obtained using chemical proteasome inhibitors that cannot be directed to specific tissues.

Conclusions and significance: K6W-Ub provides a novel, genetic means to study functions of the UPP because it can be targeted to specific cells and tissues. A fully functional UPP is required to execute most stages of lens differentiation, specifically removal of cell nuclei. In the absence of a functional UPP, small aggregate prone, cataractous lenses are formed.

Show MeSH
Related in: MedlinePlus