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Protein kinase B phosphorylates AHNAK and regulates its subcellular localization.

Sussman J, Stokoe D, Ossina N, Shtivelman E - J. Cell Biol. (2001)

Bottom Line: We show that AHNAK is a phosphorylation substrate of protein kinase B (PKB) in vitro and in vivo.Nuclear exclusion of AHNAK is mediated through a nuclear export signal (NES) in a manner that depends on the phosphorylation of serine 5535 of AHNAK by PKB, a process that also plays a major role in determining extranuclear localization of AHNAK.AHNAK is a new PKB substrate whose function, though unknown, is likely to be regulated by its localization, which is in turn regulated by PKB.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research Institute, University of California at San Francisco, San Francisco, CA 94143, USA.

ABSTRACT
AHNAK is a ubiquitously expressed giant phosphoprotein that was initially identified as a gene product subject to transcriptional repression in neuroblastoma. AHNAK is predominantly nuclear in cells of nonepithelial origin, but is cytoplasmic or associated with plasma membrane in epithelial cells. In this study we show that the extranuclear localization of AHNAK in epithelial cells depends on the formation of cell-cell contacts. We show that AHNAK is a phosphorylation substrate of protein kinase B (PKB) in vitro and in vivo. Nuclear exclusion of AHNAK is mediated through a nuclear export signal (NES) in a manner that depends on the phosphorylation of serine 5535 of AHNAK by PKB, a process that also plays a major role in determining extranuclear localization of AHNAK. AHNAK is a new PKB substrate whose function, though unknown, is likely to be regulated by its localization, which is in turn regulated by PKB.

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Localization of endogenous AHNAK in cells at different densities and nuclear translocation after inhibition of PI 3-kinase pathway. Cells were processed for indirect immunofluorescence 1 d after plating (sparse) or 3 d after plating (dense). Dense cultures were treated for the last 1 h before fixation with the PI 3-kinase inhibitor LY294002 at 30 mM. Cells were stained as described in the legend to Fig. 2. Treatment of sparse cultures with LY294002 did not affect localization of AHNAK (unpublished data).
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fig3: Localization of endogenous AHNAK in cells at different densities and nuclear translocation after inhibition of PI 3-kinase pathway. Cells were processed for indirect immunofluorescence 1 d after plating (sparse) or 3 d after plating (dense). Dense cultures were treated for the last 1 h before fixation with the PI 3-kinase inhibitor LY294002 at 30 mM. Cells were stained as described in the legend to Fig. 2. Treatment of sparse cultures with LY294002 did not affect localization of AHNAK (unpublished data).

Mentions: Next, we examined the effect of inhibition of PKB activity on localization of AHNAK. Cells were treated for different lengths of time with LY294002 before immunofluorescent staining. These experiments were performed with HeLa and MDCK cells derived from immortalized canine kidney epithelium. AHNAK was nuclear in freshly plated (24 h or less) MDCK cells, but redistributed to plasma membrane in cultures allowed to reach confluence for 48–72 h (Fig. 3) .


Protein kinase B phosphorylates AHNAK and regulates its subcellular localization.

Sussman J, Stokoe D, Ossina N, Shtivelman E - J. Cell Biol. (2001)

Localization of endogenous AHNAK in cells at different densities and nuclear translocation after inhibition of PI 3-kinase pathway. Cells were processed for indirect immunofluorescence 1 d after plating (sparse) or 3 d after plating (dense). Dense cultures were treated for the last 1 h before fixation with the PI 3-kinase inhibitor LY294002 at 30 mM. Cells were stained as described in the legend to Fig. 2. Treatment of sparse cultures with LY294002 did not affect localization of AHNAK (unpublished data).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196192&req=5

fig3: Localization of endogenous AHNAK in cells at different densities and nuclear translocation after inhibition of PI 3-kinase pathway. Cells were processed for indirect immunofluorescence 1 d after plating (sparse) or 3 d after plating (dense). Dense cultures were treated for the last 1 h before fixation with the PI 3-kinase inhibitor LY294002 at 30 mM. Cells were stained as described in the legend to Fig. 2. Treatment of sparse cultures with LY294002 did not affect localization of AHNAK (unpublished data).
Mentions: Next, we examined the effect of inhibition of PKB activity on localization of AHNAK. Cells were treated for different lengths of time with LY294002 before immunofluorescent staining. These experiments were performed with HeLa and MDCK cells derived from immortalized canine kidney epithelium. AHNAK was nuclear in freshly plated (24 h or less) MDCK cells, but redistributed to plasma membrane in cultures allowed to reach confluence for 48–72 h (Fig. 3) .

Bottom Line: We show that AHNAK is a phosphorylation substrate of protein kinase B (PKB) in vitro and in vivo.Nuclear exclusion of AHNAK is mediated through a nuclear export signal (NES) in a manner that depends on the phosphorylation of serine 5535 of AHNAK by PKB, a process that also plays a major role in determining extranuclear localization of AHNAK.AHNAK is a new PKB substrate whose function, though unknown, is likely to be regulated by its localization, which is in turn regulated by PKB.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research Institute, University of California at San Francisco, San Francisco, CA 94143, USA.

ABSTRACT
AHNAK is a ubiquitously expressed giant phosphoprotein that was initially identified as a gene product subject to transcriptional repression in neuroblastoma. AHNAK is predominantly nuclear in cells of nonepithelial origin, but is cytoplasmic or associated with plasma membrane in epithelial cells. In this study we show that the extranuclear localization of AHNAK in epithelial cells depends on the formation of cell-cell contacts. We show that AHNAK is a phosphorylation substrate of protein kinase B (PKB) in vitro and in vivo. Nuclear exclusion of AHNAK is mediated through a nuclear export signal (NES) in a manner that depends on the phosphorylation of serine 5535 of AHNAK by PKB, a process that also plays a major role in determining extranuclear localization of AHNAK. AHNAK is a new PKB substrate whose function, though unknown, is likely to be regulated by its localization, which is in turn regulated by PKB.

Show MeSH
Related in: MedlinePlus