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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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The region in the COOH terminus of AHNAK between amino acids 4826 and 5068 is necessary for the extranuclear localization of AHNAK and might contain NES. (A) Localization of the indicated EGFP fusion constructs in transiently transfected HeLa cells 24 h after transfection. Cells shown in the right panels were treated with 20 ng/ml LMB for 1 h. (B) Quantitative analysis of the subcellular distribution of transfected AHNAK polypeptides in dense HeLa cells was performed as described in legend to Fig. 6. Gray bars represent localization in untreated dense cells; black bars represent cultures treated with LMB at 20 ng/ml for 1 h.
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fig7: The region in the COOH terminus of AHNAK between amino acids 4826 and 5068 is necessary for the extranuclear localization of AHNAK and might contain NES. (A) Localization of the indicated EGFP fusion constructs in transiently transfected HeLa cells 24 h after transfection. Cells shown in the right panels were treated with 20 ng/ml LMB for 1 h. (B) Quantitative analysis of the subcellular distribution of transfected AHNAK polypeptides in dense HeLa cells was performed as described in legend to Fig. 6. Gray bars represent localization in untreated dense cells; black bars represent cultures treated with LMB at 20 ng/ml for 1 h.

Mentions: To further analyze the specific role of phosphorylation by PKB in nuclear exclusion of AHNAK, we have conducted experiments aimed at determining the smallest domain of AHNAK whose localization is still regulated by PKB. We have constructed three additional EGFP–AHNAK fusion constructs encoding progressively shorter COOH-terminal fragments of AHNAK (Fig. 7) . EGFP–C817 assumed predominantly nuclear localization upon transfection into sparsely plated cells, but in dense cells was found in cytoplasm of most cells (Fig. 7) behaving essentially as EGFP–C998. Introduction of serine 5535 to aspartate substitution into EGFP–C817 had the same effect on its localization as on localization of the longer EGFP–C998 resulting in nuclear exclusion of most of the protein irrespective of cell density (Fig. 7). Two shorter EGFP fusion constructs were prepared containing the last 575 and 213 amino acids of AHNAK. Both of them produced polypeptides that were overwhelmingly nuclear upon transfection into HeLa cells. Density of transfected cells had no effect on the nuclear localization of shorter EGFP fusions (Fig. 7).


Protein kinase B phosphorylates AHNAK and regulates its subcellular localization.

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

The region in the COOH terminus of AHNAK between amino acids 4826 and 5068 is necessary for the extranuclear localization of AHNAK and might contain NES. (A) Localization of the indicated EGFP fusion constructs in transiently transfected HeLa cells 24 h after transfection. Cells shown in the right panels were treated with 20 ng/ml LMB for 1 h. (B) Quantitative analysis of the subcellular distribution of transfected AHNAK polypeptides in dense HeLa cells was performed as described in legend to Fig. 6. Gray bars represent localization in untreated dense cells; black bars represent cultures treated with LMB at 20 ng/ml for 1 h.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: The region in the COOH terminus of AHNAK between amino acids 4826 and 5068 is necessary for the extranuclear localization of AHNAK and might contain NES. (A) Localization of the indicated EGFP fusion constructs in transiently transfected HeLa cells 24 h after transfection. Cells shown in the right panels were treated with 20 ng/ml LMB for 1 h. (B) Quantitative analysis of the subcellular distribution of transfected AHNAK polypeptides in dense HeLa cells was performed as described in legend to Fig. 6. Gray bars represent localization in untreated dense cells; black bars represent cultures treated with LMB at 20 ng/ml for 1 h.
Mentions: To further analyze the specific role of phosphorylation by PKB in nuclear exclusion of AHNAK, we have conducted experiments aimed at determining the smallest domain of AHNAK whose localization is still regulated by PKB. We have constructed three additional EGFP–AHNAK fusion constructs encoding progressively shorter COOH-terminal fragments of AHNAK (Fig. 7) . EGFP–C817 assumed predominantly nuclear localization upon transfection into sparsely plated cells, but in dense cells was found in cytoplasm of most cells (Fig. 7) behaving essentially as EGFP–C998. Introduction of serine 5535 to aspartate substitution into EGFP–C817 had the same effect on its localization as on localization of the longer EGFP–C998 resulting in nuclear exclusion of most of the protein irrespective of cell density (Fig. 7). Two shorter EGFP fusion constructs were prepared containing the last 575 and 213 amino acids of AHNAK. Both of them produced polypeptides that were overwhelmingly nuclear upon transfection into HeLa cells. Density of transfected cells had no effect on the nuclear localization of shorter EGFP fusions (Fig. 7).

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