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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 the COOH-terminal domain of AHNAK fused to EGFP is regulated by the phosphorylation status of serine 5535. (A) Amino acid sequence of the region of AHNAK around the PKB phosphorylation site and mutations introduced into it. (B) Localization of the EGFP–COOH-terminal AHNAK fusion proteins in transiently transfected HeLa cells.
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fig5: Localization of the COOH-terminal domain of AHNAK fused to EGFP is regulated by the phosphorylation status of serine 5535. (A) Amino acid sequence of the region of AHNAK around the PKB phosphorylation site and mutations introduced into it. (B) Localization of the EGFP–COOH-terminal AHNAK fusion proteins in transiently transfected HeLa cells.

Mentions: To directly demonstrate a role for PKB phosphorylation in localization of AHNAK we examined localization of site-specific AHNAK mutants affecting PKB site. We have subcloned the COOH terminus of AHNAK into vector pEGFP.C1 to create a fusion of the enhanced green fluorescent protein (EGFP) with the last 998 amino acids of AHNAK (EGFP-C998). Site-specific mutations of AHNAK shown in Fig. 5 A were introduced into EGFP–C998.


Protein kinase B phosphorylates AHNAK and regulates its subcellular localization.

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

Localization of the COOH-terminal domain of AHNAK fused to EGFP is regulated by the phosphorylation status of serine 5535. (A) Amino acid sequence of the region of AHNAK around the PKB phosphorylation site and mutations introduced into it. (B) Localization of the EGFP–COOH-terminal AHNAK fusion proteins in transiently transfected HeLa cells.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Localization of the COOH-terminal domain of AHNAK fused to EGFP is regulated by the phosphorylation status of serine 5535. (A) Amino acid sequence of the region of AHNAK around the PKB phosphorylation site and mutations introduced into it. (B) Localization of the EGFP–COOH-terminal AHNAK fusion proteins in transiently transfected HeLa cells.
Mentions: To directly demonstrate a role for PKB phosphorylation in localization of AHNAK we examined localization of site-specific AHNAK mutants affecting PKB site. We have subcloned the COOH terminus of AHNAK into vector pEGFP.C1 to create a fusion of the enhanced green fluorescent protein (EGFP) with the last 998 amino acids of AHNAK (EGFP-C998). Site-specific mutations of AHNAK shown in Fig. 5 A were introduced into EGFP–C998.

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