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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 subject to regulation by cell density only when the PKB phosphorylation site is intact. (A) The indicated EGFP fusion constructs were transfected into dense HeLa cultures, plated for 2 d, and fixed 24 h later. (B) Quantitative analysis of the subcellular distribution of transfected COOH terminus of AHNAK in dense versus sparse HeLa cells. Experiments were performed at least three times with each construct and at least 200 fluorescent cells were counted per each experiment. White bars represent data obtained with cells plated at low density; gray bars represent data obtained with cells plated at high density. Error is expressed as a standard deviation of the mean values.
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fig6: Localization of the COOH-terminal domain of AHNAK fused to EGFP is subject to regulation by cell density only when the PKB phosphorylation site is intact. (A) The indicated EGFP fusion constructs were transfected into dense HeLa cultures, plated for 2 d, and fixed 24 h later. (B) Quantitative analysis of the subcellular distribution of transfected COOH terminus of AHNAK in dense versus sparse HeLa cells. Experiments were performed at least three times with each construct and at least 200 fluorescent cells were counted per each experiment. White bars represent data obtained with cells plated at low density; gray bars represent data obtained with cells plated at high density. Error is expressed as a standard deviation of the mean values.

Mentions: To examine whether the COOH-terminal domain of AHNAK changes its localization depending on formation of cell–cell contacts, we have performed transient transfection experiments of dense cultures of HeLa cells. As shown in Fig. 6 , we found that the EGFP–C998 fusion of COOH terminus of AHNAK undergoes a change in subcellular localization depending on culture density similar to that shown for endogenous AHNAK. Whereas predominantly nuclear in subconfluent cultures, it is found in cytoplasm of densely plated cells (Fig. 6). However, neither of the two constructs with alterations in the PKB phosphorylation site nor the construct with a mutated NLS are capable of assuming a different localization depending on cell density (Fig. 6). The S/A mutant that cannot be phosphorylated by PKB is mostly retained in the nuclei of transfected cells, whereas the S/D mutant mimicking constitutive phosphorylation and the NLS mutant are retained in the cytoplasm independently of cell density of transfected cultures (Fig. 6 B). These results confirm that shuttling of AHNAK as a function of cell density is regulated at least partially through phosphorylation at the PKB site and the adjacent nuclear localization signal.


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 subject to regulation by cell density only when the PKB phosphorylation site is intact. (A) The indicated EGFP fusion constructs were transfected into dense HeLa cultures, plated for 2 d, and fixed 24 h later. (B) Quantitative analysis of the subcellular distribution of transfected COOH terminus of AHNAK in dense versus sparse HeLa cells. Experiments were performed at least three times with each construct and at least 200 fluorescent cells were counted per each experiment. White bars represent data obtained with cells plated at low density; gray bars represent data obtained with cells plated at high density. Error is expressed as a standard deviation of the mean values.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Localization of the COOH-terminal domain of AHNAK fused to EGFP is subject to regulation by cell density only when the PKB phosphorylation site is intact. (A) The indicated EGFP fusion constructs were transfected into dense HeLa cultures, plated for 2 d, and fixed 24 h later. (B) Quantitative analysis of the subcellular distribution of transfected COOH terminus of AHNAK in dense versus sparse HeLa cells. Experiments were performed at least three times with each construct and at least 200 fluorescent cells were counted per each experiment. White bars represent data obtained with cells plated at low density; gray bars represent data obtained with cells plated at high density. Error is expressed as a standard deviation of the mean values.
Mentions: To examine whether the COOH-terminal domain of AHNAK changes its localization depending on formation of cell–cell contacts, we have performed transient transfection experiments of dense cultures of HeLa cells. As shown in Fig. 6 , we found that the EGFP–C998 fusion of COOH terminus of AHNAK undergoes a change in subcellular localization depending on culture density similar to that shown for endogenous AHNAK. Whereas predominantly nuclear in subconfluent cultures, it is found in cytoplasm of densely plated cells (Fig. 6). However, neither of the two constructs with alterations in the PKB phosphorylation site nor the construct with a mutated NLS are capable of assuming a different localization depending on cell density (Fig. 6). The S/A mutant that cannot be phosphorylated by PKB is mostly retained in the nuclei of transfected cells, whereas the S/D mutant mimicking constitutive phosphorylation and the NLS mutant are retained in the cytoplasm independently of cell density of transfected cultures (Fig. 6 B). These results confirm that shuttling of AHNAK as a function of cell density is regulated at least partially through phosphorylation at the PKB site and the adjacent nuclear localization signal.

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