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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 between amino acids 4875 and 4884 serves as an NES. (A) Sequence of the putative NES in AHNAK. The amino acid residues substituted by alanine residues are in bold. (B) Removal of the putative NES or introduction of mutations into Leu 4879 and Ile 4882 result in nuclear retention of AHNAK. Constructs were transfected into dense HeLa cells. (C) Quantitative analysis of the subcellular distribution of transfected AHNAK polypeptides in dense HeLa cells was performed as described in legend to Fig. 6.
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fig9: The region between amino acids 4875 and 4884 serves as an NES. (A) Sequence of the putative NES in AHNAK. The amino acid residues substituted by alanine residues are in bold. (B) Removal of the putative NES or introduction of mutations into Leu 4879 and Ile 4882 result in nuclear retention of AHNAK. Constructs were transfected into dense HeLa cells. (C) Quantitative analysis of the subcellular distribution of transfected AHNAK polypeptides in dense HeLa cells was performed as described in legend to Fig. 6.

Mentions: To verify the presence of NES between positions 4875 and 4884 in the AHNAK protein, fusion protein EGFP–C755 was generated spanning the COOH-terminal sequences of AHNAK starting immediately after the putative NES at position 4887. This fusion protein was overwhelmingly nuclear in both sparsely or densely growing HeLa cells (Fig. 9) . Because localization of the EGFP–C817 was cytoplasmic in densely growing cells and responsive to treatment with LMB, whereas EGFP–C755 was nuclear under all conditions, the possibility of a functional NES within the first 62 amino acids of C817 was further strengthened. To examine whether constitutive phosphorylation of AHNAK at serine 5535 could interfere with nuclear localization of EGFP–C755, an aspartate substitution was introduced into this construct. EGFP–C755S/D was found predominantly in the nuclei of transfected cells similar to shorter fusions EGFP–C581 and C213 with serine 5535 to asparate substitution described above (Fig. 9). This is compatible with the explanation that the putative NES absent from EGFP–C755 is necessary for the nuclear export of PKB-phosphorylated AHNAK.


Protein kinase B phosphorylates AHNAK and regulates its subcellular localization.

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

The region between amino acids 4875 and 4884 serves as an NES. (A) Sequence of the putative NES in AHNAK. The amino acid residues substituted by alanine residues are in bold. (B) Removal of the putative NES or introduction of mutations into Leu 4879 and Ile 4882 result in nuclear retention of AHNAK. Constructs were transfected into dense HeLa cells. (C) Quantitative analysis of the subcellular distribution of transfected AHNAK polypeptides in dense HeLa cells was performed as described in legend to Fig. 6.
© Copyright Policy
Related In: Results  -  Collection

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

fig9: The region between amino acids 4875 and 4884 serves as an NES. (A) Sequence of the putative NES in AHNAK. The amino acid residues substituted by alanine residues are in bold. (B) Removal of the putative NES or introduction of mutations into Leu 4879 and Ile 4882 result in nuclear retention of AHNAK. Constructs were transfected into dense HeLa cells. (C) Quantitative analysis of the subcellular distribution of transfected AHNAK polypeptides in dense HeLa cells was performed as described in legend to Fig. 6.
Mentions: To verify the presence of NES between positions 4875 and 4884 in the AHNAK protein, fusion protein EGFP–C755 was generated spanning the COOH-terminal sequences of AHNAK starting immediately after the putative NES at position 4887. This fusion protein was overwhelmingly nuclear in both sparsely or densely growing HeLa cells (Fig. 9) . Because localization of the EGFP–C817 was cytoplasmic in densely growing cells and responsive to treatment with LMB, whereas EGFP–C755 was nuclear under all conditions, the possibility of a functional NES within the first 62 amino acids of C817 was further strengthened. To examine whether constitutive phosphorylation of AHNAK at serine 5535 could interfere with nuclear localization of EGFP–C755, an aspartate substitution was introduced into this construct. EGFP–C755S/D was found predominantly in the nuclei of transfected cells similar to shorter fusions EGFP–C581 and C213 with serine 5535 to asparate substitution described above (Fig. 9). This is compatible with the explanation that the putative NES absent from EGFP–C755 is necessary for the nuclear export of PKB-phosphorylated AHNAK.

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