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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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Relocalization of AHNAK to the cytoplasm in dense cultures correlates with an increase in PKB activity. (A) Sparsely growing (top) or dense (bottom) HeLa cells were stained for endogenous AHNAK with a polyclonal anti-AHNAK antiserum against the repeated domain described previously (Shtivelman and Bishop, 1993) and goat anti–rabbit Cy-3–conjugated antibodies. (B) Extracts from HeLa cells grown at normal (subconfluent) density with or without LY294002 treatment (30 μM for 1 h), at low density (sparse), and confluent culture (dense) were subjected to Western blotting with an antibody specific to phosphoserine 473 of PKB (pS473-PKB) to detect activated PKB or antibody to PH domain (PH-PKB) to detect PKB protein.
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fig2: Relocalization of AHNAK to the cytoplasm in dense cultures correlates with an increase in PKB activity. (A) Sparsely growing (top) or dense (bottom) HeLa cells were stained for endogenous AHNAK with a polyclonal anti-AHNAK antiserum against the repeated domain described previously (Shtivelman and Bishop, 1993) and goat anti–rabbit Cy-3–conjugated antibodies. (B) Extracts from HeLa cells grown at normal (subconfluent) density with or without LY294002 treatment (30 μM for 1 h), at low density (sparse), and confluent culture (dense) were subjected to Western blotting with an antibody specific to phosphoserine 473 of PKB (pS473-PKB) to detect activated PKB or antibody to PH domain (PH-PKB) to detect PKB protein.

Mentions: We have shown previously that AHNAK is a predominantly nuclear protein in different cultured cells of nonepithelial origin by subcellular fractionation and immunofluorescent staining (Shtivelman and Bishop, 1993). However, a plasma membrane localization of AHNAK was reported in epithelial cells (Hashimoto et al., 1993, 1995). We have performed immunofluorescent staining of endogenous AHNAK in a HeLa cervical carcinoma cell line. In freshly plated cultures of HeLa cells that have not reached high density, AHNAK was found in the nuclei with a weak diffuse cytoplasmic staining (Fig. 2 A), confirming our previous observations. However, we have noticed that in dense cultures of HeLa cells that have been plated for >2 d, AHNAK assumes mostly cytoplasmic localization (Fig. 2 A).


Protein kinase B phosphorylates AHNAK and regulates its subcellular localization.

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

Relocalization of AHNAK to the cytoplasm in dense cultures correlates with an increase in PKB activity. (A) Sparsely growing (top) or dense (bottom) HeLa cells were stained for endogenous AHNAK with a polyclonal anti-AHNAK antiserum against the repeated domain described previously (Shtivelman and Bishop, 1993) and goat anti–rabbit Cy-3–conjugated antibodies. (B) Extracts from HeLa cells grown at normal (subconfluent) density with or without LY294002 treatment (30 μM for 1 h), at low density (sparse), and confluent culture (dense) were subjected to Western blotting with an antibody specific to phosphoserine 473 of PKB (pS473-PKB) to detect activated PKB or antibody to PH domain (PH-PKB) to detect PKB protein.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Relocalization of AHNAK to the cytoplasm in dense cultures correlates with an increase in PKB activity. (A) Sparsely growing (top) or dense (bottom) HeLa cells were stained for endogenous AHNAK with a polyclonal anti-AHNAK antiserum against the repeated domain described previously (Shtivelman and Bishop, 1993) and goat anti–rabbit Cy-3–conjugated antibodies. (B) Extracts from HeLa cells grown at normal (subconfluent) density with or without LY294002 treatment (30 μM for 1 h), at low density (sparse), and confluent culture (dense) were subjected to Western blotting with an antibody specific to phosphoserine 473 of PKB (pS473-PKB) to detect activated PKB or antibody to PH domain (PH-PKB) to detect PKB protein.
Mentions: We have shown previously that AHNAK is a predominantly nuclear protein in different cultured cells of nonepithelial origin by subcellular fractionation and immunofluorescent staining (Shtivelman and Bishop, 1993). However, a plasma membrane localization of AHNAK was reported in epithelial cells (Hashimoto et al., 1993, 1995). We have performed immunofluorescent staining of endogenous AHNAK in a HeLa cervical carcinoma cell line. In freshly plated cultures of HeLa cells that have not reached high density, AHNAK was found in the nuclei with a weak diffuse cytoplasmic staining (Fig. 2 A), confirming our previous observations. However, we have noticed that in dense cultures of HeLa cells that have been plated for >2 d, AHNAK assumes mostly cytoplasmic localization (Fig. 2 A).

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