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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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Related in: MedlinePlus

AHNAK is a phosphorylation substrate of PKB in vitro. Purified PKB was tested with the GST fusion proteins indicated as described in the Materials and methods. GST–Bad served as a positive control for phosphorylation and GST as a negative control. GST–C213 contains the last 213 amino acids of AHNAK fused to GST. The S/A construct contains a substitution of serine to alanine within the PKB consensus site. GST–M466 contains part of the AHNAK middle domain consisting of four repeats.
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fig1: AHNAK is a phosphorylation substrate of PKB in vitro. Purified PKB was tested with the GST fusion proteins indicated as described in the Materials and methods. GST–Bad served as a positive control for phosphorylation and GST as a negative control. GST–C213 contains the last 213 amino acids of AHNAK fused to GST. The S/A construct contains a substitution of serine to alanine within the PKB consensus site. GST–M466 contains part of the AHNAK middle domain consisting of four repeats.

Mentions: Examination of the amino acid sequence of AHNAK revealed the presence of a single consensus phosphorylation site for PKB surrounding serine residue 5535 (RHRSNSF) in the COOH-terminal domain of AHNAK. We have examined the functionality of the putative PKB phosphorylation site at serine 5535 in the in vitro phosphorylation assays. Three glutathione S-transferase (GST)–AHNAK fusion polypeptides were produced and purified from bacteria to test as PKB substrates. GST–C213 encompassed the last 213 amino acids of AHNAK (between positions 5431 and 5643), including the PKB consensus phosphorylation site at 5535. In GST–C213S/A serine 5535, the hypothetical phosphorylation target of PKB was converted to an alanine. The third GST fusion (GST–M466) contained 466 amino acids from the middle domain of AHNAK, encompassing three and a half AHNAK repeats that contain multiple PKC and casein kinase II consensus phosphorylation sites but no PKB consensus sites (unpublished data). These polypeptides and the GST–Bad fusion polypeptide (positive control for PKB phosphorylation) were used in the in vitro kinase assays with active PKB purified from insect cells. Out of the three GST–AHNAK polypeptides only GST–C213 was phosphorylated in vitro; substitution of serine to alanine in GST–S/A completely abolished the appearance of a phosphorylated band (Fig. 1) . The unrelated AHNAK polypeptide GST–M466 was not phosphorylated either. These results show that serine 5535 of AHNAK functions as an in vitro substrate of PKB.


Protein kinase B phosphorylates AHNAK and regulates its subcellular localization.

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

AHNAK is a phosphorylation substrate of PKB in vitro. Purified PKB was tested with the GST fusion proteins indicated as described in the Materials and methods. GST–Bad served as a positive control for phosphorylation and GST as a negative control. GST–C213 contains the last 213 amino acids of AHNAK fused to GST. The S/A construct contains a substitution of serine to alanine within the PKB consensus site. GST–M466 contains part of the AHNAK middle domain consisting of four repeats.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: AHNAK is a phosphorylation substrate of PKB in vitro. Purified PKB was tested with the GST fusion proteins indicated as described in the Materials and methods. GST–Bad served as a positive control for phosphorylation and GST as a negative control. GST–C213 contains the last 213 amino acids of AHNAK fused to GST. The S/A construct contains a substitution of serine to alanine within the PKB consensus site. GST–M466 contains part of the AHNAK middle domain consisting of four repeats.
Mentions: Examination of the amino acid sequence of AHNAK revealed the presence of a single consensus phosphorylation site for PKB surrounding serine residue 5535 (RHRSNSF) in the COOH-terminal domain of AHNAK. We have examined the functionality of the putative PKB phosphorylation site at serine 5535 in the in vitro phosphorylation assays. Three glutathione S-transferase (GST)–AHNAK fusion polypeptides were produced and purified from bacteria to test as PKB substrates. GST–C213 encompassed the last 213 amino acids of AHNAK (between positions 5431 and 5643), including the PKB consensus phosphorylation site at 5535. In GST–C213S/A serine 5535, the hypothetical phosphorylation target of PKB was converted to an alanine. The third GST fusion (GST–M466) contained 466 amino acids from the middle domain of AHNAK, encompassing three and a half AHNAK repeats that contain multiple PKC and casein kinase II consensus phosphorylation sites but no PKB consensus sites (unpublished data). These polypeptides and the GST–Bad fusion polypeptide (positive control for PKB phosphorylation) were used in the in vitro kinase assays with active PKB purified from insect cells. Out of the three GST–AHNAK polypeptides only GST–C213 was phosphorylated in vitro; substitution of serine to alanine in GST–S/A completely abolished the appearance of a phosphorylated band (Fig. 1) . The unrelated AHNAK polypeptide GST–M466 was not phosphorylated either. These results show that serine 5535 of AHNAK functions as an in vitro substrate of PKB.

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