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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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Characterization of phosphospecific antibodies against PKB-phosphorylated AHNAK and immunofluorescent detection of phosphorylated AHNAK. (A) Purified phosphospecific antibodies (pS5535-AHNAK) were used for Western blot analysis of HEK293 cells transiently transfected with two COOH-terminal expression constructs of AHNAK differing only in one amino acid residue; serine 5535 was either intact (wt) or substituted by alanine (S5535A). As a control, an identical Western blot was detected with antibody fraction from the same rabbit before immunoaffinity purification on phosphopeptide column. (B) Reactivity of phosphospecific antibodies with the endogenous AHNAK is greatly reduced in cells treated with LY294002. Western blot analysis of AHNAK was performed with cells extracts prepared before and after treatment with 30 μM LY294002 for 1 h using purified phosphospecific antibodies. Antiserum against repeat region of AHNAK was used as a control for the total AHNAK protein levels (anti-AHNAK). Activity of PKB in cell extracts was monitored by anti-PKB antibodies as described in the legend to Fig. 2. (C) Western blot analysis of AHNAK phosphorylation on serine 5535 in MDCK neo (control) and caPKB clone expressing caPKB. Cells were either untreated or treated with 30 μM LY294002 for 4 h. Blots were probed with antibodies indicated as in B. (D) Purified phosphospecific antibodies detect preferentially plasma membrane–associated AHNAK in dense MDCK cells but react only weakly with the nuclear AHNAK in cells treated for 1 h with LY294002. Inclusion of the immunizing phosphopeptide abolished reactivity of antibodies with cells (unpublished data). (E) Western blot analysis of AHNAK contents in subcellular fractions prepared from sparsely and densely growing cells. HeLa cells were fractionated into nuclear (N) and cytoplasmic (C) fractions by hypotonic lysis. Aliquots of fractions and total lysates (T) representing approximately equal cell numbers were subjected to electrophoresis and Western blotting with the indicated anti-AHNAK antibodies.
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fig4: Characterization of phosphospecific antibodies against PKB-phosphorylated AHNAK and immunofluorescent detection of phosphorylated AHNAK. (A) Purified phosphospecific antibodies (pS5535-AHNAK) were used for Western blot analysis of HEK293 cells transiently transfected with two COOH-terminal expression constructs of AHNAK differing only in one amino acid residue; serine 5535 was either intact (wt) or substituted by alanine (S5535A). As a control, an identical Western blot was detected with antibody fraction from the same rabbit before immunoaffinity purification on phosphopeptide column. (B) Reactivity of phosphospecific antibodies with the endogenous AHNAK is greatly reduced in cells treated with LY294002. Western blot analysis of AHNAK was performed with cells extracts prepared before and after treatment with 30 μM LY294002 for 1 h using purified phosphospecific antibodies. Antiserum against repeat region of AHNAK was used as a control for the total AHNAK protein levels (anti-AHNAK). Activity of PKB in cell extracts was monitored by anti-PKB antibodies as described in the legend to Fig. 2. (C) Western blot analysis of AHNAK phosphorylation on serine 5535 in MDCK neo (control) and caPKB clone expressing caPKB. Cells were either untreated or treated with 30 μM LY294002 for 4 h. Blots were probed with antibodies indicated as in B. (D) Purified phosphospecific antibodies detect preferentially plasma membrane–associated AHNAK in dense MDCK cells but react only weakly with the nuclear AHNAK in cells treated for 1 h with LY294002. Inclusion of the immunizing phosphopeptide abolished reactivity of antibodies with cells (unpublished data). (E) Western blot analysis of AHNAK contents in subcellular fractions prepared from sparsely and densely growing cells. HeLa cells were fractionated into nuclear (N) and cytoplasmic (C) fractions by hypotonic lysis. Aliquots of fractions and total lysates (T) representing approximately equal cell numbers were subjected to electrophoresis and Western blotting with the indicated anti-AHNAK antibodies.

Mentions: The unusually large size of AHNAK and the presence of numerous phosphorylation sites for a variety of kinases within its sequence precluded phosphopeptide mapping as a means of identification of the PKB-phosphorylated residue. To determine whether PKB phosphorylates AHNAK in vivo, we generated antisera to phosphopeptide containing serine 5535. Antibodies specifically recognizing AHNAK phosphopeptide containing phosphorylated serine 5535 were purified and their specificity was examined. Fig. 4 A shows that purified phosphospecific antibodies specifically recognize an exogenously expressed COOH-terminal polypeptide of AHNAK, but not a version that has serine 5535 mutated to an alanine residue.


Protein kinase B phosphorylates AHNAK and regulates its subcellular localization.

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

Characterization of phosphospecific antibodies against PKB-phosphorylated AHNAK and immunofluorescent detection of phosphorylated AHNAK. (A) Purified phosphospecific antibodies (pS5535-AHNAK) were used for Western blot analysis of HEK293 cells transiently transfected with two COOH-terminal expression constructs of AHNAK differing only in one amino acid residue; serine 5535 was either intact (wt) or substituted by alanine (S5535A). As a control, an identical Western blot was detected with antibody fraction from the same rabbit before immunoaffinity purification on phosphopeptide column. (B) Reactivity of phosphospecific antibodies with the endogenous AHNAK is greatly reduced in cells treated with LY294002. Western blot analysis of AHNAK was performed with cells extracts prepared before and after treatment with 30 μM LY294002 for 1 h using purified phosphospecific antibodies. Antiserum against repeat region of AHNAK was used as a control for the total AHNAK protein levels (anti-AHNAK). Activity of PKB in cell extracts was monitored by anti-PKB antibodies as described in the legend to Fig. 2. (C) Western blot analysis of AHNAK phosphorylation on serine 5535 in MDCK neo (control) and caPKB clone expressing caPKB. Cells were either untreated or treated with 30 μM LY294002 for 4 h. Blots were probed with antibodies indicated as in B. (D) Purified phosphospecific antibodies detect preferentially plasma membrane–associated AHNAK in dense MDCK cells but react only weakly with the nuclear AHNAK in cells treated for 1 h with LY294002. Inclusion of the immunizing phosphopeptide abolished reactivity of antibodies with cells (unpublished data). (E) Western blot analysis of AHNAK contents in subcellular fractions prepared from sparsely and densely growing cells. HeLa cells were fractionated into nuclear (N) and cytoplasmic (C) fractions by hypotonic lysis. Aliquots of fractions and total lysates (T) representing approximately equal cell numbers were subjected to electrophoresis and Western blotting with the indicated anti-AHNAK antibodies.
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fig4: Characterization of phosphospecific antibodies against PKB-phosphorylated AHNAK and immunofluorescent detection of phosphorylated AHNAK. (A) Purified phosphospecific antibodies (pS5535-AHNAK) were used for Western blot analysis of HEK293 cells transiently transfected with two COOH-terminal expression constructs of AHNAK differing only in one amino acid residue; serine 5535 was either intact (wt) or substituted by alanine (S5535A). As a control, an identical Western blot was detected with antibody fraction from the same rabbit before immunoaffinity purification on phosphopeptide column. (B) Reactivity of phosphospecific antibodies with the endogenous AHNAK is greatly reduced in cells treated with LY294002. Western blot analysis of AHNAK was performed with cells extracts prepared before and after treatment with 30 μM LY294002 for 1 h using purified phosphospecific antibodies. Antiserum against repeat region of AHNAK was used as a control for the total AHNAK protein levels (anti-AHNAK). Activity of PKB in cell extracts was monitored by anti-PKB antibodies as described in the legend to Fig. 2. (C) Western blot analysis of AHNAK phosphorylation on serine 5535 in MDCK neo (control) and caPKB clone expressing caPKB. Cells were either untreated or treated with 30 μM LY294002 for 4 h. Blots were probed with antibodies indicated as in B. (D) Purified phosphospecific antibodies detect preferentially plasma membrane–associated AHNAK in dense MDCK cells but react only weakly with the nuclear AHNAK in cells treated for 1 h with LY294002. Inclusion of the immunizing phosphopeptide abolished reactivity of antibodies with cells (unpublished data). (E) Western blot analysis of AHNAK contents in subcellular fractions prepared from sparsely and densely growing cells. HeLa cells were fractionated into nuclear (N) and cytoplasmic (C) fractions by hypotonic lysis. Aliquots of fractions and total lysates (T) representing approximately equal cell numbers were subjected to electrophoresis and Western blotting with the indicated anti-AHNAK antibodies.
Mentions: The unusually large size of AHNAK and the presence of numerous phosphorylation sites for a variety of kinases within its sequence precluded phosphopeptide mapping as a means of identification of the PKB-phosphorylated residue. To determine whether PKB phosphorylates AHNAK in vivo, we generated antisera to phosphopeptide containing serine 5535. Antibodies specifically recognizing AHNAK phosphopeptide containing phosphorylated serine 5535 were purified and their specificity was examined. Fig. 4 A shows that purified phosphospecific antibodies specifically recognize an exogenously expressed COOH-terminal polypeptide of AHNAK, but not a version that has serine 5535 mutated to an alanine residue.

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