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The protein Nalpha-terminal acetyltransferase hNaa10p (hArd1) is phosphorylated in HEK293 cells.

Målen H, Lillehaug JR, Arnesen T - BMC Res Notes (2009)

Bottom Line: The NatA complex is associated with ribosomes and cotranslationally acetylates human proteins with Ser-, Ala-, Thr-, Val-, and Gly- N-termini after the initial Met- has been removed.A direct or indirect role of GSK-3 kinase in regulating hNaa10p phosphorylation is supported by the observed effects of Wortmannin and LiCl, a GSK-3 activator and inhibitor, respectively.We demonstrate that hNaa10p protein is phosphorylated in cell culture potentially pointing at phosphorylation as a means of regulating the function of one of the major Nalpha-terminal acetyltransferases in human cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biology, University of Bergen, N-5020 Bergen, Norway. hiwa.malen@gades.uib.no

ABSTRACT

Background: The hNaa10p (hArd1) protein is the catalytic subunit of the human NatA Nalpha-terminal acetyltransferase complex. The NatA complex is associated with ribosomes and cotranslationally acetylates human proteins with Ser-, Ala-, Thr-, Val-, and Gly- N-termini after the initial Met- has been removed. In the flexible C-terminal tail of hNaa10p, there are several potential phosphorylation sites that might serve as points of regulation.

Findings: Using 2D-gel electrophoresis and hNaa10p specific antibodies, we have investigated whether hNaa10p is phosphorylated in HEK293 cells. Several differently charged forms of hNaa10p are present in HEK293 cells and treatment with Calf Intestine Alkaline Phophatase (CIAP) strongly suggests that hNaa10p is phosphorylated at multiple sites under various cell culture conditions. A direct or indirect role of GSK-3 kinase in regulating hNaa10p phosphorylation is supported by the observed effects of Wortmannin and LiCl, a GSK-3 activator and inhibitor, respectively.

Conclusion: We demonstrate that hNaa10p protein is phosphorylated in cell culture potentially pointing at phosphorylation as a means of regulating the function of one of the major Nalpha-terminal acetyltransferases in human cells.

No MeSH data available.


Related in: MedlinePlus

The phosphorylation of hNaa10p caused by activation of GSK-3 kinase is reversed by a GSK-3 inhibitor. Cells were stimulated in vivo with Wortmannin or both LiCl + Wortmannin. Cells stimulated with neither LiCl nor Wortmannin served as negative control. 50 μg of the resuspended proteins in rehydration buffer were analyzed by 2D-SDS PAGE using 13 cm IPG pH 4–7 for IEF, and hNaa10p was detected by immunoblotting using anti-hNaa10p. The molecular size markers are indicated to the left. (A) negative control, cells were unstimulated, (B) cells were stimulated by Wortmannin in vivo (1 μM/8 hours), and (C) cells were stimulated with both Wortmannin (1 μM/8 hours) and LiCl (50 mM) in vivo.
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Figure 3: The phosphorylation of hNaa10p caused by activation of GSK-3 kinase is reversed by a GSK-3 inhibitor. Cells were stimulated in vivo with Wortmannin or both LiCl + Wortmannin. Cells stimulated with neither LiCl nor Wortmannin served as negative control. 50 μg of the resuspended proteins in rehydration buffer were analyzed by 2D-SDS PAGE using 13 cm IPG pH 4–7 for IEF, and hNaa10p was detected by immunoblotting using anti-hNaa10p. The molecular size markers are indicated to the left. (A) negative control, cells were unstimulated, (B) cells were stimulated by Wortmannin in vivo (1 μM/8 hours), and (C) cells were stimulated with both Wortmannin (1 μM/8 hours) and LiCl (50 mM) in vivo.

Mentions: Further and to confirm GSK-3 involvement in hNaa10p phosphorylation, we applied in vivo treatment of cells with LiCl, an inhibitor of GSK-3 and one of the most effective drugs for the treatment of bipolar (manic-depressive) disorder [13]. To further explore the possibility that hNaa10p protein is phosphorylated by GSK-3 kinase, control cells (Figure 3A) were compared to cells treated with Wortmannin (Figure 3B) or Wortmannin and LiCl in combination (Figure 3C). The Wortmannin treatment results (Figure 3B) confirmed the results presented in Figure 2, that Wortmannin indirectly activates GSK-3 and induces acidic shifts of several hNaa10p isoforms. In this experiment, however, we only observed a Wortmannin-induced mobility shift corresponding to 1–2 phosphorylation sites. Addition of the GSK-3 inhibitor LiCl to cell cultures receiving Wortmannin counteracted the Wortmannin-mediated phosphorylation of hNaa10p (Figure 3C). This result strengthens further that GSK-3 phosphorylates hNaa10p in HEK293 cells.


The protein Nalpha-terminal acetyltransferase hNaa10p (hArd1) is phosphorylated in HEK293 cells.

Målen H, Lillehaug JR, Arnesen T - BMC Res Notes (2009)

The phosphorylation of hNaa10p caused by activation of GSK-3 kinase is reversed by a GSK-3 inhibitor. Cells were stimulated in vivo with Wortmannin or both LiCl + Wortmannin. Cells stimulated with neither LiCl nor Wortmannin served as negative control. 50 μg of the resuspended proteins in rehydration buffer were analyzed by 2D-SDS PAGE using 13 cm IPG pH 4–7 for IEF, and hNaa10p was detected by immunoblotting using anti-hNaa10p. The molecular size markers are indicated to the left. (A) negative control, cells were unstimulated, (B) cells were stimulated by Wortmannin in vivo (1 μM/8 hours), and (C) cells were stimulated with both Wortmannin (1 μM/8 hours) and LiCl (50 mM) in vivo.
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Figure 3: The phosphorylation of hNaa10p caused by activation of GSK-3 kinase is reversed by a GSK-3 inhibitor. Cells were stimulated in vivo with Wortmannin or both LiCl + Wortmannin. Cells stimulated with neither LiCl nor Wortmannin served as negative control. 50 μg of the resuspended proteins in rehydration buffer were analyzed by 2D-SDS PAGE using 13 cm IPG pH 4–7 for IEF, and hNaa10p was detected by immunoblotting using anti-hNaa10p. The molecular size markers are indicated to the left. (A) negative control, cells were unstimulated, (B) cells were stimulated by Wortmannin in vivo (1 μM/8 hours), and (C) cells were stimulated with both Wortmannin (1 μM/8 hours) and LiCl (50 mM) in vivo.
Mentions: Further and to confirm GSK-3 involvement in hNaa10p phosphorylation, we applied in vivo treatment of cells with LiCl, an inhibitor of GSK-3 and one of the most effective drugs for the treatment of bipolar (manic-depressive) disorder [13]. To further explore the possibility that hNaa10p protein is phosphorylated by GSK-3 kinase, control cells (Figure 3A) were compared to cells treated with Wortmannin (Figure 3B) or Wortmannin and LiCl in combination (Figure 3C). The Wortmannin treatment results (Figure 3B) confirmed the results presented in Figure 2, that Wortmannin indirectly activates GSK-3 and induces acidic shifts of several hNaa10p isoforms. In this experiment, however, we only observed a Wortmannin-induced mobility shift corresponding to 1–2 phosphorylation sites. Addition of the GSK-3 inhibitor LiCl to cell cultures receiving Wortmannin counteracted the Wortmannin-mediated phosphorylation of hNaa10p (Figure 3C). This result strengthens further that GSK-3 phosphorylates hNaa10p in HEK293 cells.

Bottom Line: The NatA complex is associated with ribosomes and cotranslationally acetylates human proteins with Ser-, Ala-, Thr-, Val-, and Gly- N-termini after the initial Met- has been removed.A direct or indirect role of GSK-3 kinase in regulating hNaa10p phosphorylation is supported by the observed effects of Wortmannin and LiCl, a GSK-3 activator and inhibitor, respectively.We demonstrate that hNaa10p protein is phosphorylated in cell culture potentially pointing at phosphorylation as a means of regulating the function of one of the major Nalpha-terminal acetyltransferases in human cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biology, University of Bergen, N-5020 Bergen, Norway. hiwa.malen@gades.uib.no

ABSTRACT

Background: The hNaa10p (hArd1) protein is the catalytic subunit of the human NatA Nalpha-terminal acetyltransferase complex. The NatA complex is associated with ribosomes and cotranslationally acetylates human proteins with Ser-, Ala-, Thr-, Val-, and Gly- N-termini after the initial Met- has been removed. In the flexible C-terminal tail of hNaa10p, there are several potential phosphorylation sites that might serve as points of regulation.

Findings: Using 2D-gel electrophoresis and hNaa10p specific antibodies, we have investigated whether hNaa10p is phosphorylated in HEK293 cells. Several differently charged forms of hNaa10p are present in HEK293 cells and treatment with Calf Intestine Alkaline Phophatase (CIAP) strongly suggests that hNaa10p is phosphorylated at multiple sites under various cell culture conditions. A direct or indirect role of GSK-3 kinase in regulating hNaa10p phosphorylation is supported by the observed effects of Wortmannin and LiCl, a GSK-3 activator and inhibitor, respectively.

Conclusion: We demonstrate that hNaa10p protein is phosphorylated in cell culture potentially pointing at phosphorylation as a means of regulating the function of one of the major Nalpha-terminal acetyltransferases in human cells.

No MeSH data available.


Related in: MedlinePlus