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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

Activation of GSK-3 kinase leads to a phosphorylation induced acidic shift of hNaa10p. Cells were in vivo stimulated with Wortmannin 1 μM for 8 hours, cells which remained untreated served as control. Furthermore, Wortmannin treated cells were divided in to two aliquots, one was treated with 20 U/ml CIAP enzyme, while the other remained untreated and served as negative control for this experiment. 50 μg of resuspended proteins were analyzed by 2D-SDS PAGE using 13 cm IPG pH 4–7 for IEF, and hNaa10p was detected by immunoblotting using anti-hNaa10p. Molecular size markers are indicated to the left. (A) control, unstimulated cells, and (B) cells were stimulated with 1 μM/8 hours Wortmannin. (C) Cells were stimulated with 1 μM/8 hours Wortmannin in vivo (control for D), and (D) cells were stimulated with 1 μM/8 hours Wortmannin in vivo, and the purified proteins from these cells were treated with CIAP in vitro.
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Figure 2: Activation of GSK-3 kinase leads to a phosphorylation induced acidic shift of hNaa10p. Cells were in vivo stimulated with Wortmannin 1 μM for 8 hours, cells which remained untreated served as control. Furthermore, Wortmannin treated cells were divided in to two aliquots, one was treated with 20 U/ml CIAP enzyme, while the other remained untreated and served as negative control for this experiment. 50 μg of resuspended proteins were analyzed by 2D-SDS PAGE using 13 cm IPG pH 4–7 for IEF, and hNaa10p was detected by immunoblotting using anti-hNaa10p. Molecular size markers are indicated to the left. (A) control, unstimulated cells, and (B) cells were stimulated with 1 μM/8 hours Wortmannin. (C) Cells were stimulated with 1 μM/8 hours Wortmannin in vivo (control for D), and (D) cells were stimulated with 1 μM/8 hours Wortmannin in vivo, and the purified proteins from these cells were treated with CIAP in vitro.

Mentions: The ELM search suggested that hNaa10p might be phosphorylated at 7 residues by GSK-3 (Table 1). We therefore investigated whether inhibition or activation of GSK-3 would result in changes in hNaa10p 2D-PAGE mobility consistent with a change in its phosphorylation state. GSK-3 is a highly conserved and widely expressed serine/threonine protein kinase that plays a central role in the regulation of several physiological processes [13]. Inhibition of phosphatidyl inositol 3 kinases (PI3Ks) by Wortmannin inactivates Akt kinase, which results in the indirect activation of GSK-3 kinase [14]. Thus, addition of Wortmannin should alter the hNaa10p 2D-PAGE mobility pattern if hNaa10p is a GSK-3 substrate. There was an acidic shift of the hNaa10p protein isoforms as a consequence of Wortmannin treatment in vivo as compared to control cells (Figure 2A–B), suggesting that hNaa10p is more extensively phosphorylated in the Wortmannin treated cells than in the non-treated cells. The mobility shift corresponded to the addition of 4–6 phosphate groups, estimated using bioinformatic tools . To further investigate the acidic shift caused by Wortmannin treatment, the experiment was combined with in vitro treatment with Calf Intestine Alkaline Phosphatase (CIAP). HEK293 cells were treated with Wortmannin; the protein content of the cells was divided into two aliquots, one aliquot was added CIAP, while the other remained untreated serving as control. The results indicate that in vivo stimulation of the cells by Wortmannin induces an acidic shift of the protein as expected (Figure 2C), while in vitro treatment with CIAP caused reversal of the acidic shift (Figure 2D). This indicates that the acidic shift was mainly caused by phosphorylation induced by GSK-3 activation, suggesting that GSK-3 kinase is involved in the phosphorylation of hNaa10p.


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

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

Activation of GSK-3 kinase leads to a phosphorylation induced acidic shift of hNaa10p. Cells were in vivo stimulated with Wortmannin 1 μM for 8 hours, cells which remained untreated served as control. Furthermore, Wortmannin treated cells were divided in to two aliquots, one was treated with 20 U/ml CIAP enzyme, while the other remained untreated and served as negative control for this experiment. 50 μg of resuspended proteins were analyzed by 2D-SDS PAGE using 13 cm IPG pH 4–7 for IEF, and hNaa10p was detected by immunoblotting using anti-hNaa10p. Molecular size markers are indicated to the left. (A) control, unstimulated cells, and (B) cells were stimulated with 1 μM/8 hours Wortmannin. (C) Cells were stimulated with 1 μM/8 hours Wortmannin in vivo (control for D), and (D) cells were stimulated with 1 μM/8 hours Wortmannin in vivo, and the purified proteins from these cells were treated with CIAP in vitro.
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Figure 2: Activation of GSK-3 kinase leads to a phosphorylation induced acidic shift of hNaa10p. Cells were in vivo stimulated with Wortmannin 1 μM for 8 hours, cells which remained untreated served as control. Furthermore, Wortmannin treated cells were divided in to two aliquots, one was treated with 20 U/ml CIAP enzyme, while the other remained untreated and served as negative control for this experiment. 50 μg of resuspended proteins were analyzed by 2D-SDS PAGE using 13 cm IPG pH 4–7 for IEF, and hNaa10p was detected by immunoblotting using anti-hNaa10p. Molecular size markers are indicated to the left. (A) control, unstimulated cells, and (B) cells were stimulated with 1 μM/8 hours Wortmannin. (C) Cells were stimulated with 1 μM/8 hours Wortmannin in vivo (control for D), and (D) cells were stimulated with 1 μM/8 hours Wortmannin in vivo, and the purified proteins from these cells were treated with CIAP in vitro.
Mentions: The ELM search suggested that hNaa10p might be phosphorylated at 7 residues by GSK-3 (Table 1). We therefore investigated whether inhibition or activation of GSK-3 would result in changes in hNaa10p 2D-PAGE mobility consistent with a change in its phosphorylation state. GSK-3 is a highly conserved and widely expressed serine/threonine protein kinase that plays a central role in the regulation of several physiological processes [13]. Inhibition of phosphatidyl inositol 3 kinases (PI3Ks) by Wortmannin inactivates Akt kinase, which results in the indirect activation of GSK-3 kinase [14]. Thus, addition of Wortmannin should alter the hNaa10p 2D-PAGE mobility pattern if hNaa10p is a GSK-3 substrate. There was an acidic shift of the hNaa10p protein isoforms as a consequence of Wortmannin treatment in vivo as compared to control cells (Figure 2A–B), suggesting that hNaa10p is more extensively phosphorylated in the Wortmannin treated cells than in the non-treated cells. The mobility shift corresponded to the addition of 4–6 phosphate groups, estimated using bioinformatic tools . To further investigate the acidic shift caused by Wortmannin treatment, the experiment was combined with in vitro treatment with Calf Intestine Alkaline Phosphatase (CIAP). HEK293 cells were treated with Wortmannin; the protein content of the cells was divided into two aliquots, one aliquot was added CIAP, while the other remained untreated serving as control. The results indicate that in vivo stimulation of the cells by Wortmannin induces an acidic shift of the protein as expected (Figure 2C), while in vitro treatment with CIAP caused reversal of the acidic shift (Figure 2D). This indicates that the acidic shift was mainly caused by phosphorylation induced by GSK-3 activation, suggesting that GSK-3 kinase is involved in the phosphorylation of hNaa10p.

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