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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

hNaa10p in confluent and active cells. 50 μg proteins of each sample resuspended in rehydration buffer and analyzed by 2D-SDS PAGE using 13 cm IPG pH 4–7 for IEF. hNaa10p was detected by immunoblotting using anti-hNaa10p. The molecular size markers are indicated to the left. (A) Resting cells were grown for near 6 days without medium change. (B) Active cells were splitted into fresh medium 16 hours before harvesting.
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Figure 5: hNaa10p in confluent and active cells. 50 μg proteins of each sample resuspended in rehydration buffer and analyzed by 2D-SDS PAGE using 13 cm IPG pH 4–7 for IEF. hNaa10p was detected by immunoblotting using anti-hNaa10p. The molecular size markers are indicated to the left. (A) Resting cells were grown for near 6 days without medium change. (B) Active cells were splitted into fresh medium 16 hours before harvesting.

Mentions: hNaa10p was then investigated in HEK293 cells grown to confluence without medium change (for 6 days), these cell cultures are in a steady state with respect to cell number per dish and most cells are presumably in the G0-phase (Figure 5A). These cultures showed no signs of apoptosis as observed by light microscopy. For comparison the hNaa10p status was also analysed in extracts from rapidly dividing HEK293 cells subcultivated with fresh medium 16 hours before harvest (Figure 5B). The results indicate that hNaa10p is more phosphorylated in resting cells than in actively dividing cells.


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

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

hNaa10p in confluent and active cells. 50 μg proteins of each sample resuspended in rehydration buffer and analyzed by 2D-SDS PAGE using 13 cm IPG pH 4–7 for IEF. hNaa10p was detected by immunoblotting using anti-hNaa10p. The molecular size markers are indicated to the left. (A) Resting cells were grown for near 6 days without medium change. (B) Active cells were splitted into fresh medium 16 hours before harvesting.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2654568&req=5

Figure 5: hNaa10p in confluent and active cells. 50 μg proteins of each sample resuspended in rehydration buffer and analyzed by 2D-SDS PAGE using 13 cm IPG pH 4–7 for IEF. hNaa10p was detected by immunoblotting using anti-hNaa10p. The molecular size markers are indicated to the left. (A) Resting cells were grown for near 6 days without medium change. (B) Active cells were splitted into fresh medium 16 hours before harvesting.
Mentions: hNaa10p was then investigated in HEK293 cells grown to confluence without medium change (for 6 days), these cell cultures are in a steady state with respect to cell number per dish and most cells are presumably in the G0-phase (Figure 5A). These cultures showed no signs of apoptosis as observed by light microscopy. For comparison the hNaa10p status was also analysed in extracts from rapidly dividing HEK293 cells subcultivated with fresh medium 16 hours before harvest (Figure 5B). The results indicate that hNaa10p is more phosphorylated in resting cells than in actively dividing 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