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Akt phosphorylation on Thr308 but not on Ser473 correlates with Akt protein kinase activity in human non-small cell lung cancer.

Vincent EE, Elder DJ, Thomas EC, Phillips L, Morgan C, Pawade J, Sohail M, May MT, Hetzel MR, Tavaré JM - Br. J. Cancer (2011)

Bottom Line: Akt activation involves the phosphorylation of two residues: threonine 308 (Thr308) in the activation loop and serine 473 (Ser473) in the C-terminal hydrophobic motif.The phosphorylation status of Thr308 and Ser473 was compared with that of three separate Akt substrates - PRAS40, TSC2 and TBC1D4 - in fresh frozen samples of early-stage human non-small cell lung cancer (NSCLC).Akt Thr308 phosphorylation correlated with the phosphorylation of each Akt substrate tested, whereas Akt Ser473 phosphorylation did not correlate with the phosphorylation of any of the substrates examined.

View Article: PubMed Central - PubMed

Affiliation: School of Biochemistry, Medical Sciences Building, University of Bristol, Bristol BS8 1TD, UK.

ABSTRACT

Background: The activity of the protein kinase Akt is frequently dysregulated in cancer and is an important factor in the growth and survival of tumour cells. Akt activation involves the phosphorylation of two residues: threonine 308 (Thr308) in the activation loop and serine 473 (Ser473) in the C-terminal hydrophobic motif. Phosphorylation of Ser473 has been extensively studied in tumour samples as a correlate for Akt activity, yet the phosphorylation of Thr308 or of downstream Akt substrates is rarely assessed.

Methods: The phosphorylation status of Thr308 and Ser473 was compared with that of three separate Akt substrates - PRAS40, TSC2 and TBC1D4 - in fresh frozen samples of early-stage human non-small cell lung cancer (NSCLC).

Results: Akt Thr308 phosphorylation correlated with the phosphorylation of each Akt substrate tested, whereas Akt Ser473 phosphorylation did not correlate with the phosphorylation of any of the substrates examined.

Conclusion: The phosphorylation of Thr308 is a more reliable biomarker for the protein kinase activity of Akt in tumour samples than Ser473. Any evaluation of the link between Akt phosphorylation or activity in tumour samples and the prediction or prognosis of disease should, therefore, focus on measuring the phosphorylation of Akt on Thr308 and/or at least one downstream Akt substrate, rather than Akt Ser473 phosphorylation alone.

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Examination of the correlation between Akt Thr308 and Ser473 phosphorylation and the phosphorylation of Akt substrates. Triplicate samples of normal and patient-matched tumour tissues were separated on SDS–PAGE gels. Phosphorylation of Akt (on Thr308 and Ser473) and three downstream substrate proteins (PRAS40-Thr246, TSC2-Ser939 and TBC1D4-Thr642) was determined by western blotting with phospho-specific antibodies. The data were quantified by densitometry and the strength of evidence of difference in phosphorylation between the normal and tumour samples was determined by Kruskal–Wallis test. Phosphorylation of each protein/site was then coded according to whether it increased (black), decreased (grey) at P⩽0.05 or remained unchanged (white; P>0.05) in the tumour samples relative to normal tissue. The coded data were then grouped according to the direction of change in Thr308 phosphorylation: patients in group I showed an increase, group 2 a decrease and group 3 no change. Group I were further subdivided according to the change in Akt Ser473 phosphorylation (group Ia showing an increase or no change, and group Ib a decrease).
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fig3: Examination of the correlation between Akt Thr308 and Ser473 phosphorylation and the phosphorylation of Akt substrates. Triplicate samples of normal and patient-matched tumour tissues were separated on SDS–PAGE gels. Phosphorylation of Akt (on Thr308 and Ser473) and three downstream substrate proteins (PRAS40-Thr246, TSC2-Ser939 and TBC1D4-Thr642) was determined by western blotting with phospho-specific antibodies. The data were quantified by densitometry and the strength of evidence of difference in phosphorylation between the normal and tumour samples was determined by Kruskal–Wallis test. Phosphorylation of each protein/site was then coded according to whether it increased (black), decreased (grey) at P⩽0.05 or remained unchanged (white; P>0.05) in the tumour samples relative to normal tissue. The coded data were then grouped according to the direction of change in Thr308 phosphorylation: patients in group I showed an increase, group 2 a decrease and group 3 no change. Group I were further subdivided according to the change in Akt Ser473 phosphorylation (group Ia showing an increase or no change, and group Ib a decrease).

Mentions: We next coded the data for Thr308 and Ser473 phosphorylation according to whether they increased (black) or decreased (grey) at P<0.05. The coded data are represented within the chart in Figure 3 and are grouped according to the direction of change in Thr308 phosphorylation. Of the 12 cases where Thr308 phosphorylation was increased relative to normal tissue (termed group Ia tumours in Figure 3), Ser473 phosphorylation was increased in parallel in only 6 cases. In three tumours (designated group Ib), Ser473 phosphorylation was regulated in a diametrically opposite manner to Thr308 phosphorylation.


Akt phosphorylation on Thr308 but not on Ser473 correlates with Akt protein kinase activity in human non-small cell lung cancer.

Vincent EE, Elder DJ, Thomas EC, Phillips L, Morgan C, Pawade J, Sohail M, May MT, Hetzel MR, Tavaré JM - Br. J. Cancer (2011)

Examination of the correlation between Akt Thr308 and Ser473 phosphorylation and the phosphorylation of Akt substrates. Triplicate samples of normal and patient-matched tumour tissues were separated on SDS–PAGE gels. Phosphorylation of Akt (on Thr308 and Ser473) and three downstream substrate proteins (PRAS40-Thr246, TSC2-Ser939 and TBC1D4-Thr642) was determined by western blotting with phospho-specific antibodies. The data were quantified by densitometry and the strength of evidence of difference in phosphorylation between the normal and tumour samples was determined by Kruskal–Wallis test. Phosphorylation of each protein/site was then coded according to whether it increased (black), decreased (grey) at P⩽0.05 or remained unchanged (white; P>0.05) in the tumour samples relative to normal tissue. The coded data were then grouped according to the direction of change in Thr308 phosphorylation: patients in group I showed an increase, group 2 a decrease and group 3 no change. Group I were further subdivided according to the change in Akt Ser473 phosphorylation (group Ia showing an increase or no change, and group Ib a decrease).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3111153&req=5

fig3: Examination of the correlation between Akt Thr308 and Ser473 phosphorylation and the phosphorylation of Akt substrates. Triplicate samples of normal and patient-matched tumour tissues were separated on SDS–PAGE gels. Phosphorylation of Akt (on Thr308 and Ser473) and three downstream substrate proteins (PRAS40-Thr246, TSC2-Ser939 and TBC1D4-Thr642) was determined by western blotting with phospho-specific antibodies. The data were quantified by densitometry and the strength of evidence of difference in phosphorylation between the normal and tumour samples was determined by Kruskal–Wallis test. Phosphorylation of each protein/site was then coded according to whether it increased (black), decreased (grey) at P⩽0.05 or remained unchanged (white; P>0.05) in the tumour samples relative to normal tissue. The coded data were then grouped according to the direction of change in Thr308 phosphorylation: patients in group I showed an increase, group 2 a decrease and group 3 no change. Group I were further subdivided according to the change in Akt Ser473 phosphorylation (group Ia showing an increase or no change, and group Ib a decrease).
Mentions: We next coded the data for Thr308 and Ser473 phosphorylation according to whether they increased (black) or decreased (grey) at P<0.05. The coded data are represented within the chart in Figure 3 and are grouped according to the direction of change in Thr308 phosphorylation. Of the 12 cases where Thr308 phosphorylation was increased relative to normal tissue (termed group Ia tumours in Figure 3), Ser473 phosphorylation was increased in parallel in only 6 cases. In three tumours (designated group Ib), Ser473 phosphorylation was regulated in a diametrically opposite manner to Thr308 phosphorylation.

Bottom Line: Akt activation involves the phosphorylation of two residues: threonine 308 (Thr308) in the activation loop and serine 473 (Ser473) in the C-terminal hydrophobic motif.The phosphorylation status of Thr308 and Ser473 was compared with that of three separate Akt substrates - PRAS40, TSC2 and TBC1D4 - in fresh frozen samples of early-stage human non-small cell lung cancer (NSCLC).Akt Thr308 phosphorylation correlated with the phosphorylation of each Akt substrate tested, whereas Akt Ser473 phosphorylation did not correlate with the phosphorylation of any of the substrates examined.

View Article: PubMed Central - PubMed

Affiliation: School of Biochemistry, Medical Sciences Building, University of Bristol, Bristol BS8 1TD, UK.

ABSTRACT

Background: The activity of the protein kinase Akt is frequently dysregulated in cancer and is an important factor in the growth and survival of tumour cells. Akt activation involves the phosphorylation of two residues: threonine 308 (Thr308) in the activation loop and serine 473 (Ser473) in the C-terminal hydrophobic motif. Phosphorylation of Ser473 has been extensively studied in tumour samples as a correlate for Akt activity, yet the phosphorylation of Thr308 or of downstream Akt substrates is rarely assessed.

Methods: The phosphorylation status of Thr308 and Ser473 was compared with that of three separate Akt substrates - PRAS40, TSC2 and TBC1D4 - in fresh frozen samples of early-stage human non-small cell lung cancer (NSCLC).

Results: Akt Thr308 phosphorylation correlated with the phosphorylation of each Akt substrate tested, whereas Akt Ser473 phosphorylation did not correlate with the phosphorylation of any of the substrates examined.

Conclusion: The phosphorylation of Thr308 is a more reliable biomarker for the protein kinase activity of Akt in tumour samples than Ser473. Any evaluation of the link between Akt phosphorylation or activity in tumour samples and the prediction or prognosis of disease should, therefore, focus on measuring the phosphorylation of Akt on Thr308 and/or at least one downstream Akt substrate, rather than Akt Ser473 phosphorylation alone.

Show MeSH
Related in: MedlinePlus