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Updated: Apr 30, 2026
Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach
Published on: February 21, 2019
Differential phosphorylation of Akt1 and Akt2 by protein kinase CK2 may account for isoform specific functions
Cristina Girardi1, Peter James2, Sofia Zanin1
1Department of Biomedical Sciences, University of Padova, Via U. Bassi 58/b, 35131 Padova, Italy; CNR Institute of Neurosciences, Via U. Bassi 58/b, 35131 Padova, Italy.
Abstract:
Akt (also known as PKB) is a survival kinase frequently up-regulated in cancer; three isoforms of Akt exist, and among them Akt1 and Akt2 are the most widely and highly expressed. They share the same structure and activation mechanism and have many overlapping functions; nevertheless isoform-specific roles and substrates have been reported, which are expected to rely on sequence diversities. In particular, a special role in differentiating Akt1 and Akt2 isoforms has been assigned to the linker region, a short segment between the PH and the catalytic domains. We have previously found that a residue in the linker region (Ser129) is directly phosphorylated by protein kinase CK2 in Akt1; the phosphorylation of the homologous residue in Akt2 (Ser131) has never been analyzed. Here we show that Akt2, endogenously or ectopically expressed in different cell lines, is not phosphorylated on Ser131 by CK2, while in vitro recombinant Akt2 is a CK2 substrate. These data support the hypothesis that in vivo a steric hindrance occurs which prevents the access to the CK2 site. Additionally, we have found that Ser129 phosphorylation is involved in the recognition of the Akt1-specific substrate palladin; this observation provides an explanation of why Akt2, lacking Ser131 phosphorylation in the linker region, has a low efficiency in targeting palladin. CK2-dependent phosphorylation is therefore a crucial event which, discriminating between Akt1 and Akt2, can account for different substrate specificities, and, more in general, for fine tuning of Akt activity in the control of isoform-dependent processes.
Insights
Protein kinase CK2 phosphorylation of Akt1, but not Akt2, in vivo influences substrate specificity. This isoform-specific phosphorylation fine-tunes Akt activity for cellular processes.
Area of Science:
- Molecular Biology
- Biochemistry
- Cancer Research
Background:
- Akt (also known as Protein Kinase B or PKB) is a crucial survival kinase often overexpressed in various cancers.
- Three Akt isoforms (Akt1, Akt2, Akt3) exist, with Akt1 and Akt2 being the most prevalent, sharing structural similarities and activation pathways.
- Despite functional overlap, isoform-specific roles suggest sequence variations, particularly in the linker region, dictate distinct substrate interactions.
Purpose of the Study:
- To investigate the phosphorylation of Akt2 at Ser131 by protein kinase CK2 and compare it with Akt1's Ser129 phosphorylation.
- To elucidate the mechanism behind potential differences in CK2-mediated phosphorylation between Akt1 and Akt2 isoforms in vivo.
- To determine the functional consequence of differential Akt isoform phosphorylation on substrate recognition, specifically for palladin.
Main Methods:
- Analysis of endogenous and ectopically expressed Akt2 phosphorylation status at Ser131 in various cell lines.
- In vitro kinase assays using recombinant Akt2 to assess direct CK2 phosphorylation.
- Investigation of the role of Akt1 Ser129 phosphorylation in the interaction with the substrate palladin.
Main Results:
- Endogenous and ectopically expressed Akt2 is not phosphorylated by CK2 on Ser131 in vivo, unlike Akt1 on Ser129.
- Recombinant Akt2 is phosphorylated by CK2 in vitro, suggesting steric hindrance prevents CK2 access to the site in vivo.
- Akt1 Ser129 phosphorylation is essential for recognizing the specific substrate palladin, explaining Akt2's lower efficiency.
Conclusions:
- CK2-dependent phosphorylation acts as a critical discriminator between Akt1 and Akt2 isoforms in vivo.
- This differential phosphorylation influences substrate specificity and contributes to fine-tuning isoform-dependent cellular processes.
- The findings highlight the importance of post-translational modifications in regulating Akt isoform function and downstream signaling in cancer.
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