ATM kinase phosphorylates Ser15 of p53 in a pH-dependent manner

Year 2024, Volume: 25 Issue: 2, 177 - 186, 15.10.2024

Serap Pektaş

https://doi.org/10.23902/trkjnat.1499251

Abstract

The phosphorylation of Ser15 in the transactivation domain (TAD) of the tumor suppressor protein 53 (p53) by ataxia-telangiectasia mutated (ATM) kinase is a crucial step in the tumor suppressor function of p53. An understanding of the factors that affect the rate of Ser15 phosphorylation may provide new strategies for the manipulation of the ATM-p53 pathway in cancer therapy. In this study, the effect of electrostatic interactions between ATM and p53 was investigated by measuring the phosphorylation of Ser15 at varying pH ranges from 5 to 9. To achieve this, two different kinase assay methods were utilized: the ELISA technique, which directly quantifies the phosphorylated Ser15, and the Universal Kinase Assay, which assesses the formation of ADP. The results revealed that Ser15 phosphorylation was pH-dependent, with higher phosphorylation rates observed in the alkaline range. To ascertain whether the lower phosphorylation rates observed at acidic pH were due to protein denaturation, a pH-dependent solubility profile was generated using the CamSol server. The obtained results demonstrated comparable solubility rates within the pH range of the kinase assays performed. Furthermore, the significance of negatively charged residues in TAD1-39 was evaluated by substituting Asp and Glu residues with hydrophobic and uncharged hydrophilic residues in TAD1-39 using ChimeraX and subsequently comparing their interactions with the ATM using the protein-protein docking server HADDOCK2.4. The results of the docking simulations indicated that the alteration of negatively charged residues with uncharged ones resulted in a reduction in the efficiency of the interaction between the ATM and TAD1-39. In conclusion, it can be stated that electrostatic interactions between the ATM and TAD are important for optimal Ser15 phosphorylation.

Keywords

ATM kinase, enzyme-substrate interactions, kinase assays, p53

Ethical Statement

Since the article does not contain any studies with human or animal subject, its approval to the ethics committee was not required.

Supporting Institution

Recep Tayyip Erdogan University Scientific Research Projects Unit and The Scientific and Technological Research Council of Türkiye (TUBİTAK)

Project Number

RTEU-BAP FB-2019 , TUBITAK 116Z360

Thanks

The author would like to thank Professor Ali O. Kılıç (Trabzon, Türkiye) for kindly allowing the use of the cell culture laboratory of Karadeniz Technical University, Medical Microbiology Department, for the production of ATM kinase.

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