Protein Phosphorylation

• p53 protein must be activated by phosphorylation for transcribing genes that inhibit the cell cycle, activate DNA repair, and in some cases, cause programmed cell death or apoptosis. Any alteration in phosphorylation can inactivate the protein, making the cell cancerous. 
• Phosphorylation of histone modifies the structure of chromatin, altering its protein-protein and DNA-protein interactions. Usually, it occurs when DNA is damaged, opening up space around broken DNA so that repair can happen.

The protein kinases belong to the family of kinases and are responsible for the mechanism of phosphorylation. Activation or deactivation of kinase happens in different ways: through phosphorylation of the kinase itself, binding with activators or inhibitors, or checking their cell localization concerning their substrate.

The catalytic domain of protein kinase has one N- and a C-terminal subdomain, connected by a peptide strand, forming an active site with a front catalytic pocket and a back pocket. The catalytic domain is activated through the phosphorylation of the activation loop or an allosteric mechanism. The kinases also have non-catalytic domains where substrates bind and recruit other signaling proteins.

Mechanism of Protein Phosphorylation

The amino acids undergoing modification have a nucleophilic hydroxyl (-OH) group that attacks the terminal γ-phosphate (PO3^2-) of ATP. Thus, As a result, the phosphate group gets transferred to the amino acid side chain forming the phosphoprotein. For example, the phosphorylation of tyrosine forms phosphotyrosine. Magnesium ions facilitate this transfer, releasing a large amount of free energy when the phosphate–phosphate bond in ATP is broken.

This addition makes the protein hydrophilic polar from hydrophobic apolar, allowing the protein to change conformation when interacting with other molecules.

During the addition of the phosphate group, the nature of the bond formed varies greatly depending on the amino acid.

• In amino acids serine, threonine, and tyrosine, it occurs through phosphodiester bond formation
• In aspartic acid and glutamic acid, it happens through mixed anhydride linkages
• In histidine, lysine, and arginine, it occurs through phosphoramidite bonds

It changes the conformation of the protein, thus making it activated or deactivated. Once activated, the proteins can alter gene expression, cell signaling, and differentiation in cascade-like steps. It affects cellular processes like protein synthesis, cell division, signal transduction, cell growth, development, and aging.

There are several ways in which phosphorylating a protein affects the functioning of that protein:

1. Acting as a Molecular Switch

The activation of protein kinase B through phosphorylation of its Ser and Thr residues regulates cell survival.

2. Regulating Signaling Pathways

Temporary protein-protein interaction helps to regulate many signaling pathways. A typical example is glomerular podocyte protein nephrin 1 (Neph1), which on phosphorylation interacts with an adaptor protein involved, thus activating a cascade of signaling pathways.

In addition, the phosphorylation of a protein regulates signal transduction since it stimulates the subcellular translocation of the phosphorylated protein. The phosphorylation of serine/threonine-protein kinase (Ser350) residue of the death-associated protein (DAP) causes translocation from the cytoplasm to the nucleus of the protein, apoptosis-inducing kinase 2 (DRAK2).

3. Production and Recycling of ATP

The formation of ATP occurs through site-specific phosphorylation of ADP. It supplies the cell with the energy required to perform all cellular activities.

4. Therapeutic Functions

Protein phosphorylation is now a central focus of drug discovery due to the identification of therapeutic targets like protein kinases, protein phosphatases, and the binding domains of phosphoproteins.