Glycogen_Synthase_Kinase_3

The alpha and beta monomers each have an adenosine triphosphate (ATP) binding site between its N- and C-terminal lobes (1). In each of the subunits the N-lobe consists of beta sheets and the C-lobe consists of alpha helices (1). In between the two lobes is the short hinge domain of random coils and is also where the ATP binding site of the protein is located (1, 2). GSK3B also has a paralog, glycogen synthase kinase 3 alpha (PDB ID: 4EKK), which is encoded by a different gene (1, 2). Glycogen synthase kinase 3 alpha (GSK3A) is 51000 daltons but GSK3B only has a molecular weight of 46000 daltons (5). Each of the subunits performs similar catalytic functions, but a major difference is that the function and activity of GSK3B are dependent on the phosphorylation of the Tyr-216 and Tyr-279 while GSK3A is not (5). GSKA and GSKB also function in similar cell signaling pathways and have a significant amount of functional overlap, but each of the proteins have distinct functions as well (2).

GSK3 has been shown to be a master regulator of insulin dependent glycogen synthesis, neurotrophic factor signaling, Wnt signaling, neurotransmitter signaling, and microtubule dynamics in the cell (2). GSK3A and GSK3B both have a significant effect on the cells of the human heart in early development and past the prenatal phase of life (6). Experiments have been done that show that by phosphorylating the Ser-21 residue of GSKA resulted in increased heart failure in mice, but inhibiting GSK3B by phosphorylation of the Ser-9 residue caused a decrease in the hypertrophy of the heart cells (6). In early development GSK3B is involved in the apoptosis signaling cycle of the cell and when it is inhibited the hearts of the mice were shown to be much larger than the control group (6).

GSK3A and GSK3B have also been shown to regulate the insulin dependent glycogen synthesis so that blood sugar levels can e properly maintained (2). When GSK3A and GSK3B are hyperactive it leads to multiple human disorders or neurological development disorders because GSK3 is most often a negative regulator and stops cell signals from going any further (2). The prospect of finding a way to deactivate GSK3 to stop the attenuation of the cellular signaling and has caused GSK3 to become a target of many drugs like SB-216763, AR-A014418, and CHIR99021 (2). The only problem with these drugs is that they are general inhibitors and each inhibit at least nine other kinases in the human body (2). So far these drugs have been shown to help stop disorders like depression, bipolar disorder, and certain types of cancer (2).

The Ser-9 residue of GSK3B is functionally significant because it directly inhibits the substrate from binding to the active site of GSK3B (2). The phosphorylated N-terminal changes its configuration on the protein and blocks the access to the catalytic center and serves as a pseudosubstrate (2). GSK3B also has Ser-389 can also cause inhibition by phosphorylation (6). One main ligand that forms a complex with GSK3 is CHIR99021, but it is a general inhibitor and inhibits a total of eighteen other known kinases. The drug BRD3937 can also bind to the ATP binding domain and directly inhibits protein activity by acting like CHIR99021 but with a greater specificity to GSK3 (2). It is able to bind to the ATP binding site and inhibit protein function because of the hydrogen bonds it can form with the backbone of the carboxyl group on Asp-133 (2). Similarly, another hydrogen bond forms between the backbone carbonyl group of Val-135 and BRD3937 to effectively cover the hinge region of the protein and block the ATP substrates from effectively binding (2). Both CHIR99021 and BRD3937 mimic the binding of ATP, which is why they are so effective in inhibiting the ATP substrate from binding because they both directly compete for the same active site (2).

Magnesium divalent cations are ligands with GSK3B functioning to balancing the negative charge that is on the aspartic acid side chains. Asp-200 and Asp-264 both have side chains with net negative charges so the magnesium cation is placed between the two so it can neutralize their negative charge (1, 5). This process can be inhibited by adding lithium cations to the solution because those cations directly compete with the magnesium cations, inhibiting the activity of GSK3B by interrupting the ligand complex that would normally be formed with magnesium cations (2). It has also been shown that when another divalent cation is introduced to the solution, it inhibits the activity of GSK3 by competing with the magnesium cations (8). The extraneous divalent cation competes with the magnesium cation and has been shown to be more efficient in inhibiting the activity of GSK3 than lithium (8). Lithium cations have been used to inhibit GSK3 and are used as the primary treatment for bipolar disorder, but the efficiency of divalent cations to inhibit GSK3 will open more possibilities for drugs combating bipolar disorder (2, 8).

Kinases phosphorylate their substrates and cause many different effects for cell signaling pathways. Cyclin dependent kinase 2 (CDK2), another protein naturally found in Homo sapiens, was shown to be similar to the GSK3, but its function is to phosphorylate substrates involved in the cell cycle (3). The similarity of the two proteins was calculated by performing a bioinformatics search using the Dali server and PSI-Blast. The Dali server calculates the similarity of the proteins on their tertiary structures using a sum of pairs method and their intermolecular distances. The Z-score is a quantitative measure of the similarity of the two proteins. A Z-score above two indicates significant similarity between their tertiary structures. The Z-score between GSK3 and CDK2 is 34.1, so the two proteins have significantly similar tertiary structures (9). One of the main differences is that CDK2 has a tertiary structure with four subunits as opposed to GSK3, which is a homodimer (1). The four subunits of CDK2 are two pairs of homozygous dimers which explains the Z-score being 34.1 because CDK2 and GSK3 have tertiary structures that consist of homodimers and the proteins have similar folds as well (1). Another comparison of similarities was made using PSI-Blast in order to check the similarity of the primary structures of the proteins. For this, the E value should be below 0.05 to represent a similarity between the structures (10). The E value for CDK2 is 1e-48. The similarities between their sequences show that they both have a tyrosine in one of their subunits that needs to be phosphorylated in order for the proteins to be activated (2, 11). Similarities in the sequence of GSK3 and CDK2 also explain why both proteins form a complex with magnesium divalent cations and ATP. CDK2 also has a ligand complex with a benzene derivative that contains carboxyl amines and tertiary butyl groups (1). The whole ligand is structurally similar to MES and it functions to balance the negative charges from the carboxylic acids on the Asp-86 and Asp-145 side chains of the CDK2 subunits (1). Since CDK2 is a tetramer it allows for more hydrophobic residues in its subunits compared to GSK3 (1). Phe-80 and Phe-186 are in one of the subunits of CDK2 because the other two subunits are able to orient their hydrophobic residues, like Leu-58, so that they interact and leave hydrophilic amino acids with polar side chains, like His-125, exposed on the surface (1). Since GSK3 is only a dimer it is limited in the amount of hydrophobic amino acids that can be in one of the subunits because of the amount of its surface that will be exposed to polar molecules, which leads to some of these differences in the structure between GSK3 and CDK2 (1).

GSK3B is an important serine/threonine kinase that is used throughout the body in cell signaling pathways. GSK3B is a protein that is regulated by phosphorylation at the Tyr-216 and Tyr-279 residues to activate it. It is also a paralog with GSK3A and both the proteins are coded by different genes and fulfill distinct functions in the cell signaling pathways in which they are involved (2, 6). Being able to inhibit GSK3B will lead to drugs that can combat a variety of disorders and diseases including Alzheimer’s disease and depression (2). Many different drug complexes have been tested in order to find a more specific inhibitor instead of the general inhibitors that work on a large variety of serine/ threonine kinases (2). Once a specific drug complex has been found it will be possible to combat diseases caused by the hyperactivity of GSK3B and save many lives.